.an..u)n|v.w'.

§.._.,. .

41.1“?‘34:

;

1‘49 Qua; vain.» f

 

f' . .—.r.4~ ,. u- ’v '

o ‘23:. .IL‘

4"

. . -\
‘ -'f'--v::.l
u I“: i"{u'n

|- ...
. .._. -
W 5 I 1” “- 5Jo“:
Emanélax: «"1
hear." in

‘5 .
“=23? tax?"

*1.
.5?
J

r.r1
QQ‘J‘J
rafl‘luqu'
I ‘3‘". ‘.. _
' }

r2 “

1").'
a A

‘l
_ "“KJ‘ .
. “3 ‘ .."’% F‘”-’
2:33:37! ._ . g; “'1
.VI‘ - '2

    
   
  
  

    
     
  
 
 
       

x
L If? y’rw
I" V O 1 1
. 1 ."~"L1" “’
- L - - ‘. .t. A: Mr .
WSW?! £95» « ' ”

' , .l v
‘71C-“‘ " )xvl‘fl‘. ”.1
. ‘1"-

* l““'l.
h. I

«'1 fit

4

11" “"1

.5”

1f,-I"-n'.J—“l{.v-;5{. 'J‘
‘-.L ‘2.

 

4'4

     

   
 

\' n w ‘ 1;. .‘ W .yh

‘1 I. \. “-2 '. . ‘ .l .l A 1 u‘ 3 I l' " H ¢

I'J'I‘ H I "1‘11": ,':.| ' IJ“‘!,I . 1"" n I ‘ll,l,,1(|'l‘ LI ' I",
"it"! ‘ ‘ g u; ‘ - . > _ . .. 2 J)? '; ‘..‘..,‘l ' hi/q’d J. ,‘
Hf". ,) .v. .‘ .I 1y) |, ‘ _ ‘ ‘ H -_ , . .‘ . ' ‘i- .W H. Inn-y: ”05v“ 1 I"';l

I I ‘. u ' ‘ ‘ ‘ ‘ ‘ A . V f, ‘. ’ L . v > I . '| [HI p ."

:Il "ml'l‘l‘ ‘l ‘. " .."‘::‘ "W t h :2" ", , “ H ‘ . :7. . I M . ‘ ‘t- p. ‘ - l‘ I . ‘ 6". ‘ )‘n‘! I. v "if 1". .ku."‘l.| :16... db
" ., " I... ‘ ‘ .‘ " ." , “ . .‘ ' - I”'\ "4‘. .-'. M ' ' "' -l' 1‘ l'Ev' ”A ',

- In I " “ | . a". W ' ' ' - t . |- ’ ‘ I

““31 fili’fflfl-Ju“f:|.-;'.E.m W 1. " I’ ‘ .‘ -- ' ‘

I 1'“ ‘nw

m. .2. *l v.‘. '
‘,‘.L:"."M...:"}.JQ “MEG "‘x‘t :-

Via, a- .

,‘”,_W-_ I

This is to certify that the

thesis entitled
Prostaglandin Metabolism and Function in Canine
Cortical Collecting Tubule Cells Isolated

Using a Monoclonal Antibody
presented by

Arlyn Garcia-Perez

has been accepted towards fulfillment
of the requirements for

Ph . D. degree in Biochemistry

 

 

WW AM

Major professor
Dr. William L. Smith

[hue May 11, 1984

 

0-7639 MSU is an Affirmative Action/Equal Opportunity Institution

 

 

 

  
   
  
  
  
 
  
  
  
  
  
 
 
 
 
  
  

 

kimono: Alto FUNCTION IN emits cocht
‘ = WE céttS‘ ISOLATED USING A‘ minnow Mmoov

By
Arlyn Garci a-Perez

”Mrwm 1m- . »
NW routs w": ~

 

' ‘n ‘EbviO W03 "“3" "'.A’ DISSERTATION V i.‘ ' t |~ ’ t \"u:

. in. the put“ too
:10? (Oiifltlttg up.- .-

aflils premium: .2; "“i'r'~'f ‘

Submitted to ' _ y ‘ .
MW) mm‘gm Statétfiflvet‘siti ‘* “NM-'-
U {flannel fulfillfintw of the reguirements

".>I wit"
.

1

"WV

‘
V d i‘.‘ many of the nwrprn.o:!ch. an- J‘OI‘Pfizfé‘ ;rooe"ties a!

We cotts Ln situ. LC‘CT

"m“ g9ii‘5 10".»

u to urm,i9%i%“. WWW???

f 3559'.“ “runoff-0&1 ‘ V“

”apnea”

mkmmmmmm '4“ ”at" "It“

. "Act of asmtry seen with intent colic-fling

amine“ «on and in studies «signs to mm

 

  
   
  
  
  
  
  
  
  
  
 
 
 
 
  

A

T ”“131”? ’
& M'W.HBRDSTAGLANDIN METABOLISM AND FUNCTION IN CANINE CORTICAL
H 7"

ABSTRACT

I7 '5atDuECTINs TUBULE CELLS ISOLATED USING A MONOCLONAL ANTIBODY
v

.v_ ‘dfi’d t '

I ?r ‘1

By

Arlyn Garcia-Perez

i‘fiigcthat reacts with an ecto-antigen of the canine renal
:gflecting tubule was selected and cloned. Plastic culture dishes
‘ :«=;=« uith the purified monoclonal antibody were routinely used to
godaoob 107 collecting tubule cells from a mixture of 109 renal
“Sqftdcol cells prepared by treatment of the canine renal cortex with
ifa jgenase. Primary, monoiayer cultures of canine cortical collecting
f: .e (CGCT) cells were established from the absorbed cells. These
V gfiflfls‘ exhibited many of the morphological and biochemical properties of
V'WiLJdess4na»tubuie cells in situ. CCCT cells also formed immunoreactive

.tdwRImsponse to bradykinin, (Asu1'5, Arge) vasopressin

Ifl‘éharacteristics of asymmetry seen with intact coliecting
if. 'These monolayers were used in studies designed to determine

-:€T15 an apical-basalaterai asymmetry to the release of

 

 

   
  
  
 
 
 
  
 
  
 
  
 
  
   
 
 
 
  

. ATthough AVP caused CAMP release only when added to the

it? fioiateral side of CCCT cells, AVP caused the release of PGE2 when
1iiad'ed to either the apical or basolateral surface. Bradykinin caused

'1 PBEZ release only when added to the apical surface of CCCT cells.

{PBEZ was released in comparable amounts on each side of the monolayer
‘l

”.t' in response both to AVP and to bradykinin.

" 5 High concentrations (ZJO's M) of PGE2 added to either side
l

“rhf lief the monolayer caused the release of CAMP. However, at concentra-
{Mentions (10'10-10'12 M) at which PGEZ had no independent

1" *_}effect on CAMP release, PGEZ inhibited the release of CAMP normally
Iii) .occurring in response to AVP. This inhibition occurred with PGE2
" vii .added to either the apical or basolateral surface of the CCCT cell
nonoiayer. PGEZ (10'11 M) also inhibited the AVP- induced

~ j.huecllnuliltion of intracellular CAMP by CCCT cells seeded on culture
~tt

_-. Q ddshes. This inhibition was only observed when the cells were

-\';‘:preincubated with PGE2> >20 min. The results presented are
i- -‘F

‘ h}l5 eonSistent with the concept that inhibition by prostaglandins of the
9W1
Bigdroosmotic effect of AVP is due to inhibition of AVP induced CAMP

 

   
  
 
  
 
  
 
   
 
 
  
  
 
 
  
  
       

11.
, jcantinuen pra'u_-’ .- 1'

U. I am crczw":’ .. r ‘ '
I £11. ‘
t y!“ :\

Jinn-tr. .w :i -,

“it i 1 I 1

h i:

1"““""“’ 11mm»: ‘1 swim. FOR CREER
at thrquwuui '1.

1'”? Day. F"ar‘b Trentw,
. ”803M merit inflnl'iua.‘ '1 :'-" . ' ,1
”t Miy intellectua“; radar-.1119, a
£931 ’1
.f Jun? thanks go to «2» 541mm Riv":

1"1‘ ‘WW and Carol. . Hold”, who hear '1'. ‘1 -. .~e < r
"Infill"!!! fidelity and saifless menu-r" ,

)g ,.Wor inc-snows in natura.

~Q pedal frienos. ios VQNM, Lu ea mains»;

. Wt Swift. Shawn Fun”. »hfl~~ ”imprint,

than Manon. John mm at: in?! m7 3”“ :_ x

ACKNOWLEDGEMENTS

  
  
  
 
  
  
  
  
   
  
  
  
  
  
  

%' Bfu- w
‘,: ’0'1'J thank Dr. William L. Smith, advocatus diaboli per excellentia,
;u.§pr his continued professional guidance and financial support. Most
f;,ifi90rtantly, I am grateful to him for relentlessly endeavoring to
'23 instill in me the value of the persistent pursuit of excellence.
'aniQi I thank the members of my guidance committee, Drs. William S.
‘ '.§pielman, John L. Wang, J. Throck Watson, and William M. Wells, for
(their demonstrated interest in this research project and their sincere
wehcouragement throughout it.
,i'$" Jeffrey Day, Frank Grenier, Sherry Huey, Yasuhito Tanaka and
'.l"Tsuyoshi Hatanabe merit individual kudos. They made my experience in
- Qhe lab not only intellectually rewarding, but also emotionally
titaining.

"l". Particular thanks go to my sisters, Maria de Lourdes
It"

1 thank my special friends, los verdaderos, los de siempre:
ianila-Barreto, Robert Swift, Shawn Farrell, Marco Villanueva,

‘ a'Fillwock. Susan Uselton, John Burczak and Paul Boyer. Each one

111

 

  

Ififli, and Mary Dunn. With humor and affection they brightened
guorking day.
'effibove all, I thank my family. They have been my inspiration, my

‘ my strength, mi fabrica de ilusiones, the beginning and end of

{x'nurfidrcig ‘
l: ,L .-‘ »

,'.!“ .83 1‘13. '
'3. ‘ 1
51G” ’zr;L
. ‘ T ,
ENE-n "5

':«+ ‘ .3"

. 111W€ylh '
“3.3,".

xul'hjirs.‘ jff
5- j

‘-
-\'

,Qi .Lu CWOLJ.

--M I:
51‘.

 

COPYRIGHT ACKNOWLEDGEMENTS

   
  
   
  
  
  
   
  
  
  
  

)1; w‘ Figure 1 is reproduced from Smith, w. L., Minerai Electroiyte
.,;I. §-;
1 -?'f ab. 6, 10-26, 01981 S. Karger AG. Figures 3 and 5 are reproduced,

M.fln modified form, from Smith et a1., Prostagiandins and the Kidney,
94f.:%3unn, M. 0., Patrono, c., and Cinotti, G.A., Eds.), 01983 Plenum

I .:.Tf]ishing Corporation. Chapters 11, III, and IV inciude the text and
éfires. 1n expanded and modified form, of the foliowing publications
A Garcia-Perez. A. and Smith, H. L.: Am. J. Physio]. 244, C211- 6220,
:‘,g983 American Physiologicai Society and J. Clin. Invest., in press,
335984 Rockefeiler University Press.
»7.$:“: The work described in this dissertation was supported in part by a
, “a?£el1ouship from the Graduate Professionai Opportunity Program of the
‘MU‘B. Office of Education and by a U. S. P. H. S. Predoctora] Traineeship

we
‘ 5n) manna.

 

  
  
  
 
  
 
 
  
 
 
  
 
 
  
 
  
  
 
  
 
 
   
  
 
 
   
  
  
 

i .
‘Jjnt' TABLE OF CONTENTS

:‘LL‘I :ST w TABLESI I I I I I I I I I I I I I I I I I I I I I I I I I Vii

‘ H‘tIST OF FIGURES I I I I I I I I I I I I I I I I I I I I I I I I I VIII

J'v

-,MNCLATURE‘ AND ABBREVIATIONSI I I I I I I I I I I I I I I I I I x

r ‘moDUcTIONI I I I I I I I I I I I I I I I I I I I I I I I I I I 1
CHAPTER

LITERATURE REVIEW. . . . . . . . . . . . . . . . ; . . . . 4

Prostaglandin Biochemistry . . . . . . . . . . . . . . . . 4
Renal Structure and Function . . . . . . . . . . . . . . . 11
Prostaglandins and the Kidney. . . . . . . . . . . . . . . 15
Collecting Tubule Function . . . . . . . . . . . . . . . . 19
Prostaglandins and Water Resorption. . . . . . . . . . . . 19

ISOLATION AND CHARACTERIZATION OF CANINE CORTICAL
COLLECTING TUBULE (CCCT) CELLS: AVP INDUCES PGE2
RELEASE I I I I I I I I I I I I I I I I I I I I I I I I I I 22

Mayt‘eri a] s and Methods I I I I I I I I I I I I I I I I I I I 23
Results. I I I I I I I I I I I I I I I I I I I I I I I I I 34
D1SCU551°n I I I I I I I I I I I I I I I I I I I I I I I I 64

APICAL-BASOLATERAL MEMBRANE ASYMMETRY IN CANINE
CORTICAL COLLECTING TUBULE (CCCT) CELLS:
BRADYKININ, AVP, PGEZ INTERRELATIONSHIPS . . . . . . . . . 67

Materials and Methods. . . .V. . . . . . . . . . . . . . . 68
Resu] ts I I I I I I I I I I I I I I I I I I I I I I I I I I 77
Discussion I I I I I I I I I I I I I I I I I I I I I I I I 111

g A AVP-PGE INTERACTIONS IN CANINE CORTICAL COLLECTING
‘ tuputE fccCT) CELLS. . . . . . . . . . . . . . . . . . . . 117

Anatéria1s and Methods. . . . . . . . . . . . . . . . . . 118
1 Resu‘ts I I I I I I I I I I I I I I I I I I I I I I I I I I 120
‘ ~ DISCUSSION 0 I o o o o o o o o o o e o c I a o I o o o o o 135

I

¥:"IH.‘I. I I I I I I I I I I I I I I I I I I I I I I I I I I 141

 

.vi

 

Table

LIST OF TABLES

Page
Differential Binding of MDCK Cells and Swiss Mouse 3T3
Cells to Culture Dishes Coated with Rat Anticanine
Collecting Tubule Antibody. . . . . . ..... . . . . . 38

Inhibition by Aspirin of Hormone-induced Synthesis
of iPGEz by CCCT Cells. . . . . . . . . . . . . . . . . . 63

Release of Prostaglandins by CCCT Cells on Culture
Dishes: Effect of Flurbiprofen . . . . . . . . . . . . . 85

Release of Prostaglandins by CCCT Cells on Culture

Dishes: Effect of Aspirin. . . . . . . . . . . . . . 86

Release of Prostaglandins by CCCT Cells on Millipore
Filters . . . . . . . . . . . . . . . . . . . . . . . . . 87

Time Course of Bradykinin-induced Release of PGE2
by CCCT Cells on Millipore Filters. ..... . . . . . . 105

Effector-induced Release of PGE2 and cAMP by CCCT
Cells on Millipore Filters. . . . . . . . . . . . . . . . 106

Inhibition by PGEZ of AVP-induced CAMP Release
from CCCT Cell Monolayers on Millipore Filters in
the Presence of Inhibitors of PG Synthesis. . . . . . . . 126

Effect of Preincubation Time on the Inhibition by
PGE of AVP-induced Formation of CAMP by CCCT
Cel s on Culture Dishes . . . . . . . . . . . . . . . . . 127

Inhibition by PGEZ of AVP-induced Formation of
cAMP by CCCT Cells on Culture Dishes. . . . . . . . . . . 129

Inhibition by PGEZ of AVP-induced Formation of
CAMP by CCCT Cells in the Presence of Flurbiprofen. . . . 130

Effect of Low Concentrations of PGE on Basal
Levels of Intracellular cAMP in CCC Cells. . . . . . . . 131

v11

 

r——_———_— ‘

LIST OF FIGURES

Figure Page
1 Prostaglandin biosynthetic pathway. . . . . . . ..... 6
2 Basic nephron structure . . . . . . . . ......... 13
3 Renal sites of prostaglandin synthesis. . . . ...... 17

4 Photomicrographs of canine collecting tubule
immunofluorescently stained with cct-l. . . . . . . . . . 35

5 Isolation of canine cortical collecting tubule (CCCT)

cells using polystyrene culture dishes precoated with

CCt-l o o o oooooo o o o o o I o oooooooooo 41
6 Photomicrograph of confluent monolayer of CCCT cells. . . 43
7 Growth of CCCT cells in monolayer culture ..... . . . 45

8 Photomicrograph of hemicysts formed by confluent
CCCT cells. . . . . . . ..... . . ........ . 48

9 Electron photomicrograph of canine cortical
collecting tubule in situ . . . . . . . . . . . . . . . . 50

10 Electron photomicrograph of an isolated intercalated
Ce] 1. O O I I I O O 0 O O O I O O 0 O O O O O I O O I I D 52

11 Electron photomicrograph of an isolated principal
C81]. 0 o c o o o o I o n a a o c nnnnn o o o n u o o 54

 

: 12 Formation of cAMP by CCCT cells in response to
‘ hormones. . . . . ..... . . . . . . . . . . . . . . . 57

13 Time course for formation of intracellular cAMP by
I CCCT C9115. a o o I o o o I o n o o I o o o o o o o o o a 59
i
D

14 Formation of immunoreactive PGE by CCCT cells in
response to bradykinin, AVP, an DD-AVP ..... . . . . 62

I 15 CCCT cell monolayer system for studying functional
1 asymmetry of the cortical collecting tubule . . . . . . . 72

16 Electron photomicrograph of principal and

intercalated CCCT cells seeded on Millipore
filters in chambers . . . . . . . . . . . . . . . . . . . 79

mi K
“mgr

 

 

ylIIF=_‘——__—_’—________________‘___""””’”’"i

Figure

17 Permeability to [3H]-inulin of Millipore filters
seeded with CCCT cells, MDCK cells, 3T3 cells, or no
cells. . . . . . . . . . . ................

18 Permeability of confluent CCCT cell monolayers to
[3HJ-inulin, [3H]-PGE2, and 22Ma* ..... . . . . . . . .

19 Permeability to [3H]-inulin of CCCT cell monolayers
on Millipore filters in the presence of effectors .....

20 Sidedness of DD-AVP effect on cAMP release from
CCCT cell monolayers on Millipore filters. . . . . . . . .

21 Time course of cAMP release from CCCT cells seeded
on Millipore filters . . . . . . . . . ..........

22 Concentration dependence for the DD-AVP-induced
release of cAMP from CCCT cell monolayers on
Millipore filters. . . . . . ..... . . . ...... .

23 Concentration dependence for the DD-AVP-induced
release of iPGEZ by CCCT cells on Millipore
filters. 0 I I I I I I I O I C I O I I I I I I I O I D O I

24 Sidedness of DD-AVP effect on the release of iPGE2
from CCCT cells on Millipore filters . . . . . . . . . . .

25 Sidedness of bradykinin effect on the release of
iPGEz from CCCT cells on Millipore filters . . . . . . . .

26 Concentration dependence for the PGEz-induced release
of cAMP from CCCT cell monolayers on Millipore filters . .

27 Sidedness of PGE2 effect on cAMP release from CCCT
cell monolayers on Millipore filters . . . . . . . . . . .

28 Apical-basolateral asymmetry of effector actions in
CCCT cells . . . . . . . . . . . . . . . . . . . . . . . .

29 Inhibition by PGEZ of DD-AVP-induced cAMP release
from CCCT cell monolayers on Millipore filters . . . . . .

30 Concentration dependence for the inhibition by PGEZ
of DD-AVP-induced release of CAMP. . . . . . . . . . . . .

31 Concentration dependence for the inhibition by PGFZa
0f AVP-indUCEd CAMP formation. I I I I I I I I I I I I I I

32 Model for AVP, bradykinin, PGEZ interrelationships
in CCCT cells. . . . . . . . . . . . . . . . . . . . . . .

Page

84

89

91

93

96

99

101

104

108

110

115

122

124

134

138

 

 

NOMENCLATURE AND ABBREVIATIONS

A terminological convention should be noted. In absorptive
epithelia (e.g., the renal tubule), the basolateral surface of the
cells corresponds to the region facing the capillaries. This surface
is also referred to as the blood or serosal side of the tubule. The
apical surface of the cells corresponds to the region where absorption
occurs. This region is the lumen of the renal tubule and is also known

as the urine or mucosal side.

Abbreviations used are: ADH, antidiuretic hormone; AVP, arginine
vasopressin; BK, bradykinin; cAMP, adenosine-3',5'-cyclic
monophosphate; CCCT, canine cortical collecting tubule; DD-AVP,
(Asu1'6,Ar98) vasopressin or desmopressin acetate; DMEM,

Dulbecco's modified Eagle's medium; IBMX, isobutylmethylxanthine; MDCK,
Madin-Darby canine kidney; PG, prostaglandin, RPCT, renal papillary

collecting tubule.

 

W—_—‘

INTRODUCTION

It has been recognized for almost twenty years that the kidneys
synthesize prostaglandins. The different experimental approaches
historically utilized to identify the agents involved in the overall
function of the kidney have also been used to study renal prostaglandin
metabolism. These approaches include: whole animal clearance methods,
perfusion of isolated kidneys, "stop flow" and micropuncture
techniques, and the use of kidney slices and homogenates (1,2). These
procedures have helped to define that prostaglanins are involved in the
modulation of a variety of renal functions. However, only after the
development by Burg gt_al. of techniques for perfusing microdissected
tubules (3), has it been possible to study the contributions of

prostaglandins to individual functions of discrete nephron segments.

 

E Although the microdissection technique has provided valuable

‘ information about prostaglandin metabolism in single nephron segments,
I it is not practical for detailed biochemical analyses. This procedure
is laborious and yields segments only 1-2 mm in length (approximately
103 cells). Hundreds of dissected tubules (106 cells) would be
required to make biochemical measurements (e.g., PGE2 and cAMP
radioimmunoassays) which can be readily performed with cultured cells
using one semi-confluent 24-well dish. This problem has been partially
circumvented by the development of renal cell lines such as Madin-Darby

canine kidney (MDCK) cells (4-6) and LLC-PK1 (7,8), which are

 

 

apparently derived from the distal and proximal tubules, respectively.
These cells retain many, but not all, of the properties of the parent
cells. Consequently, it has become important to prepare homogeneous
cell populations from different parts of the renal tubule which can be
grown and manipulated in monolayer culture in a differentiated state.

Interest in our laboratory in studying prostaglandin metabolism in
isolated collecting tubule cells first arose when Smith gt_al. immuno—
histochemically localized the prostaglandin-forming enzyme, PGH
synthase, in the kidney (9,10). The collecting tubule was the only
tubular segment that stained for this enzyme. This observation, in
conjunction with the report by Grantham gt_al. that PGE1 inhibited
the hydroosmotic effect of antidiuretic hormone (ADH) in perfused,
microdissected cortical collecting tubules (11), suggested an important
role for prostaglandins in the regulation of collecting tubule
function. Suspensions and cultures of cells enriched in rabbit
papillary collecting tubule (RPCT) cells were subsequently isolated and
studied by Grenier gt 31. in our laboratory (12-14). Although these
studies contributed to our understanding of prostaglandin metabolism in
the papillary collecting tubule, RPCT cells did not form prostaglandins
in response to AVP. Moreover, prostaglandins did not inhibit
AVP-induced cAMP accumulation.

The procedure used to isolate RPCT cells (hypotonic lysis of other
cells present) could not be used to obtain other tubular cell types in
which prostaglandins may play a role. This was especially true of
cells present in the cortex, which contains 4-5 different tubular cell
types. Therefore, it was necessary to develop a simple and convenient

procedure for the selective isolation of homogeneous populations of

\“

   
   

‘The approach chosen for this undertaking was the development

’”10nal antibodies that interact wnth cell surface determinants

' ihg tubule (CCCT) cells by immunodissection and studies on the
"ifilahdin metabolism and function of these cells is the subject of

3,: dissertation.
q‘. «or \f‘)4‘_" 1,

 

 

Tw—__—'

LITERATURE REVIEW

Prostaglandin Biochemistry. Prostaglandins are oxygenated fatty
acid derivatives. The pathway for the biosynthesis of prostaglandins
is illustrated in Figure 1. These compounds share a common set of
precursors so that the synthetic pathway for each prostaglandin differs
only in the final step. The major precursor of prostaglandins in
humans is all gig 5,8,11,14-eicosatetraenoic acid or arachidonic acid,
which gives rise to prostaglandins containing two carbon-carbon double
bonds in their side chains: PGEZ, PGFZG, PGDZ, prostacyclin
(PGIZ) and thromboxane A2 (TxAz). Arachidonic acid can be

obtained from meat or is formed by elongation and desaturation of the

 

essential fatty acid, linoleic acid, present in vegetables (15).
Arachidonic acid is found primarily esterified at the sflfZ position of
cellular phosphoglycerides (16).

l Prostaglandin formation can be conveniently discussed in three
stages (Figure 1)(17). In the first stage, a stimulus, usually a
hormone, interacts with the cell surface to activate phospholipases
which catalyze the hydrolysis of arachidonate from phosphoglycerides.
The major control point for prostaglandin formation appears to be at
this stage of arachidonic acid release, since the concentrations of
free arachidonate in cells are normally quite low (<10'6 M). The
reactions involved in the release of arachidonic acid are still

subjects of controversy (17,18). Arachidonate release has been

 

  
     
    
  
 
 

\ —'l
m - _ “ ‘
l
x
c , |
g.
.' ‘
‘
3; .
. I _ ‘ID I
7' A.
. I
a I ;
i
L‘! 3 ‘
a a ? L. ’
$3'\‘-.| .. _ ~‘
is
g ’ -
I _.
" . r' 7 .
A x ' .-- ’ -" A
i / ‘

l

/

.'

i .-
«swim; i:9d1rvzoid wag-62m? .Lgewugu

+

u

l’
J
A

E; ‘- Va ‘ " f:
g f "x . .',

83310.! A”

m.”

 

 

 

;

“Hiram“. "as, ' I . . L1
. ,- 3' "L .»-.‘* _ ‘

“W‘s ' (-

   

 

.V"r>

Figure 1. Prostaglandin biosynthetic pathway.

 

 

.mx»

.19. .52
-

    

.l
d
n
0% .- A 5:982 a
m noon xo
:8
_.| .68
oz
.83 {nun
mm x o:
H
.
zoou
ox

Figure 1

 

U_2m<4aoozm_

 

23.50;wm
to

6:32.- 0.5.5
o 294828.295

3000
A ”at: tofu up
. ‘llllll
19:38:85 _ «oflmwfia 9 Stats...
46:35- 0.:

UOEHUHGE
.
46535-2:

I ~ JSVNBQAXOO-DAD

9

U

4

E

g _.

5

4

I

<

20_»<>:U<

mngbghm

 

studied most extensively in platelets, which form TxAz as their major
prostaglandin (17). Arachidonate is released mainly from two
phospholipids, phosphatidylinositol (PI) and phosphatidylcholine (PC).
In the current view of arachidonate release, a cytosolic
phosphatidylinositol (PI)-specific phospholipase C cleaves PI to yield
diglyceride and inositol phosphate (19-22). The diglyceride formed
seems to have a dual purpose. Diglyceride lipase(s) (23,24) hydrolyzes
some diglyceride to yield arachidonate (90% of platelets' PI is
1-stearoyl-2-arachidonoyl PI)(25) and a 1-monoglyceride. Remaining
diglyceride serves to activate a diglyceride-dependent protein kinase
C, which in turn activates a phospholipase A2 that specifically
cleaves arachidonate from PC (26-29). Phospholipase A2 activities
have been found in cells and tissues that are active in prostaglandin
generation, such as platelets, neutrophils, fetal membranes and
macrophages (27).

The second stage of prostaglandin formation involves the
conversion of arachidonic acid to the endoperoxide PGHZ by PGH
synthase. This enzyme was the first of the prostaglandin biosynthetic
enzymes to be extensively purified and characterized (30,31). It
catalyzes two distinct and sequential events: (a) oxygenative
cyclization of arachidonic acid to produce P662 and (b) reduction of
the hydroperoxy group at C-lS of P662 to produce PGH2 (18). The
activity that catalyzes PGGZ formation is commonly referred to as the
cyclooxygenase. It is this activity which is inhibited by nonsteroidal
antiinflammatory drugs like aspirin. The reduction of P662 is
catalyzed by a hydroperoxidase activity. Both activities require heme

(30,32). PGH synthase is membrane-bound (18). Purified PGH synthase

- - ..--—-,-._—:——,....-..,_- -zmfiw
..__ o... ___-1

has a subunit molecular weight of approximately 70,000 daltons (31).
Cyclooxygenase and hydroperoxidase activities copurify (32,33).
Furthermore, both activities are precipitated by monoclonal antibodies
directed against the purified PGH synthase (34). Thus, it has been
concluded that both cyclooxygenase and hydroperoxidase activities
reside in the same protein chain (18).

In the third stage of biosynthesis, PGHZ is converted to what
are considered to be the biologically active forms of prostaglandins--
PGEZ, PGan, PGDZ, TxAz and P612. Each of these deriva—
tives, with the possible exception of PGan, can be formed from
PGH2 in a single enzymatic step (18). Synthesis of PGE2 and P602
is catalyzed, respectively, by PGH-PGE isomerase (35,36) and PGH-PGD
isomerase (37,38). Conversions of PGH2 to PGI2 and TxAz are
catalyzed by PGIZ synthase and TxAz synthase, respectively (39,40).
It is unclear whether PGan is formed directly from PGH2 by a
PGan reductase (18). No heat-labile activity involved in this
conversion has ever been detected (41,42). Two alternative pathways
for the synthesis of PGFZG have been proposed (18). One involves
the reduction of P002 mediated by an ll-keto-PGDZ reductase (not
shown in Figure 1)(43-45). This enzyme has been purified to apparent
homogeneity from rabbit liver (43). The second pathway involves the
reduction of PGE2 catalyzed by a 9-keto-PGE2 reductase (46).

PGH-PGD isomerase and 9-keto-PGE2 reductase are soluble enzymes
(17). Two different PGH-PGD isomerases have been purified to
electrophoretic homogeneity from rat spleen (37,47) and rat brain
(38,48). These are the only known isoenzymes in prostaglandin

biosynthesis. Both are composed of a single polypeptide chain (18).

 

The spleen isomerase is cytosolic and has a subunit molecular weight of
26,000-34,000 and an absolute requirement for reduced glutathione
(37,47). The brain enzyme is also cytosolic, but has a subunit
molecular weight of 80,000-85,000 and no requirement for reduced
glutathione (38,48).

All other enzymes that utilize PGHZ as substrate are integral
membrane proteins. PGH-PGE isomerase has only been partially purified
(35,50) and little is known of its physical or chemical properties.
PGIZ synthase has been solubilized and purified to electrophoretic
homogeneity from bovine aorta (51,52) and porcine aorta (53). Studies
with both preparations indicate that the enzyme is a hemoprotein with a
subunit molecular weight of approximately 50,000. TxAz synthase has
not been purified to homogeneity (18).

Biologically active prostaglandins are inactivated by several
types of chemical modifications depending on the prostaglandin.
Catabolism can be enzymatic or nonenzymatic (54). TxAz is hydrolyzed
nonenzymically to Tsz. The t1/2 of TxAz in aqueous solution
at pH 7.4 and 37° is 30 sec (55). Tsz has no appreciable biological
activity. Enzymatic inactivation of other prostaglandins is effected
via a catabolic cascade (18), the first step of which is oxidation of
the 15-hydroxyl substituent by a 15-hyroxyprostaglandin dehydrogenase
(15-PGDH). The resulting 15-keto products have less than one tenth the
biological activity of the parent molecules (47). Two types of 15-PGDH
have been described, differing in their specificities for the pyridine
nucleotide cosubstrate. Type I 15-PGDH uses NAD+ (56), while Type II
uses NADP+ (57). lg_ijg, pulmonary 15-PGDH (Type 1) activity is

responsible for the rapid (30 sec) inactivation of PGE2 and PGFZG

I" 10

that enter the circulation (58). P612 and P602, which are not

taken up by the lung, are probably inactivated by renal (Type I)
15-PGDH (18). The majority of prostaglandin metabolites found in urine
contain a 15-keto group, the apparent result of catabolism by 15-PGDH
(59,60).

The second step in catabolism is reduction of the double bond
between C-13 and C-14 by 15-ketoprostaglandin A13 reductase
(13-PGR). 13-PGR is coupled metabolically to 15-PGDH; it is found in
every tissue that contains 15-PGDH (18). 13-PGR from bovine lung has
recently been purified to homogeneity (61). The purified enzyme is
specific for 15-ketoprostaglandins, uses NADH+ as cofactor, and has
an approximate subunit molecular weight of 39,500 (61).

Another enzyme involved in prostaglandin catabolism is 9-hydroxy-
prostaglandin-dehydrogenase (9-PGDH), which catalyzes the oxidation of
the 9-hydroxyl substituent of 15-keto—13,14-dihydro-PGan, the
PGFZG catabolic product of 15-PGDH and 13-PGR (62,63). 9-PGDH, a

 

cytosolic enzyme which has been purified to homogeneity, uses NAD+ as
cofactor and has a subunit molecular weight of 34,000 (64). 9-PGDH is
not essential for prostaglandin inactivation since it mainly modifies
already inactive prostaglandin catabolites.

Prostaglandins are considered to be autocoids, acting on the cells
in which they are synthesized or on closely neighboring cells. This
concept originated with the observations that circulating PGEZ and
PGFZQ are rapidly catabolized in the lung (58) and are present in
blood at very low concentrations (65). It was also shown that

prostaglandins, unlike classical hormones, are synthesized by virtually

11

all mammalian tissues (66,67), although not all cells within a given
organ synthesize prostaglandins.

Newly formed prostaglandins are not stored, but rather exit cells
quickly (68,69). Because biological membranes are not freely permeable
to prostaglandins (70-72), carrier mechanisms must exist to transport
these newly formed prostaglandins. At the present time nothing is
known about the nature or regulation of these putative carriers.
Finally, with regards to function, prostaglandins can act through both
CAMP-dependent and CAMP-independent pathways to influence biological

processes (17).

Renal Structure and Function. The mammalian kidney performs two
major functions during the process of urine formation: (a) it excretes
all nonessential or noxious products of bodily metabolism and (b) it
regulates the volume and composition of body fluids. These actions of
the kidney provide for the conservation of water and essential

substances and the maintenance of acid-base balance. There are more

 

' than one million nephrons in each human kidney and each nephron is
capable of forming urine by itself.
. The kidney is composed of three general regions (Figure 2):
cortex, medulla, and papilla or inner medulla. Most nephrons traverse
1 these three areas. Figure 2 illustrates the basic anatomy of the
nephron, which consists of a vascular component (the glomerulus) and a
tubular component. Throughout its course, the tubule is composed of a
single layer of epithelial cells which differ in structure and function
from portion to portion.
Blood enters the glomerulus (G) from the interlobular artery (IA)

through the afferent arteriole (AA), forcing fluid to filter into

zrv
.1. -

   
  
 
  

~ 4 .E-iviv'; :
u ,' ‘
.9"; ’9'“ '2' go‘xrmon :‘rst
_ _ 11.; msg#51015 ,AA mien-3
-'. I , l!" mu . . W {10 ‘5 13°19. .. ,,
.. at, . » ' . . 336 ,U?fi<imr?nffl$
? 7 i W on 13m: in ' gm 351i?! puma—sets
rt' 9'2: " catholics 15311103 .703 :14»: hawk-woo;- Tsieih
‘ I 5 'tvfll'lqoq , ’ esfa'ooa'jsiium, 13M
5 b...

sini ' fl“?

3,;

.5 91'0“?

 
 
  

  

            

g
3.
3' I .
gm.

 

 

Figure 2.

12

Basic nephron structure. G, glomerulus; IA, interlobular
artery; AA, afferent arteriole; EA, efferent arteriole; PCT,
proximal convoluted tubule; PR, pars recta; DTL, descending
thin limb; ATL, ascending thin limb; MTAL, medullary thick
ascending limb; CTAL, cortical thick ascending limb; DCT,
distal convoluted tubule; CCT, cortical collecting tubule;
MCT, medullary collecting tubule; PCT, papillary collecting
tubule; and IC, interstitial cells.

 

PCT

  

cc1
ocr comx
Lem _ _ _ _
IA
PR MEDULLA
~\MCI
MTAL

 

 

 

 

DTl
ATl
jéa? RAPIlLA
IC
PCT

Figure 2

 

Bowman's capsule, and leaves through the efferent arteriole (EA).
Bowman's capsule opens into the first portion of the tubule, the proxi-
mal convoluted tubule (PCT). Along with glomeruli, PCTs lie in the
cortical region of the kidney. From the PCT, the fluid passes through
the pars recta (PR) into Henle's loop. The PR is found in both corti-
cal and medullary regions. The loop of Henle is subdivided structur-
ally into descending thin limb (DTL), ascending thin limb (ATL),
medullary thick ascending limb (MTAL) and cortical thick ascending limb
(CTAL). Some DTL is found in the medulla, but most is confined to the
papillary region, as is the ATL. From the loop of Henle the fluid
flows next into the distal convoluted tubule (DCT), which is in the
renal cortex. Finally, the fluid flows into the collecting tubule,
which traverses all three regions of the kidney, cortical (CCT),
medullary (MCT) and papillary (PCT). The fluid from collecting tubules
of several nephrons merge into collecting ducts, exclusively found in
the papilla, on its way to the renal pelvis for excretion (73).

Blood filtered in the glomerulus passes into the efferent
arteriole. Most of this blood then flows through a peritubular
capillary network that surrounds the cortical portions of the tubules.
The remainder of the blood from the efferent arterioles flows into
straight capillary loops called the vasa recta that extend downward
into the medulla and papilla, enveloping the tubules, before looping
back upward to empty into the cortical veins.

As the glomerular filtrate flows through the tubule, most of its
water and varying amounts of its solutes are reabsorbed into the
surrounding capillaries. Tubular segments are very diverse

structurally; consequently, there is great heterogeneity in tubular

fl

 

15

transport processes. A general observation is that each substance is
often transported by more than one segment of the nephron and that the

mechanism of transport differs from segment to segment (74).

Prostaglandins and the Kidney. In 1965 Lee gt a1. (75) reported

the isolation from the rabbit renal medulla of two prostaglandin—like
compounds which were later identified as PGEZ and PGFZG. The
presence of PGH synthase within the renal medulla was first
demonstrated by Hamberg (76) by adding tritiated arachidonic acid to
medullary homogenates. The bulk of the radioactivity recovered was
identified as PGEZ. Smaller amouns of PGan were also detected.

It is now well established that all of the biologically active
prostaglandin derivatives, with the possible exception of PGDZ,
are synthesized in the kidney. As illustrated in Figure 3, the cell
types which in the healthy kidney have been shown to form
prostaglandins are renomedullary interstitial cells (IC)(9,77,78),
cortical and medullary collecting tubule cells (9,10,78,79),
endothelial cells of the arterial portion of the renal vasculature
(10,80) and glomerular mesangial and epithelial cells (10,81,82). It
is not known in all cases which products are formed by which renal
cells or the extent to which species differences exist.

At the outset of the research described in this dissertation, it
was clear that PGEZ was the major product of the papillary collecting
tubule; however, it was only presumed, not established, that this was
also the case for other regions of the collecting tubule. The studies
with CCCT cells described in this thesis and additional recent reports
by Jackson gt_al. on isolated rat medullary collecting tubules (83) and

by Kirschenbaum t l. on isolated rabbit cortical collecting tubules

E“

.-

     
   
      

    
      
 

393'? 15.05:! .E 1:1“qu

(R M30103) eieedhngripngl‘pamr w
1.1 s

- e um... --_

   

v.4) - "i; .7 3,3,.
1'6“ r‘ 89—,” _

16

Figure 3. Renal sites of prostaglandin synthesis (colored in black).

IA

 

PR

MTAI.

17

 

 

 

CCT
CORTEX
MEDULLA
AACT
I—I’ '
PAPIllA
PCT

Figure 3

 

 

18

(84), strongly suggest that PGEZ is also the major prostaglandin
product of the cortical and medullary collecting tubules. PGEZ also
appears to be the major prostaglandin formed by renal medullary
interstitial cells (77,85,86). However, the nature of the hormonal
stimuli triggering PGEz synthesis differs between collecting tubules
and interstitial cells (17). PGIZ is the principal prostaglandin
synthesized by homogenates of interlobular arteries and afferent
arterioles (80), but PGE2 and perhaps TxA2 (87-90) may also be
formed by at least certain regions of the vasculature.

Regarding renal catabolism of prostaglanins, there exist in the
kidney both Types I and II 15-PGDH (91). In early studies using
histochemical methods, a prostaglandin dehydrogenase present in
cortical tissue was localized to the distal convoluted tubule (92).
15-PGDH Type I activity has more recently been shown to be concentrated
in the cortex, but also found in variable quantities throughout the
nephron and vasculature (47,91,92). 13-PGR and 9-PGDH are also present
in the kidney (17).

The concentrations of active prostaglandin in arterial blood
entering the kidney are relatively low (e.g., <15 pg/ml for PGEZ in
dogs)(65), and even these prostaglandins are probably metabolized by
the renal vasculature before they have any significant effects.
Therefore, prostaglandins acting on the kidney under normal conditions
are most likely produced intrarenally. Furthermore, the major
physiological effects ascribed to prostaglandins acting in the kidney
can be explained by intrarenal synthesis. Renin release in
juxtaglomerular cells can be affected by prostaglandins synthesized

in neighboring glomerular arterioles (93,94), decreases in renal blood

 

19

flow occurring in response to vasoconstrictors can be attenuated by
prostaglandins formed in the renal vasculature (95), and sodium and
water resorption in the collecting tubule can be modulated by
prostaglandins synthesized in the collecting tubule itself and possible

the renomedullary interstitial cells (10,11,96).

Collecting Tubule Function. The collecting tubule is the site for
the final regulation of urinary sodium, potassium, hydrogen ion, and
water excretion (97). It is the main target of action for antidiuretic
hormone (ADH). The collecting tubule, which is poorly permeable to
water in the absence of ADH, becomes highly permeable in its presence
(98). This effect of ADH is seen only when this hormone is added to
the peritubular side (basolateral side) of the collecting tubule (99).

It is well established that the action of ADH is mediated by CAMP
and that ADH induces physiological changes in the luminal cell membrane
(99). The nature of the events that link an elevation of intracellular
cAMP to an increase in water permeability at the apical cell membrane
is ill-defined. The only mechanism by which cAMP has been shown to
mediate hormonal effects involves protein phosphorylation (100),
catalyzed by the active form of CAMP-dependent protein kinase. That
cAMP elicits ADH-induced changes in membrane permeability by activation
of phosphorylation of membrane proteins (or other proteins) is an

attractive idea, but one with little direct support (101).

Prostaglandins and Water Resorptio . A variety of approaches,

both jg_vivo and in vitro, have demonstrated that prostaglandins

 

 

_———_—_-- L...—

20

antagonize the effect of ADH on water resorption. In several mammals
studied, pretreatment with cyclooxygenase inhibitors such as aspirin,
indomethacin, and meclofenamate increased urine osmolality in response
to arginine vasopressin (AVP), the naturally-occurring fonn of ADH
(102-108). Indomethacin pretreatment also potentiated AVP-stimulated
CAMP synthesis, suggesting that prostaglandins may block water
resorption by inhibiting CAMP synthesis (108). Using isolated,
perfused, rabbit collecting tubules, Grantham gt_gl. (11) showed that
AVP-stimulated water resorption is inhibited by 50% with 10"9 M

PGEl. The phosphodiesterase inhibitor, theophylline, by itself
caused water resorption, an effect that PGEl potentiated. PGEl

alone caused a small increase in water resorption. These results
suggested that PGEl, like AVP, could cause CAMP synthesis in cortical
collecting tubules which then leads to water resorption. Grantham
further postulated that PGE1 and AVP may compete for a common
receptor coupled to adenylate cyclase and that PGE1 inhibits
AVP-stimulated water resorption when both are present simultaneously.
Later, on the basis of similar experiments in toad bladder, other
authors concluded that PGE1 does not alter ADH-receptor binding, but
rather 'uncouples' the receptor from adenylate cyclase (109). Most
recently, studies have specifically evaluated the effect of
prostaglandins on ADH-receptor binding (110,111). These studies were
performed in human mononuclear phagocytes, cells which display
AVP-PGEZ effects on CAMP metabolism similar to those observed in the
collecting tubule (110,111). It was demonstrated that the presence of

PGE2 did not modify AVP specific binding to the cells.

 

Direct evidence for an inhibition of AVP-induced CAMP synthesis by
prostaglandins has been inconsistent at best. One important cause of
this problem is the more-recently-apparent biphasic nature of the

effect of PGE on CAMP accumulation. Marumo gt_al, (112) and Beck gt

31. (113) noted that the inhibitory effect of PGE1 was apparent at

low concentrations (10'8 M), whereas at high concentrations no
inhibition was apparent. Studies in which an additive effect was
observed (114,115) have used extremely high concentrations of PGE2
(>10'4 M). PGEZ used alone at these high concentrations
unequivocally stimulates adenylate cyclase activity (115,116), making
any inhibitory effect on ADH-stimulated cyclase difficult to detect.
The current hypothesis proposed to explain the relationship
between AVP and prostaglandins is that prostaglandins act as negative
feedback modulators of the antidiuretic action of AVP on the kidney.
If this is so, one would expect AVP to stimulate renal prostaglandin
synthesis. Only very recent studies, by Kirschenbaum gt_al. (84),
concurrent with those described in this dissertation, directly
demonstrate that ADH does stimulate prostaglandin synthesis in the

cortical collecting tubule of the rabbit and the dog, respectively.

 

CHAPTER II

ISOLATION AND CHARACTERIZATION OF CANINE CORTICAL
COLLECTING TUBULE (CCCT) CELLS: AVP INDUCES PGE2 RELEASE

This chapter describes the preparation of monoclonal antibodies
specific for the canine collecting tubule and the use of these
antibodies to isolate cortical cells. These cells proliferate in
culture and have many of the histochemical and hormonal properties
expected for cortical collecting tubule cells. In addition, the
results of experiments designed to determine the effects of bradykinin,
arginine vasopressin (AVP), and DD-AVP on prostaglandin synthesis by

CCCT cells are presented.

22

 

 

MATERIALS AND METHODS

Materials. Hypoxathine, penicillin, streptomycin sulfate,
aminopterin, thymidine, bradykinin triacetate, isoproterenol,
calcitonin, bovine serum albumin, arginine vasopressin (AVP),
3-isobutyl-1-methylxanthine (IBMX), and trypsin were purchased from
Sigma Chemical. Parathyroid hormone (PTH) and [Asu1’6,Ar98]
vasopressin (DD-AVP) were obtained from Beckman. Collagenase (CLS II)
was obtained from Worthington Biochemicals. Dulbecco's Modified
Eagle's Medium (DMEM), antibiotic-antimycotic (100X), and DMEM
containing D-glucose (4.5 g/l) and L-glutamine (2 mM) were purchased
from KC Biologicals. NCTC 109 medium was from MA Bioproducts,
Bethesda, MD. Normal horse serum was from Flow Laboratories. Sea
Plaque Agarose was from Marine Colloid Division, FMC. Fluorescein
isothiocyanate (FITC)—labeled rabbit antirat immunoglobulin (IgG), goat
antirat IgGZC, sheep antirat IgGl, goat antirat IgGZa, rabbit
antirat 1962b, and rabbit antiprostaglandin (PG) PGE2 were
obtained from Miles Laboratories. [3H]PGE2, [12511adenosine
3',5'-cyclic monophosphate (CAMP), and radioimmunoassay supplies for
the CAMP assay were all purchased from New England Nuclear, Boston.

PGE2 was a gift from Dr. John E. Pike of Upjohn, Kalamazoo, MI.

Cell Culture. Madin-Darby canine kidney (MDCK) cells (ATCC CCL34)

obtained frm the American Type Culture Collection were grown in DMEM

 

24

containing 10% fetal bovine serum, antibiotic-antimycotic (IX) and 2 mM
glutamine. Swiss mouse 3T3 fibroblasts (ATCC CCL92) obtained fran the
American Type Culture Collection were grown in DMEM containing 10%
fetal bovine serum. Both cell lines were grown at 37°C under a

water-saturated 7% C02 atmosphere.

Preparation of Monoclonal Antibodies. Four-week-old female

Sprague-Dawley (Spartan Laboratories) rats were immunized two times 2
wk apart (first iv then ip) with 107 MDCK cells suspended in 0.2 ml
Krebs buffer (composition in EH3 118 NaCl, 25 NaHC03, 14 glucose,
4.7 KCl, 2.5 CaClz, 1.8 M9304, and 1.8 KH2P04), pH 7.3. Three
days after the second inoculation, the rats were killed by cervical
dislocation; a blood sample was taken to determine the presence of
antitubule antibody activity in the serum; and the spleens were removed
under sterile conditions for myeloma-spleen cell fusions.

Fusions were performed by modification of the method of Galfre gt
31. (118). The spleens were placed in 5 ml of DMEM and cut into pieces
that were teased apart with forceps to release the lymphocytes. After
the mixture was vortexed, the large tissue fragments were allowed to
settle for 1 min, the supernatant was transferred to a new tube, and
the splenic lymphocytes were collected by centrifugation at 1,000 g for
5 min. Red blood cells in the pellet were lysed by addition of 5.0 ml
of 0.2% saline for 30 s followed by 5.0 ml of 1.6% saline for 30 5.
Finally, 10 ml of DMEM containing 20 EM N-2-hydroxyethylpiperazine-N'--
2-ethanesulfonic acid (HEPES), pH 7.6, were added, and the spleen cells
were again collected by centrifugation, resuspended in DMEM containing

20 mM HEPES, pH 7.6, and counted using a hemacytometer.

 

~———-\_' -p—v‘i—wu- “j
i

 

25

The mouse myeloma strain SP2/0-Agl4 (119) obtained from the Cell
Distribution Center at the Salk Institute was grown in DMEM (4.5
D-glucose/l) containing 10% fetal bovine serum and 100 ug/ml each of
penicillin and streptomycin at 37°C under a water-saturated 7% C02
atmosphere. SP2 myeloma cells (1-5 x 106), which had been washed and
resuspended in DMEM containing 20 mM HEPES, pH 7.6, were mixed with 1-5
x 107 of the isolated splenic lymphocytes. The cell mixture was
collected by centrifugation at 1,000 g for 5 min in a sterile glass
centrifuge tube. After the supernatant was removed, the fusion was
begun by gently shaking the cell pellet, largely intact, with a
solution containing 35% polyethylene glycol 1000 (Baker) and 5%
dimethylsulfoxide in DMEM for 1 min. During the ensuing 3 min the
fusion solution was diluted with 3 ml of DMEM plus 20 mM HEPES, pH 7.6;
then, over a period of 6 min, the fusion mixture was diluted further
with 12 ml of HT media [compositionz DMEM containing 10% (vol/vol)
fetal bovine serum, 10% (vol/vol) horse serum, 10% (vol/vol) NCTC 109
media, 2 mM glutamine, 100 pM hypoxanthine, 16 mM thymidine, 3 PH
glycine, 100 mg/l penicillin, and 100 mg/l streptomycin]. Finally, the
cells were collected by centrifugation, resuspended in 75 ml of HT
media and dispensed into 96-well Costar 3596 tissue-culture plates (100
ul/well). The plates were incubated at 37°C under a water-saturated 7%
C02 atmosphere. After 36 h, 100 pl of HAT medium (HT media plus 1 PH
aminopterin) was added to each well. Half of the media was replaced
with fresh HAT media after 2 additional days; 12-20 days after the cell
fusion, when the media from those wells with growing hybridomas began
to acidify (turn yellow), aliquots of media were removed to test for

the presence of anticanine kidney tubule antibody.

ra—=———————77 2,7 ,, 7 a.

26

Selection of Hybridomas Producing Antibody to Specific Canine
Kidney Cell Types. Media from growing hybridomas were screened for

antibody activity using an indirect immunocytofluorescence procedure on

canine kidney sections. Sections (10 um) of canine kidney were cut on

 

a Tissue-Tek cryotome essentially as described previously (10) and

incubated with media from hybridoma cells (1:2 dilution in phosphate--

1 buffered saline (PBS, composition in EM: 151 NaCl, 45 KH2P04, and

i 2.5 NaOH), pH 7.2, for 30 min. After the sections were washed with

' PBS, pH 7.2, FITC-labeled rabbit antirat 196 (1:20 dilution in PBS, pH

| 7.2) was added, and the samples were incubated for 30 min. The

’ sections were then washed, mounted on cover slips, and examined by

l fluorescence microscopy. Presence of antibody in the medium from

' hybridoma cells was indicated by the appearance of fluorescence in

, renal cells compared with the absence of such fluorescence in control

1 samples. HAT media, preimmune rat serum, and PBS, pH 7.2, were all

used for control staining. A Leitz Orthoplan microscope equipped with

an Orthomat camera was used to visualize fluorescent staining.

Photomicroscopy was performed using Kodak Tri-X pan film (ASA 400).
Cells from wells yielding positive responses in the indirect

immunofluorescence procedure were Cloned once in soft agar using the

procedure of Cotton gt_al. (120), but with Swiss mouse 3T3 cells as a

feeder layer (121). 3T3 cells were seeded at a density of 5 x 104

cells in a 100-nm culture dish. After the cells had adhered to the

dishes, medium was removed and the 3T3 cells were overlayed with 5 ml

of Sea Plaque Agarose working solution (6 ml of 5% Sea Plaque Agarose

in PBS, pH 7.4, plus 90 ml of HT medium). The dishes were Chilled at

4°C to gel the agar layer and then warmed at 37°C for 10-15 min. For

 

 

 

 

27

each plate, 2 x 103 hybridoma cells were resuspended in 1 ml of Sea
Plaque Agarose working solution and dripped evenly over the plate. The
plates were Chilled for 15 min at 4°C and then placed at 37°C under a
water-saturated 7% C02 atmosphere. After 10-12 days, symmetrically
shaped colonies were picked from the agarose-medium suspension using
sterile micropipettes. Cells from individual clones were cultured in
HT media, and the media was retested for antitubule antibody activity

as described above.

Ouchterlony Double-Diffusion Analysis. Media (0.04 ml) from

positive hybridoma cultures was tested against sheep antirat 1961
(0.04 ml), goat antirat Inga (0.04 ml), rabbit antirat IgGZb
(0.04 ml), and goat antirat Ingc (0.04 ml) in 1.5% agar at 24°C
for 16-24 h.

Purification of Rat Ingc from Hybridoma Culture Media. The

cct-l hybridoma line (which secretes antibody to collecting tubule
cells) was grown in IgG-free HT medium (121). Antibody-containing
medium was decomplemented for 5-10 min at 56°C, adjusted to pH 8.2, and
applied to a Protein A-Sepharose CL-4B column (1 x 5 cm). Absorbed
material was eluted stepwise using 0.1 M_buffers of pH 8.0 (sodium
phosphate), pH 6.0, pH 4.5, and pH 3.5 (sodium citrate)(121,122). The
absorbance at 280 nm of each fraction was measured to determine the
location of protein peaks. Anticollecting tubule antibody activity in
the peak fractions was detected by the indirect immunofluorescence
procedure. 196 (Ingc) secreted by the cct-1 line was eluted at pH

4.5. Fractions containing Ingc were pooled, titrated to pH 7.4,

 

 

28

sterilized by filtration using a Millex sterile disposable filter unit

(0.45 um pore size), and stored in glass screw-top tubes at -20°C.

Adsorption of Rat Iggzp to Culture Dishes. Polystyrene

 

culture dishes (100 mm) were coated under sterile conditions with 9 ml
of purified Ingc (cct-l; 10 ug/ml of PBS, pH 7.4) for 3 h at 24°C

with intermittent swirling or overnight at 4°C. Unbound antibody was
removed by aspiration, and each antibody-coated dish was then washed
three times with 3 ml of a 1% solution (wt/vol) of bovine serum albumin
in PBS, pH 7.4. The dishes were allowed to dry face down on a layer of

sterile absorbing material.

Selective Adsorption of MDCK Cells to Antibody-Coated Culture

 

Plgpgs. MDCK cells (105) or 106 Swiss mouse 3T3 cells, each

suspended in 1 ml of PBS, pH 7.4, were added to different
antibody-coated culture dishes. The dishes were then washed five times
with PBS, pH 7.4, to remove nonadherent cells. The application pro-
cedure was always timed to last less than 3 min to minimize nonspecific
binding of the cells to the plates. To test for nonspecific binding of
MDCK cells to antibody-coated dishes and untreated dishes. Antibody-
coated dishes that had not been washed with 1% bovine serum albumin
(BSA) and uncoated dishes that had been washed with 1% BSA were also
tested using both 3T3 cells and MDCK cells to determine the effect of
the washing procedure on nonspecific binding of cells. Binding was
quantitated by detaching the cells from dishes with 0.1% (wt/vol)

trypsin containing 0.05% (wt/vol) ethylenediaminetetraacetic acid

 

29

(EDTA) in PBS, pH 7.2, and then counting the cells with a

hemacytometer.

Isolation of Canine Cortical Collecting Tubule (CCCT) Cells Using

 

Antibody-Coated Plates. Under sterile conditions, renal cortical

 

tissue (5 g) was carefully dissected from canine kidneys (obtained
immediately postmortem from mongrel dogs) and washed gently with 5 ml
of PBS, pH 7.4, to remove excess blood. The tissue was minced in a
petri dish with a sterile razor blade and transferred to a plastic
culture tube containing 24 ml of 0.1% (wt/vol) collagenase (CLS II) in
Krebs buffer, pH 7.3. The minced tissue was incubated for 40 min at
37°C under a water-saturated 7% C02 atmosphere. The tissue was then
gently agitated by drawing it up and down five to ten times in a
largebore (10 ml) pipette. The partially dispersed tissue was
incubated at 37°C under a 7% coz atmosphere for an additional 20 min.
The resulting cell suspension was centrifuged at 1,000 g for 5-10 min.
Red blood cells in the pellet were removed by hypotonic lysis with 10.0
ml of 0.2% saline for 30 s followed by 10.0 ml of 1.6% saline for 30 s,
and the suspension was filtered through several Gelman stainless steel
meshes (0.25 mm pore size). The remaining cortical cells were
collected by centrifugation at 800 g for 10 min on a tabletop centri-
fuge. The cell pellet was resuspended in 10% BSA in PBS, pH 7.2, and
centrifuged again for 10 min to collect the cells and remove cell
debris (13).

The cells (~109) were resuspended in 10 ml of PBS, pH 7.4, and
overlayed on antibody-coated dishes (1 ml/dish) for 3 min. The dishes

were then washed three to five times with 5 ml of PBS, pH 7.4. Bound

30

cells were detached by treatment with 3 ml of 0.1% (wt/vol) trypsin
containing 0.05% (wt/vol) EDTA in PBS, pH 7.2. The sample was
centrifuged, the supernatant removed by aspiration, and the cells
resuspended in DMEM containing 10% decomplemented fetal bovine serum,
antibiotic-antimycotic (IX), and 2 mM glutamine. The cells were then
seeded on 100 mm culture dishes, 24-well Costar 3524 tissue culture
plates, or sterile glass slips, as required for subsequent

experiments.

Incubation of Cells with Effectors. Treatment of monolayer

 

cultures of nonconfluent CCCT cells with effectors (i.e., bradykinin,
AVP, DD-AVP, isoproterenol, PTH, calcitonin) was done in triplicate
using 24-well culture dishes seeded at a density of 5 x 104
cells/well. All experiments were performed 6-10 days following
isolation of the cells. Cells were first rinsed free of media with
Krebs buffer, pH 7.3, and then 0.3-0.5 ml of buffer containing an
effector was added to the cells. The cells were incubated for the
desired time at 37°C under a water-saturated 7% C02 atmosphere.
Typically, 0.05-0.10 ml of buffer was removed from each well for
radioimmunoassays of extracellular CAMP or prostaglandin (PG) E2.

The remaining buffer was removed and discarded, and 0.20 ml of 0.1%
(wt/vol) sodium dodecyl sulfate (SDS) was added to each well. The
plate was incubated at 37°C for 15 min, and the solubilized protein was
assayed by the Lowry procedure (123) using BSA as a standard. All the
effectors used were checked to determine whether they had an
independent effect on the radioimmunoassays. Catabolism of PGE2

formed by CCCT cells that could occur during the incubations with

31

effectors was determined as previously described (126). There was no
detectable catabolism.

For intracellular CAMP measurements, the solution in each well was
aspirated and 0.5 ml of cold 6% TCA was added to each well. The wells
were incubated at -80° for 20 min, thawed at 24° for 25 min and
incubated for 2 hrs at 0-4°. The liquid in each well was then
transferred to a test tube and extracted four times with 10 volumes of
diethyl ether. Residual ether was evaporated in a 60° water-bath for
10 min. The remaining aqueous phase was lyophilized. The lyophilized
samples were resuspended in 0.125-0.3 ml of buffer and assayed for CAMP

by radioimmunoassay.

Statistical Analysis. All experiments involving an

 

effector-induced response were done using a minimum of three replicates
per treatment. A completely random analysis of variance was used to
test for differences between sample means at P<0.05 (124). Dunnett's
test was used for comparing differences between effector means and the

control mean (124).

Enzyme Histochemistry. Histochemical staining for succinate

 

dehydrogenase, glycerol-B-phosphate dehydrogenase, and NADH diaphorase
(125) was performed on CCCT cells that had been cultured on glass cover
slips and quick frozen in isopentane (-70°C) and on sections (10 pm) of
canine kidney cut on Ames Lab-Tek cryotome. Light microscopy was

performed on a Leitz Orthoplan microscope.

Electron Microscopy. Samples examined by transmission electron

microscopy included canine renal cortical tissue and CCCT cells grown

32

in monolayers attached to culture dishes. Primary fixation of all
samples were performed for 24 h at 4°C with 2% glutaraldehyde in 0.1 M
sodium phosphate, pH 7.2. In the case of cortical tissue, cubes (about
1 mm3) were cut from fresh tissue and immersed in fixative. Cultured
CCCT cells were overlayed with the fixative, and the fixation was
performed in culture dishes. After fixation in glutaraldehyde all
samples were washed for 1 h with frequent changes of 0.1 M sodium
phosphate, pH 7.2. The samples were then postfixed overnight at 4°C in
1% 0504 for the culture cells and 2% 0504 for the cortical

tissue. The samples were washed and then dehydrated by sequential
exposure to 25, 50, 70, 80, 95, and 100% (3 times each) solutions of
ethanol. Infiltration and embedding procedures were slightly different
for the two types of samples. After the third treatment with 100%
ethanol, CCCT cells were removed from culture dishes by scraping with a
rubber policeman, transferred to 4-dram screw-top vials and collected
by centrifugation at 500 g for 2 min. Propylene oxide was added to the
cell pellet and, after a brief incubation period, mixed with an
equivalent volume of Epon-Araldite resin (Epon 812:Araldite
502:dodecenyl succinic anhydride, 25:20:60, vol/vol/vol) and agitated
for 4 h at 24°C. Extra resin was then added to provide a ratio of
resin to propylene oxide of 2, and the mixture was agitated overnight.
The cells were then collected by centrifugation and resuspended in
Epon-Araldite resin to which had been added 0.024 volumes of
2,4,6-tri(dimethylaminomethyl)phenol. The samples were placed in Beem
capsules and incubated for 72 h at 60°. After the third treatment with
100% ethanol, a gradual transition to acetone was carried out for the

cortical tissue by exposing it to a mixture of 100% ethanol:acetone

33

(2:1) for 15 min, then 100% ethanol:acetone (1:2) for 15 min, and
finally two Changes of acetone alone, 30 min each Change. The cortical
tissue in acetone was then mixed with Epon-Araldite resin to a final
acetone-to-resin ratio of 3 and agitated for 3 h at 24°C. More resin
was then added to a final acetone-to-resin ratio of 1, and the samples
were again agitated for 3 h at 24°C. The cortical tissue was then
agitated with resin only, overnight at 24°C. The tissue segments were
placed in flat embedding molds (1/8 x 1/4 x 1/2 in.) filled with
Epon-Araldite resin to which had been added 0.024 volumes of
2,4,6-tri(dimethylaminomethyl)phenol and incubated for 48 h at 65°C.
The capsules and blocks were sectioned. The sections were
counterstained with 2% uranyl acetate in H20 and examined and
photographed using a Phillips Model 201 transmission electron

microscope. Kodak EM 4463 film was used for photography.

RESULTS

Selection and Characterization of Monoclonal Antibodies Against

Canine CollectingiTubule Cells. Spleen cells from rats immunized with

 

MDCK cells were fused with mouse plasmacytoma cells (SP2/O-Agl4), and
hybridomas were cultured as detailed in MATERIALS AND METHODS. Media
from three of 47 hybridoma-containing wells (from a total of 288 wells)
Caused specific staining of collecting tubules and no other tubule
segments when tested by indirect immunofluorescence. No other
antitubule reactions were noted with other hybridomas or with the serum
from the immunized rat. Cells from the hybridoma-containing well that
yielded the most intense fluorescent staining of collecting tubule
cells (Figure 4) were Cloned, yielding a hybridoma line designated as
Egg-1. This line produced antibody, which when employed in the
immunofluorescence screening procedure, stained cells from both the
cortical and medullary collecting tubules; moreover, all collecting
tubule cells stained with similar intensities, indicating that the
antibody was not directed against a specific subpopulation of
collecting tubules. Identification of the tubular sites of staining
was based on cellular morphology, distribution of staining in the
medullary rays, and coincidence of the fluorescent staining with
histochemical staining for NADH diaphorase activity (125) in both the

cortex and medulla.

34

Figure 4.

35

Photomicrographs of canine collecting tubule immunofluores-
cently stained with cct-l. A: fluorescent photomicrograph
of a section of renal medulla stained sequentially with
media from cct-I and then with fluorescein isothiocyanate
(FITC)-labeled rabbit antirat immunoglobulin (19) 196. 8:
phase contrast photomicrograph of the same tissue shown in
A. Magnification X80.

36

 

Figure 4

37

An intense, Spackled, fluorescence staining of collecting tubules
was observed in cryotome sections of canine kidney treated with 196
secreted by 523-1 and then FITC-labeled rabbit antirat 196 (Fig. 4).
Adsorption of medium from £5371 cells (containing approximately 20 pg
of rat 196) with 107 MDCK cells for 1 h at 37°C subsequently
eliminated the indirect fluorescent staining of collecting tubules in
sections of canine kidney; in contrast, adsorption of the medium with
107 Swiss mouse 3T3 cells under the same conditions had no effect on
immunocytofluorescence. These data suggest that the antigen that
interacts with 196 (323-1) is located, at least in part, on the cell
surface. The 196 secreted by Egg-1 cells was identified as belonging '
to the Ingc subclass on the basis of 1) Ouchterlony
double-diffusion analyses using antirat IgG allotype-specific sera and
2) affinity for Protein A-Sepharose and attenuated Staphylococcus

aureus cells (122).

Isolations and Characterization of Canine Cortical Collecting

Tubule Cells. Conditions for selective adsorption of CCCT cells were

 

developed by quantitating the binding of MDCK (experimental) and Swiss
mouse 3T3 (control) cells to 100 mm culture dishes pretreated with
IgGZc (Egg-1) (Table 1). The following general observations were
made. 1) Maximal binding of MDCK cells occurred with dishes incubated
for 33 h at 4°C with 290 pg of purified Ingc (Egg-1) per plate;

2) washing the antibody coated dishes three times with 1% BSA in PBS,
pH 7.4, eliminated all nonspecific binding of 3T3 cells; and 3) drying
the antibody-coated dishes following treatment with the BSA solution

enhanced the binding of MDCK cells. Under optimal conditions 15% of

38

Table 1

Differential Binding of MDCK Cells and Swiss Mouse 3T3 Cells to Culture
Dishes Coated with Rat Anticanine Collecting Tubule Antibodya

 

 

Cell Type Treatment of Culture Dish Cell Bound/Dish
MDCK PBS wash only <1o3*

313 P85 wash only <103

MDCK BSA wash only ND

3T3 BSA wash only ND

MDCK Ingc(Cct-1); no BSA wash 1.3 x 105
313 1962C (cct-l); no BSA wash <103

MDCK IgG2c(cct-1); plus BSA wash 1.4 x 105
3T3 IngC (cct-I); plus BSA wash ND

 

aCulture dishes (100 mm) were treated I) with phOSphate-buffered
saline (PBS), pH 7.4; 2) 3 times with PBS, pH 7.4, containing 1%
bovine serum albumin (BSA); 3) with 90 ug of immunoglobulin (Ig)
I962c (cct- 1) in PBS, pH 7. 4, for 3 h at 24°C and then washed 3
times w1th PBS, pH 7. 4; or 4) with 90 ug of IgGZC (cct- 1) in PBS, pH
7. 4, for 3 h at 24°C and then washed 3 times with PBS, pH 764’
containing 1% BSA. Dishes were then dried. MDCK cells (10 ) or 3T3
cells (10 ) in 1 ml of PBS, pH 7.4, were then added to the dish and
incubated for 3 min at 24°C, and dishes were washed 5 times with PBS,
pH 7.4. Binding of cells to the dish was determined as described in
MATERIALS AND METHODS. ND, no cells were detected by microsc0pic
observation of the culture dish. A few cells were observed to be
bound to the dish when examined under a microscope but no cells were
seen in the hemacytometer.

39

106 MDCK cells were bound per 100 mm antibody-coated culture dish;
under similar conditions less than 0.1% of 106 mouse 3T3 cells were
bound (Table 1); MDCK cells were not bound to dishes that were not
coated with antibody. There was no evidence that the antibody-coated
dishes adsorbed only a subpopulation of MDCK cells as approximately 15%
of those cells not adsorbed to a first dish could be adsorbed to a
second antibody-coated culture dish.

Conditions developed for optimal selectivity in binding MDCK cells
to culture dishes were used with minor modifications for isolating CCCT
cells (Figure 5). The renal cortex was dissected and then dispersed by
a combination of mincing and treatment with collagenase as described in
detail in MATERIALS AND METHODS. Dispersed cortical cells (108) were
added to each of 10 antibody-coated culture dishes and incubated for 1
min prior to washing thoroughly with PBS, pH 7.2, to remove unbound
cells. Under these conditions 106 cells routinely bound to each
dish. Bound cells were removed from dishes by trypsinization and
transferred to untreated multiwell culture dishes; approximately 80% of
the trypsinized cells adhered to uncoated dishes. Maximal attachment
of the trypsinized cells occured within 3-6 h, but 4-5 days were
required before the attached cells completely flattened and assumed the
typical appearance of the cells in monolayer culture (Figure 6); at
about this time the cells entered a proliferative period that continued
until they reached confluency (Figure 7). Starting from the density at
which the trypsinized cells were seeded (IO/mmz), the cells underwent
a 40-fold increase in cell number and cell protein prior to reaching
confluency (Figure 7). 0n reaching confluency, the isolated cells

remained viable for up to 3 mo on 100 mm culture dishes containing 10

40

Figure 5. Isolation of canine cortical collecting tubule (CCCT) cells
using polystyrene culture dishes precoated with cct-l.

CULTURE DISH

42

Figure 6. Photomicrograph of confluent monolayer of CCCT cells.
Magnification X125.

44

Figure 7. Growth of CCCT cells in monolayer culture. Cells were
seeded at a concentration of 10 cells/mmz.

45

_._._ (,_01 X) uaewnw 1130

 

 

 

 

8 L‘.’ Q in

I I I I
r- ° --53
— —2
h- —°’
— . —m
A— \\_“D
r- \\ “7
”I. ~\¢
$4 I J J J

00 (D «r (U

-o-o- (,,01 x 6") NIBLOHd T130

Figure 7

TIME IN CULTURE (daySI

46

ml of medium with or without changes of culture media. This latter
behavior is also exhibited by MDCK cells; in contrast rabbit renal
papillary collecting tubule (RPCT) cells (13) detach from the
substratum and die shortly after reaching confluency. Confluent CCCT
cells formed hemicysts in culture (Figure 8), and hemicyst formation
was blocked by the addition of ouabain (10'5 M) to the culture

medium. When CCCT cells isolated from three different dogs were seeded
at confluency on Millipore filters (1-2 x 106 cells/cmz),
transepithelial potential differences of 1 i 0.5 mV (Millipore side
positive) developed in each case 7 days after seeding and remained for
an additional 7-10 days. In parallel experiments with MDCK cells, a
potential difference of approximately 2 mV developed 1 day after
seeding and remained for at least 3 wk.

The isolated CCCT cells stained uniformly positive for NADH
diaphorase and glycerol-B-phosphate dehydrogenase and uniformly
negative for succinate dehydrogenase under reaction conditions in which
cortical collecting tubules in cryotome sections of the canine kidney
gave a similar pattern of histochemical staining. The uniform response
to the histochemical stains given by all cells in the monolayers
suggests that the CCCT cell population is relatively homogeneous.

Inspection of primary cultures of CCCT cells from three separate
isolations by transmission electron microscopy indicated that two types
of collecting tubule cells, principal cells and intercalated cells,
were present (126)(Figures 9-11). Of 303 cells examined from both
confluent and nonconfluent cultures, 36% were classified as

intercalated cells on the basis of their dark, electrondense cytoplasms

47

Figure 8. Photomicrographs of hemicysts formed by confluent CCCT
cells. Magnification X140.

 

Figure 8

49

Figure 9. Electron photomicrograph of canine cortical collecting
tubule in situ. Magnification X10,000. Internal marker is
1 um. bm, basement membrane; pc, principal cells; ic,
intercalated cell; and l, lumen.

 

Figure 9

51

Figure 10. Electron photomicrograph of an isolated intercalated cell.
Magnification X17,500. Internal marker is 1 um. rer, rough
endoplasmic reticulum; m, mitochondria; and n, nucleus.

52

 

Figure 10

53

Figure 11. Electron photomicrograph of an isolated principal cell.
Magnification X32,000. Internal marker is 1 um. m,
mitochondria; and n, nucleus.

54

 

Figure 11

55

(Figure 10). An abundance of mitochondria and other organelles and a
high content of cytoplasmic vesicles were also observed in some of the
intercalated cells (126,127). The remainder of the cells (~64%) were
classified as principal cells (Figure 11). These latter cells had
large nuclei and clear cytoplasms with a moderate content of
mitochondria and other organelles (126-128). The number of organelles
in these "light" cells was higher than has been observed in the
medullary and papillary collecting tubules. The relative numbers of
principal and intercalated cells were approximately the same in the
three isolates examined. No cells with characteristics (126,127)
expected for tubule cells other than collecting tubule cells were
observed by electron microscopy.

Primary cultures of CCCT cells were tested for their ability to
form cAMP in response to tubule-specific hormonal effectors (Figure
12). Increases in extracellular cAMP were obServed in response to AVP,
PGEZ, and isoproterenol, but not PTH or calcitonin. Half-maximal
increases in cAMP occurred with 10'10 n AVP, 10-8 M PGEZ,
and 10'9 M_isoproterenol. Thus CCCT cells are more sensitive to
AVP and PGE2 than are MDCK cells (5). Figure 13 illustrates the time
courses for the formation of intracellular cAMP in response to
supramaximal concentrations of AVP, PGEZ, and iSOproterenol. Maximum
increases in intracellular cAMP occurred 2-5 min after the addition of

each effector.

Prostaglandin Formation by CCCT Cells. Previous immunocytochem-
ical studies had indicated that the cortical collecting tubule has the

capacity to synthesize prostaglandins (9,10). Therefore, the ability

56

Figure 12. Formation of adenosine 3',5'-cyclic monophosphate (cAMP) by
CCCT cells in response to hormones. CCCT cells (6-10 days
after isolated) were incubated with the indicated concentra-
tions of arginine vasopressin (AVP) (o), isoproterenol (o),
PGEZ (A), and parathyroid hormons (PTH) (A) at 37° for 60
min in the presence of IBMX (10‘ M). Radioimmunoassays
for extracellular cAMP were performed as described in
MATERIALS AND METHODS. Statistically significant differ-
ences from control values (i.e , absence of effector) were
obse ved at _>_10-10 M AVP, 310-9 M PGEZ, and
310' M_isoproterenol (P<0.05)."

57

 

(fl
(3

h)
C)

5

 

I J

 

EXTRACELLULAR cAMP «moles/60 min/pg cell protein)

(3

10"° 10"
EFFECIOQ (M)

Figure 12

 

58

.Amo.ovav coeooeco co ooeomaa me» e_ oe_u sow: eo>comao

we: o=_a> ewe-o on“ soce ameago beao_ceem_m oz .Nmoa eu_z e_e me cea .om .om .oH .m

.N m—ocmcmuocaom_ saw: :PE oH use .m .N ma>< saw: :PE m use N ”mmepu mcwzo_—o$ msu an ounce
mew; Aces ov mm:_m> pocucou seem mmucmcmwe_u unwo_w_cm_m »_pmu_umwumum .moozhmz oz< m4<bmwp<z
:_ vmaweommu mm xommcocaeewowumc ma umcammms mm: oz<u Lm_:PPmumgu:H .x:m~ z wiofi

we mocmmmcg msu.mw vmagomcmg mLmB.m:owumn:o:H .A<v Louomwmm o: co .Aqv mmuo z iofi on
Focmcmuocaom_ z -oH .on ¢>< z oioH ;p_3 was?» umumuwvcw cow umumnaucp mew: vmumpom_

cmuem mxmv oflimv m_—m Hugo .m__wu pogo xn oz<u cap=FPmumsucF co =o_umegou Lo» mmczou we?» .mH wczmwd

59

 

 

 

 

 

 

 

 

 

 

I I T I -#€B
e
o -—1
e
v
1 ‘I 2
/ as
N
B . .-
\ a
_,CD
N
~. .1 .acp
CD CD

 

(11191on 1190 lid/salon!» awvo uvnn'i‘laovumi

Figure 13

TIME (min)

60

of CCCT cells to synthesize immunoreactive PGEZ (iPGEZ) was
examined. As shown in Figure 14, treatment of CCCT cells with
bradykinin, AVP, or DD-AVP increased the formation of iPGEz two-to
threefold; moreover, pretreatment of CCCT cells with aspirin (10"3
.M at 37°C for 60 min) blocked the increases in iPGEZ formation
brought about by the effectors (Table 2). Half-maximal increases in
iPGEZ occurred at approximately 10'10 M_for bradykinin, AVP, and
DD-AVP. The half-maximal increases in AVP-induced iPGEz formation
occurred at a concentration of AVP similar to that which caused a

half-maximal increase in AVP-induced cAMP formation.

61

Figure 14. Formation of immunoreactive PGEZ (iPGEz) by CCCT cells
in response to bradykinin, AVP, and DD-AVP. CCCT cells
(6-10 days after isolated) were incubated with the indicated
concentrations of AVP (o), DD-AVP (A), and bradykinin (A) at
37° for 60 min. Radioimmunoassays for PGEZ were performed
as described in MATERIALS AND METHODS. Statistically
significant differences from contrpb values (i.e., absence
of effector) were observed at 310' M_for all
effectors tested (P<0.0S).

62

 

 

 

 

30
e +—-‘
'8 A
"’ 0
g o
B
3’
.E 20L
E
uj“ IO
.32

l 1, l L
10"2 10"° 16° 10'6

[EFFECTOR] (M)

Figure 14

 

63

Table 2

Inhibition by Aspirin of Hormone-induced
Synthesis of iPGEz by CCCT Cellsa

 

 

iPGEz,
Hormone fmol-ug cell protein'1-60 min“1
None 6.0 i 0.7
None + aSpirin 1.8 1 0.3
Bradykinin *12.1 t 1.3
Bradykinin + aspirin 2.5 t 0.8
AVP *12.9 1 0.4
AVP + aspirin 2.9 1 0.1
DD-AVP *12.3 i 0.4
DD-AVP + aspirin 2.7 i 0.9

 

aValues are means 1 SE. Preincubations with aspirin (10"3 M)
were performed for 60 min at 37°C in Krebs buffer, pH 7.2. Hormone
concentrations in all experiments were 10' M. Significant change
from control (none) values (P<0.05).

 

DISCUSSION

We have developed a selective immunoadsorption procedure for
isolating up to 107 collecting tubule cells from a mixture of 109
renal cortical cells prepared from a single dog kidney. Evidence that
CCCT cells are derived from the collecting tubule is as follows:

1) CCCT cells stain uniformly positive for NADH diaphorase and
glycerol-3 phosphate dehydrogenase but not for succinate dehydrogenase
activity, a pattern of staining unique for the collecting tubule in the
canine renal cortex; 2) when examined by transmission electron
microscopy, CCCT cell preparations contained only cells with
morphological characteristics of intercalated and principal collecting
tubule cells and not of cells derived from other cortical tubule
segments; 3) CCCT cells formed hemicysts when grown in culture and
exhibited a transepithelial potential difference when seeded on
Millipore filters indicating that the isolated cells are transporting
epithelia (129); 4) CCCT cells formed cAMP in response to AVP, PGEZ,
and isoproterenol but not PTH, responses expected [by analogy to other
species (130,131)] for a mixture of cortical collecting tubule cells
containing both intercalated and principal cells; 5) CCCT cells formed
iPGEz as anticipated from previous immunocytochemical studies (10);
and 6) CCCT cells were adsorbed using antibodies selective for an
antigen on the cell surface of collecting tubules. These properties

are exhibited by CCCT cells that are grown in primary monolayer

64

65

cultures. The data indicate that CCCT cells can be used as a model
system to study the biochemistry of cortical collecting tubules.
Jefferson gt 91. (129) have reported the isolation and culture of a
distal tubule line (JCK-S) from the canine renal cortex using defined
culture media. The properties of JCK-S cells, especially with respect
to hormonal responsiveness, differ considerably from CCCT cells.

We have demonstrated the utility of cell immunoselection (132) in
the special case of canine collecting tubule cells. In our studies,
homogeneous p0pulations of MDCK cells were used as the immunogen for
generating monoclonal antibodies. The broader use of cell
immunoselection in renal physiology depends on the generation of
monoclonal antibodies specific for each of a variety of different renal
cell types. It is not yet known whether this will be possible with all
tubule cells. Chan gt_gl, (133) have prepared conventional rabbit
antibodies reactive with proximal tubule segments, and Herzlinger gt
.31. (134) have prepared monoclonal antibodies to cell surface
determinants of canine thick ascending limb. We have recently been
able to prepare two different monoclonal antibodies against
ectoantigens found only on rabbit proximal tubules and on rabbit
collecting tubules, respectively (135). In related work, Savoy-Moore
._E._l- have used anti-kallikrein monoclonal antibodies in the
immunodissection procedure here described to isolate cells that contain
kallikrein as an ectoenzyme (136). Most recently, using CCCT cells as
immunogen, we have obtained a monoclonal antibody with specificity for
a suprpulation of cells of the canine cortical collecting tubule in
situ. This antibody may prove to be a useful tool in isolating

homogeneous populations of principal cells or intercalated cells.

66

Studies on the prostaglandin biosynthetic capacity of CCCT cells
indicate that these cells, like rabbit renal papillary collecting
tubule (RPCT) cells (13), form iPGEz in response to extremely low
concentrations of bradykinin. However, in contrast to RPCT cells (13),
CCCT cells formed iPGEZ in response to AVP and DD-AVP. It is not yet
clear how AVP operates to elicit iPGEz synthesis, although one would
anticipate that AVP causes the activation of a lipase system capable of
liberating arachidonic acid (19,20,26). The differences between the
AVP responses of CCCT and RPCT cells may be attributable to a segmental
variation in the source of the cells since there is evidence that AVP
also induces prostaglandin formation by cortical collecting tubules
from rabbits (84,137). It will be of considerable interest to
determine if there are other regional differences between the

AVP-prostaglandin responses in the collecting tubule.

CHAPTER III

APICAL-BASOLATERAL MEMBRANE ASYMMETRY IN CANINE CORTICAL COLLECTING
TUBULE (CCCT) CELLS: BRADYKININ, AVP, PGEZ INTERRELATIONSHIPS

Prostaglandins have been shown to have two potent effects on the
transport properties of isolated collecting tubule segments. One
effect is to inhibit water resorption occurring in response to AVP
(11,138) and the second is to inhibit Na+ resorption (96,137,138,
140,141). The studies reported in this Chapter were designed to
determine whether there is an apical-basolateral membrane asymmetry
either to the release of prostaglandins by CCCT cells or to the effects
of prostaglandins on cAMP metabolism in CCCT cells. These questions
were prompted in part by a report that PGEZ acts only from the
basolateral surface of rabbit collecting tubule segments to inhibit
Na+ resorption (96). This result suggested that PGEZ is unable to
traverse the collecting tubule and that there is an asymmetry to PGEZ

receptors in the collecting tubule.

67

MATERIALS AND METHODS

Materials. [3H]Inulin Methoxy (2-3 Ci/mmole), [3HJPGE2 (160
Ci/ mmole), [3HJPGF2a (150 Ci/mmole), [3H]6-keto-PGF1a (150
Ci/mmole), [3H]Thromboxane (Tx) B2 (155 Ci/mmole), [1251]-
adenosine 3',5'-cyclic monophosphate (cAMP) (150 Ci/mmole), 22NaCl
(20 Ci/mmole), and radioimmunoassay supplies for the cAMP assay were
all purchased from New England Nuclear, Boston. Rabbit anti-PGEZ
serum was obtained from Miles Laboratories. Rabbit anti-PGFZG,
rabbit anti-6-keto-PGF1G, and rabbit anti—TxB2 sera and
radioimmunoassay supplies for these three compounds were purchased from
Seragen, Inc. PGEZ was purchased from Upjohn Diagnostics, Kalamazoo,
MI. SEP-PAK C13 cartridges were purchased from Waters Associates,
Milford, MA. Soluble calf skin collagen was obtained from Worthington.
Dulbecco's Modified Eagle's Medium (DMEM) containing D-glucose (4.5
g/l), PSN antibiotic mixture (100X), and fetal bovine serum were
purchased from GIBCO Laboratories. (Asu1’6,ArgB)vasopressin
(DD-AVP) was obtained from Beckman. Desmopressin Acetate (DD-AVP) was
purchased from Armour Pharmaceutical Company. Arginine vasopressin
(AVP) was obtained from Calbiochem-Behring. Bradykinin triacetate,
3-isobutyl-1-methylxanthine (IBMX), and L-glutamine were purchased from
Sigma Chemical. Silica Gel 60 plates were obtained from E. Merck.
Flurbiprofen and Ibuprofen were gifts from Dr. Udo Axen of the Upjohn

Company.

68

69

Isolation and Growth of CCCT Cells on Millipore Filters. CCCT
cells were isolated by immunodissection as described in Chapter II.
The isolated cells were grown on 100 mm culture dishes in DMEM contain-
ing 10% decomplemented fetal bovine serum, PSN antibiotic mixture (1X)
(5 mg Penicillin, 5 mg Streptomycin and 10 mg Neomycin/IOO ml) and Z'mfl
glutamine under a water-saturated 7% C02 atmosphere. CCCT cells that
were to be seeded on Millipore filters were grown 3-6 weeks with weekly
changes of culture medium. Typically 3—10 x 106 cells were obtained
from each culture dish. CCCT cells were removed from dishes by
trypsinization and seeded at supraconfluent densities (2 x 106
cells/cm?) on collagen-coated Millipore filters (0.45 um; cat. no.
HAMK 024 12) bonded to hollow polycarbonate cylinders (Figure 15). The
cylinders containing the cells were incubated in 6-well culture dishes
under the culture conditions described above. Cells were tested for
the presence of a transmembrane potential difference beginning four
days after seeding. CCCT cell monolayers were used only after they
exhibited a potential difference and were found to be impermeable to
inulin (see below). Most monolayers were also tested for and found to
exhibit a Sidedness in their response to AVP. Other types of cells
used for comparison including Madin-Darby canine kidney (MDCK) cells
and Swiss mouse 3T3 fibroblasts were cultured as described previously
in Chapter II and were seeded on Millipore filters as described above

for CCCT cells.

Permeability of CCCT Cells on Millipore Filters. The permeability

 

of cell monolayers to inulin, PGEZ, and Na+ was measured as

described below. All monolayers were tested for their permeability to

7O

inulin. The medium was removed by aspiration from both the inside and
outside of the polycarbonate cylinders under sterile conditions, and
this chamber was placed in a fresh 6-well culture dish. A solution
containing a radioactive solute was added to one side of the cell
monolayer, and the same solution without the isotope was added to the
other side. Typically the solution was culture medium (DMEM containing
10% decomplemented fetal bovine serum, PSN antibiotic mixture 1X and 2
mM_glutamine). There were no appreciable differences in the
permeability of CCCT cells to inulin or PGEZ when comparing the

culture medium to the same medium without serum or to Krebs-Ringer
buffer (composition in mM: 118 NaCl, 25 NaHCO3, 14 glucose, 4.7 KCl,
2.5 CaClz, 1.8 M9504, and 1.8 KH2P04), pH 7.4. Usually 1 ml

was added to the inside of the cylinder (apical side of the monolayer)
and 3 ml was added to the culture well in which the chamber was
immersed (Figure 15). In most experiments, [3H]inulin, [3HJPGE2

and 22Na+ were used at concentrations of 10'7 M. After the

desired incubation periods, 0.05 ml aliquots were removed from inside
and outside of the cylinders and radioactivity on both sides measured
by scintillation counting. Counts for each vial were normalized to the
original volumes. Percentages of the total amount of radioactivity
added were then calculated for each side at each time period measured.
Percentage data could then be transformed, to obtain a normal
distribution for further statistical analysis, by utilizing the angular

(inverse sine) transformation (124).

Effector-induced cAMP Release. Medium surrounding CCCT cells

 

grown on Millipore filters was removed under sterile conditions. The

71

.umuupamv mew AcowuoELoe az<o uwoaucwia>< eo xguweeamm ucu .mcmnsmgo

asp mc_mcw>mcuizoccm mg» xn mg=m_e m_;» c_ umumo_ucw .:_P::_ cu »u___nwwscmaew .mmucwcmeewu
.mwucmuog cmpappwumcogu ..w.wv meansmzo mmmsa eo xuwem—oa chowuuczm may zmwpnmumm on com:
»_mcvpaoc mwcmuwcu woes» use .smwu mg=u_=o m cw mmmp omega co mucoum consmgu asp .mmumeczm
Pmcmuopomcn can Pmo_ao po:_umwv ;u_3 LmAmpocoE Fpmu acmzpwcoo m Eco» cmccw_xu mumconcmuxpoa
zo__o; a do uoeeoa eou__e ocoa____z a ea nausea A Eo\moH x NV mp_oo euuu .o_=a=»

m:_uum—_oo .muwpcou mg» we acumesxmm pmcowpocae mcwxuzum Lee sawmAW gmxupocos ppmu Home

.mH ac=m_d

72

eeaeizn

 

 

 

 

«mooixn
.26 coon 8 n.><
5:3 _®m..oa_______2 J<mwkjom<m L,“ $8
33.082
2.8 .58/
cvnEozo/v >E o._1u.om

~moo-:n
.52511n

 

 

 

 

Figure 15

73

chambers were washed with Krebs-Ringer buffer, pH 7.4 and transferred
to 12-well culture dishes (Costar). Each experiment involving only one
effector (e.g., AVP) at a single concentration typically consisted of
the exposure of each cell monolayer to three treatments: (a) no
effector on either side (control), (b) effector present on the apical
side and (c) effector on the basolateral side. Because each chamber in
a given experiment received all three treatments consecutively, the
order of the treatments was randomized. For each treatment an effector
in Krebs-Ringer buffer, pH 7.4 containing 10'4 M IBMX or only

buffer with IBMX (no effector) was added to the appropriate sides and
incubated at 37° for different times, typically one hour. Volumes were
chosen so that the fluid levels were equal on both the inside and
outside of the polycarbonate chambers: 0.5 ml for inside the chambers
and 1.2 ml for outside the chamber (inside the culture dish). After
the desired time, liquid on each side of the monolayer was collected
and lyophilized. The lyophilized samples were resuspended in equal
volumes (0.3-0.5 ml) of buffer and assayed for cAMP as described
previously (13) and in Chapter II. Effector dose-response curves and
time courses were performed using only one treatment per chamber. All

other procedures were as described above.

Effector-induced PGEZ Release. The incubation procedures
described above for measuring cAMP levels were used in experiments
designed to measure prostaglandin formation. Krebs-Ringer buffer, pH
7.4 without IBMX was used in these experiments. The fluid containing
prostaglandin released by the cells to either side of the cell

monolayer was collected in individual tubes. The samples were

74

immediately acidified to pH 4 with I‘M citric acid and extracted twice
with two volumes of ethyl ether. The ether was evaporated under a
stream of nitrogen, the samples resuspended in an appropriate buffer
and analyzed for immunoreactive PGEZ as described previously (13).

In a few instances, simultaneous measurements of effector-induced

PGE2 release and cAMP release were performed to determine whether
significant qualitative differences occurred between sets of chambers.
All effectors were used in the presence of 10'4‘M_IBMX. The fluid
containing both released compounds was collected, acidified and
extracted as described above for measurements of PGE2 alone. The
aqueous phase of the extracted samples (containing >90% of the cAMP
present) was lyophilized and subsequently resuspended as usual for cAMP
radioimmunoassays. The organic phase was evaporated under a stream of

nitrogen and resuspended for PGEZ radioimmunoassays.

Characterization of Prostaglandins Releasedgby7CCCT Cells. The
release of PGE2, PGFZG, 6-keto-PGF1G, and thromboxane 82 by
CCCT cells cultured on both Petri dishes and Millipore filters was
quantitated. For CCCT cells seeded on culture dishes radioimmunoassays
were performed either directly on the medium or after extraction and
purification. Analysis of the prostaglandins released by CCCT cells
seeded on Millipore filters was performed only on ether extracts of
culture media. Purification of prostaglandins was performed as
follows: media was extracted using SEP-PAK C18 cartridges as
described by Powell (142). The methyl formate fractions containing the
prostaglandins and thromboxanes were evaporated, and each of the

residues was resuspended in chloroform (ca. 100 pl). These samples

75

were chromatographed on Silica Gel 60 plates (E. Merck). The plates
were developed twice in the organic phase of ethyl acetate/trimethyl-
pentane/acetic acid/water (55/25/10/50; v/v/v/v)). Prostaglandin
standards were visualized with iodine vapor and regions of the plates
cochromatographing with the standards were scraped into individual
tubes. The silica gel was extracted three times with 2 ml of
chloroform/methanol (1/1; v/v). The solvent was evaporated under a
stream of nitrogen, and the samples resuspended in buffer and subjected
to highly specific radioimmunoassays. The recoveries of all four
prostaglandin derivatives were monitored throughout these procedures
using tritiated prostaglandins. They averaged 63-65% for PGEZ,

PGFZQ and Tsz, and 45-50% for 6-keto-PGFla.

Statistical Analysis. All experiments involving an effector-
induced response were done using a minimum of three replicates per
treatment. A completely random analysis of variance was used to test
for differences between sample means at P<0.05 (124). Dunnett's test
was used for comparing differences between effector means with the
control mean (124). In a few instances, when it was desired to compare
all the effector means to each other and not only to the control mean,
Student-Newman-Keuls' test for all possible comparisons was used (124).

Error bars on the figures are 1 SE.

Electron Microscopy. CCCT cells seeded on Millipore filters were
examined by transmission electron microscopy. Cells on the monolayers
were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4,
for 24 h at 4°. After fixation the chambers were washed thoroughly

with 0.1 M sodium cacodylate, pH 7.4. The filters containing the fixed

76

CCCT cells were separated from the polycarbonate cylinders. The
filters were washed for 10 minutes in 0.1 M_sodium cacodylate, pH 7.4
and postfixed in 1% 0504 in the same buffer for 4 h at 24° and then
overnight at 4°. The filters were washed with sodium cacodylate buffer
and dehydrated by sequential exposure to 25, 50, 70, 80, 95 (15 min
each) and 100% (15 min then 1 h with a change in between) solutions of
ethanol. The filters were exposed to decreasing proportions of
ethanol/ acetone: 2/1, 1/2 and finally 100% acetone (twice) for 15 min
each. They were carefully cut into small pieces suitable for embedding
and placed into glass vials for infiltration. Infiltration was as
described previously in Chapter II. The sections obtained from the
blocks were counterstained with 2% lead citrate in H20 and 2% uranyl
acetate in H20. They were examined and photographed using a Phillips
Model 201 transmission electron microscope. Kodak EM 4463 film was

used for photography.

RESULTS

Orientation of CCCT Cells on Millipore Filters. In order to use

 

CCCT cells on Millipore filters as a model to study the Sidedness of
the responses of collecting tubules to hormones, we first needed to
determine if CCCT cells, when seeded at confluency on Millipore
filters, were impermeable to small molecules and were morphologically,
electrically, and biochemically asymmetric.

CCCT cells were seeded on Millipore filters at a density of 2 x
106 per cmz. Typically, these cells developed a transepithelial
potential difference of 1 1 0.2 mV (Millipore filter side positive)
4-13 days after seeding and maintained this potential for up to an
additional 21 days. As has been reported previously for Madin-Darby
canine kidney (MDCK) cells (143), consistent results were obtained only
with cells which had previously been grown and maintained at confluency
on Petri dishes for at least 10 days prior to seeding on Millipore
filters. As shown in Figure 16, CCCT cells grown on Millipore filters
exhibited typical intercellular tight junctions and had microvilli on
their apical surfaces.

Confluent monolayers of CCCT cells on Millipore filters were
relatively impermeable to inulin in comparison to Swiss mouse 3T3
fibroblasts or to filters to which no cells had been added (Figure 17).

MDCK cells exhibited a permeability barrier which was quantitatively

77

Figure 16.

78

Electron photomicrographs of principal and intercalated
CCCT cells seeded on a Millipore filter in chambers. (A)
One principal and one intercalated cell. The basolateral
sides of the cells show a flattened appearance against the
filter, the presence of basement membrane, and loose
junctional complexes between the cells. At the apical
side, the cells have an abundance of microvilli and are
joined by a tight junction; (B) tight junction between the
two cells shown in (A). BM, basement membrane; PC,
principal cell; 10, intercalated cell; A, apical side; BL,
basolateral side; TJ, tight junction; IS, intercellular
space; MF, Millipore filter. Internal markers are 1 um for
(A) and 0.1 pm for (B).

79

 

Figure 16

8O

.mnwm pmcmum_ommn msu om »—~m_u_c_ cmvum mm: cwpzcwim: u

;o_;z cw mucmswcmaxm Lm~_e_m cw umcwmuno mgmz mu—zmmc _muPu:muH .m=Pm> mmmucmocma

m mumpsupmu o» umm: use umcwscmumu mm: muo_cma mew» umumuwucw mg» um cmxm—ocos mg» we muwm
comm co venom >u_>wpomowumc eo uczosm ms» .muwm pmuwam mg» 0» »——mwuwcw umuvm mm: Ammposa
oofiv e__==e-mzmu .mp_oo o: co .m__mo mem .mp_oo goo: sue; cocoon some meoe__e x_m eo nee
.mppmo puuu guy: umummm mLmHFFe mcoaw__wz acmcmeewu mcwc co mpcmswcmgxm m—mcwm sage um:_muoo
mmapm> come mg» ucmmmcamc mucwoa .ANEo\m__mu moH x NV Lmnsmgu Lma m__mu moH x m m:_m:
mcowuwncou _muwucmuw cmvcz umvmmm mgmz mcmnsmgo __< .A4V mPPmu o: co .A<V mppmu mpm .A-V
mppmo goo: .on mppmo Home sue: umummm mcmup_m mgoawppwz mo cwpzcwimzmu o» xuwppnmmscma

.NH ocamwo

81

 

1
l
240

1
1
l80

1
1
l20

I /

l l J I Q
o a s 8 8 3
We) HBAV'IONOW T131) DNISSOHO NI'IDNI-Hg

Figure 17

..CD
N5

 

 

 

TIME (min)

82

similar to that seen with CCCT cells. The flux of inulin across the
monolayer was found to be the same in both directions.

As shown in Figure 18, PGEZ, which is the major prostaglandin
product of CCCT cells (see Tables 3, 4 and 5 in this Chapter), crossed
the CCCT cell monolayer at the same rate as inulin. This suggests that
the only route for the movement of PGE2 across the monolayer is
pericellular. It should also be noted that less than 10% of the PGE2
added to one side of the CCCT cell monolayer is able to cross the
monolayer in 60 min; 60 min was the longest treatment time used in the
studies reported below. Neither bradykinin nor AVP affected the rates
of inulin (Figure 19) movement across the cell monolayer. PGEZ
movement across the cell monolayer was also unaffected by these
effectors (data not shown). (No metabolism of PGE2 was observed in
these experiments.) Our results on the movement of PGE2 are
consistent with the observations of other groups which indicate that
prostaglandins do not freely diffuse through cell membranes (70,72).

The sidedness of the effect of AVP on the release of cAMP by CCCT
cell monolayers is shown in Figure 20. Extracellular cAMP was measured
in all cases because it was impractical to sacrifice a monolayer for a
single measurement of intracellular cAMP. AVP added to the basolateral
surface of CCCT cells caused the release of cAMP, but even supramaximal
concentrations of AVP (10'6 M) added to the apical surface of CCCT
cells failed to elicit cAMP release. cAMP was released on both sides
of the monolayer, a result consistent with the observation of elevated
medullary cAMP levels noted when animals are treated with antidiuretic
hormone (108). To determine the time course for cAMP release (Figure

21), CCCT cells were treated for 0, 15, 30, 60, or 120 min with AVP on

83

.mewm .acoee_omea age o» ».Pa_ewee
cmvwm mm: u_ cmcz pmowucmup mm: mmuaim: u we x:_$ Lm_:P—mum:mcu mshr..mc=mwe m_;u
cw umuuwamu mucmswcmaxm mg» cw mvwm Fmo_am m

e» a» »__a_pee_ woven me: A: -oH m8.er
oofiv deepen eo_oaa_o_eaa .moozemz oz< mmqmmme<z =_ eoawcomou we Beeswaoe ace: mwxspe ops—om

.mmpapom m u we zoom mo mmxapw mg» ummu op umm: mgmz mcmnsmcu x_m mama mg» .on +mz N
eea .Aav mua flzmu .on ePF=e_-mzmm a» aco1a_oeoe __oo euum Heazpeeoo ea xoe_vaao5cma

.mH mczmwm

84

 

 

O inulin
A PGEZ
O No"

 

 

 

 

4.
l
120

1*?
I
60

 

.0

'0

I I s o

o o ofi‘ 8\

N st
We) HBAVTONOIN 1130
.1000 SNISSOHO 31.0108

Figure 18

TIME (min)

85

.00 0 100.0001 1000000000010 00

mocmmnm 00 mocmmmgq mso c0 Louum00m 0o mucmmnm ..m.0o mmapm> 1000000 5°00 acm0m0000 010000000c00m0
.ucmsummcu 0mg mmomo0pam0 0:00 0o cams mzu ocmmmgamg ucm 00m0000 ppmu Emgmogu0s 0mg cmmmmgaxm

m00 10:00:0Fm00m000 0:00 mcu 00 zoom 000 xum>oom0 wooH op um~0_oegocv 0000 mgp .mooxpmz oz<
mo<omo0<z c0 vm0000mmu m0 mmr00000 v00 umuumguxm mcmz m00u001mmumogo .m0000m00m :00: c000000000
on 00000 0msgo00mq mm: A: muo1v :m0o00090010 0o mucmmnm no mucmmmca mgu fir co0uma=uc0 :05 co <
.0000000000 N00 00 0 00000 .00 00 00000000 00 00 12 -011 000-00 .1: 0-010 0000010000 00

mucmmmca mg» :0 :05 cm 000 umpmaauc0 mcmz Agm0v mgaupzu as mo\o01 x 1o mppmo pooo 0cmzp0coucozm

 

 

 

1.0 0 0.0 1.0 0 0.1 1.0 0 0.1 0.0 0 0.0 000000000010 + 0>0-00
0.0 0 0.0 1.0 0 0.1 1.0 0 0.1 0.1 0 0.010 000-00
1.0 0 0.0 0.0 0 0.1 0.0 0 0.1 1.0 0 0.0 000000000010 + 0000000000
1.0 0 0.0 1.0 0 0.1 1.0 0 0.1 0.1 0 0.000 0000010000
0.0 0 0.0 1.0 0 0.1 1.0 0 0.1 1.0 0 0.0 000000000010 + 00000000 oz
0.0 0 0.0 1.0 0 0.1 1.0 0 0.1 0.0 0 0.0 00000000 02
01000-0001-0-0 00x00 000000 00001 000000000

1:05 oo\c0muoca Ppmo m:\mm_o50v
0mmmm1mm mc0ucmpomumoeo m>0uummLocseeo

 

 

0000000000010 00 000000
1mmsmwo mczopao :o mppmo pooo >5 mc0ucm~mmumo00 0o mmmmpma

m mpnmh

86

.mm

0 Amo.ovov A0000000 0o mucmmno 0o mucm0m00 mg0 =0 Louom00m 0o mucmmno ..m._v 0m000> 1000000 5000
0:m0m000u 0000000000o0m .0cm500m00 0mg 0m000010m0 x00 00 come mg0 0cm0m00m0 uco c0m0000 ——mu
500000005 0mg ummmmgqu mgo 0000 ms» .moozboz oz< mo<1mop<z =0 cmn00ummu mm c00000000000 00000
000500: mammmoocseswm0u00 00 vm000000 m0mz mcowuapom ocwzuoo .00000m00m :00: 00000000=0 00 00000
ums0o00ma 00: A: -o1v c000000 0o mucmmnm 00.Wocm0m00 mzo :0 gm00mnso=0 :05 am < .mgmsamosuo

moo an 0 0muzz 000 00 0ouum00m 0: 0o A: -oHV 0><ioo .Az oIOHV c00000000n 0o mocmmm00

mg0 =0 :02 so 000 umuonzoc0 m0m: Agm0u mgmupzo as oo\ooH x 1o 00000 pooo acmap0coucozm

 

 

 

1.0 0 0.0 0.1 0 0.0 0.1 0 0.0 0.0 0 0.0 0000000 + 000-00
0.1 0 0.0 0.0 0 0.0 0.0 0 0.0 0.1 0 0.000 000-00
0.1 0 1.0 0.0 0 0.0 0.1 0 0.0 0.0 0 0.0 0000000 + 0001010000
0.0 0 0.0 0.0 0 0.0 0.0 0 0.0 0.0 0 0.000 0001000000
1.0 0 0.0 0.0 0 0.0 0.0 0 0.0 1.0 0 0.0 0001000 + 00000000 oz
1.0 0 0.0 0.0 0 1.0 0.0 0 1.0 1.0 0 0.0 00000000 oz
01000-0001-0-0 00x00 000000 00001 000000000

A005 oo\:0m0000 01mg m:\mm_o50o
ammom—mm 0:0u0010000000 m>0000m0000551

 

 

00101000 00 000000
”mmsmwo m0:0_:o co m—pmo booo 00 0:0vcmpm000000 0o mmmm—mm

o m—nmh

87

.0m 0 Amo.0vav A0o0um00m 0o mucmmam ..m.0o mm000> 0000000 0°00 00m0m0000 000000000co0m0
.00m000m00 0mg mm0000—qm0 00000 00 00mg mcu 00mmm0am0 0000 m00 .mooz0oz

oz< m0<1mm0<z 00 umn0000m0 00 000000000020 00000 oaosu0z 0:00:00m000000 m>0000m0o0=EE0
000 0m000000 000 umpooa 00: gmxo—ocos mc0 0o mmm0m soon 0000 eprvmz .m0m0000000

Noo an 0 0m000 000 00 0000m00m 00 0o Azo-o1o 0><ioo .0 -o1v :000x10000
0o mucmmmga m00 00 :00 so 000 000000000 m003 00m05000 co 0mg 00:0 00—mu 0ooo0

 

 

1.1 0 1.0 1.0 0 1.0 0.0 0 0.0 0.0 0 0.0 0 000.00
0.0 0 0.1 0.0 0 0.0 0.0 0 0.0 0.0 0 0.010 0000010000
0.0 0 0.1 0.0 0 0.0 0.0 0 0.0 1.0 0 0.0 00000000 oz
01000-0000-0-0 00000 000000 00000 000000000

Agmnsozo\00a oo\0mposqv
0m00m0mm 00000010000000 m>0000m0000051

 

 

000m0100 m0000100z co 010mo 0ooo 00 00000000000000 0o mmompmm

m m—nMP

88

.0caow m0: ALouummwm 0: ..0.0V

m0mpsmsu 1001000 00 xup1_000500a 0:0 5000 00:00000_0 ucmuw0wcm_m oZI .m00050gu 000001000»
00 c005 0;» mp=0m00000 :000m gumm .0mxwpocos 0;» 0o mmvwm :uon co 2 -01 00 acmmm0a 000:
000000000 110 .0010 100100 000 00 011010101 00000 00: 1001000 0010 =11m01-nzmg .000000000
00 00:0m00a 0:1 :1 m0mu__m 00oa_11_z co m00»0_o:os 1100 Fuuu 0o :__:cwnm:mH op auw—pncms0ma

.01 000010

89

ovN 00.

.515 m8;
ON.

00 on O

 

 

0000

 

 

 

 

 

 

21.15 mi...
2.0 om. on. 8 on o
oo. 1 1 1 1 1 oo.
8 I .. oo
8 I .. o0
z_z_x>o<¢m

 

 

 

 

 

1.11.5 0.11:. 1.01.01, 0:: ‘
05 oo. 00. cm on o 000 om. 90.. cm on o
1 1 1 1 1 oo. 1 1 1 1 1 oo.
1 cm I 1 on
a><uoo cahomuum Oz

 

 

 

 

 

 

(7.) HBAV‘IONOH T130 SNISSOEO "NONI-H:

Figure 19

90

.100.0v01 1000-00

mo 0000000 ..0.0V 00:10> 1000000 5000 000000000 010000000000m0 .maozhmz oz< m0<~mmp<z

:0 000000000 00 005000000 000: 0:<0 00101100000x0 0o; 00000000052000000 .meH z 0-o1

mo 00000000 000 :0 :05 on 000 00m 00 0002 0000500000 11< .00005000 x00 yo 0005 0:0 000000000
0000 0;» .000010000 000 00 0000 1100 10000010000 00 100 100000 000 000000 00 00000 000-00

2 muo1 00 00 00000000 o: 00 00000000 :0 0000100 000 00000000 0000 0:0 .10000010000

.00 0:0 100000 .< 102<0 00 0000100 00 0000 000 m0 00x0 100000000; 050 so 000000001

.0000100 000001102 :0 0000010005 1100 0000 5000 0000100 02<0 :0 000000 0><nao we 00000000m

.00 000000

91

 

 

d
d
-
-
d
q

 

 

 

 

 

 

 

 

 

 

8 L—0 0
g $\\\\\\\\\\\\\\\\\\§<0
3 ‘ 11);-
:>; H
é M45
g '-| 0’
g 0

 

PM

I I l l l

 

y no ct
(mm os/sauowd) dwvo uv1m133w1x3

Figure 20

92

.Amo.ov0VA0o0u0000 00 0000000 ..0.0v 00:10> 1000000 5000 000000000 000000000cm0m0

.00005000 0000010000 00 0000 0:0 0000000000 0:000 £000 .moozhmz oz< m0<1mmh<z :0

000000000 00 0z<0 000 00001000 000 001000 0003 000010005 000.00 00000 5000 5000 00002 .0000
1.0 100000 000 00 100 10000010000 000.00 00000 000-00 2 0-01 000; 00 .100 00000000 00

:00: 0000000 0500 000000000 000 000 x201 z -oH 00 00000000 000 :0 00m 00 000003000 0002
0000010005 1100 .0000100 00000110: co 00000m 01100 pogo 2000 0000000 0z<0 00 000000 050»

.10 000000

93

 

 

 

 

 

 

I I 1‘ .0 t

 

 

$3 9 2 m
(‘W/‘alo‘ud’ W9 UVWOTIBOWLXE

Figure 21

l20

90

TIME (min)

60

94

either the apical or basolateral side of the cells. Maximal levels of
cAMP in the medium were seen 60 min after addition of AVP to the
basolateral surface; AVP added to the apical surface did not increase
extracellular cAMP above those levels seen without AVP. Results on the
time course of release of cAMP are typical of those observed with
cultured epithelial cells (5). Half-maximal release of cAMP occurred
at a basolateral AVP concentration of approximately 10'10 fl

(Figure 22). This result is quantitatively similar to that seen with
CCCT cells grown on plastic culture dishes (Chapter 11, Figure 12).
These results suggest that AVP can act only from the basolateral

surface of CCCT cell monolayers to cause cAMP formation.

Prostaglandin Synthesis binCCT Cells. It was shown previously

 

that immunoreactive PGE2 was formed in response to AVP and bradykinin
by CCCT cells cultured on Petri dishes (Chapter 11, Figure 14 and Table
2). To determine if PGEZ was the major prostaglandin product,

analyses of the prostaglandins formed by CCCT cells were performed.
Prostaglandins were extracted from the media surrounding CCCT cells
treated with no effector or with AVP or bradykinin. The prostaglandins
were separated by thin layer chromatography and the materials in the
regions cochromatographing with PGEZ, PGan, Tx82 and

6-keto-PGF1a standards were eluted from the silica gel and analyzed

by specific radioimmunoassays (Table 3). The results indicate that the
major product formed both in the presence and absence of AVP and
bradykinin was PGEZ. Small amounts of other prostanoids were also
detected but, unlike PGEZ, the amounts of these other products did

not increase following treatment of CCCT cells with AVP or bradykinin.

95

.Amo.0v0v A0><uoo 00 0000000 ..0._v 000_0> 0000000 5000 000000000 00000000000000 .00005000
00000_0000 00 0005 000 0000000000 00000 000m .mooxpmz oz< m0<HauH<z 0? 000w00000 00 0z<0 00»
00~00000 000 000000 0002 000000005 000 00 00000 0000 500» 0_00z .meH z -00 00 00000000

000 00 000 00 000 00m 00 000000000 0003 00000000000 .000000000 000 00 W000 .0000000000

000 00 xp0>00000x0 00000000000000 000000000 000 00 00000 003 0><u00 .0000000 00000000:

00 0000000005 0000 0000 0000 0z<0 00 0000000 000000wl0><u00 000 00» 0000000000 0000000000000

.00 000000

96

 

l
x-
1
1
IO"

 

1
no"
[BASOLATERAL oo-AVP] (M)

1
l0"'0

1
K542

 

 

 

(Demons/um oslsalowd) dwva W101130V81X3

Figure 22

97

Almost identical data were obtained when medium surrounding the cells
was analyzed by radioimmunoassays without prior extraction (Table 4).

Prostaglandin radioimmunoassays were also performed on media
pooled from the basolateral and apical surfaces of CCCT cells on
Millipore filters following addition of AVP or bradykinin to both sides
of the cell monolayer. Again, PGEz was found to be the major
prostaglandin product (Table 5). PGE2 has now been found to be the
primary prostaglandin formed by preparations of rabbit, canine and rat
cortical and papillary collecting tubules (13,84,144).

The effects on PGEZ release of AVP and of bradykinin added to
either the apical or basolateral side of CCCT cells on Millipore
filters was examined. As shown in Figure 23, AVP caused an increase in
the release of PGE2 when added to either the apical or basolateral
side of the cells. Half-maximal increases in PGE2 release were
observed at approximately 10"10 fl_AVP (Figure 23); the
half-maximal concentration for AVP-induced PGEZ release was the same
for AVP added to either side of the cell monolayer (Figure 23) and was
about the same as that observed for the release of cAMP (occurring when
AVP was added to the basolateral side of the cells)(Figure 22). The
amounts of PGE2 found on the apical and on the basolateral side of
the cells were comparable both in the case of treatment with no
effector or with AVP added to either the apical or basolateral surface
(Figure 24). Thus, PGEZ was released on both sides of the monolayer
in response to AVP added to either side of the monolayer.

Although AVP was effective in eliciting PGEZ release when added
to either side of the cell monolayer, bradykinin caused PGEz release

only when added to the apical surface of the CCCT cell monolayer

98

.0000002 oz< m0<Hmuh<z

00 000000000 00 N0000 000 00~xp000 000 000000 0003 000000005 000 00 00000 0000 5000 00002
.0000000005 0000 0000 000 00 0000 0.0 000000 00 000 00000000000 000 000000 00 00000 0><u00
00 00000000000000 000000000 000 0003 000 00 000 00m 00 000000000 0003 00005000 000 .00000_0
000000002 00 00_00 0000 00 Nmo00 00 0000000 000:00_-0><-00 000 00» 0000000000 0000000000000

.00 000000

99

 

 

l'flO
flab-AVE) (Ml

   
 

 

 

O BASOLATERAL

 

.J
<1
2
O.
“L 4 ~k
ll
1 1 1 1 1 4 1 1 o
co, 0, v. N
C) C) C) C)

(Ramona/111w oexsalowd) 33901!

Figure 23

100

.000.000 00000> 0000000 0000 0000000_0 000000_0_00000 .0000000

02< 00000000: 00 000000000 00 0003 000 000 00000000055000000 .005 00 000 00m 00 005000000
000: 00005000m0 PP< .0000000005 0.00 000 00 0000 A000 00000000000 00 A<V 000000 000 00
00000 0><100 z -00 000; 00 00000000 00 0003 000500000 00030__00 0000000005 0000 000 00
0000 A000 0000000m000 00 000 _00_00 000 00 00000005 00000 00 000050 000 00000000 0000 000
.0000000 00000.00: 00 0__00 0000 5000 00000 00 0000_00 000 00 000000 0><100 00 000000000

.00 000000

101

 

 

 

FL

 

 

 

 

 

 

 

 

 

 

 

 

 

a 0'
Z "l:k\\\\\\\\\\\<
E '—i 0
E W\\\\\\\\\\ 4
E a:
5 mq

(ugw 09/salowd) 3390!

Figure

24

SIDE OF MONOLAYER

102

(Figure 25). As shown in Table 6, PGEZ was found on both sides of

the monolayer at different times after the addition of bradykinin to
the apical side indicating that PGEZ was released on both sides of

the monolayer. Following bradykinin treatment the amount of PGE2

found on the basolateral side of the monolayer tended to be 1.5-2 times
greater than that found on the apical side (Figure 25); however, this
difference was not statistically significant, and the concentration of
PGE2 on each side of the monolayer was the same (ca. 4 x 10'9 n).

In experiments where simultaneous measurements of effector-induced

PGEZ release and cAMP release were performed, qualitatively

equivalent results as described above were obtained (Table 7).

PGEz-induced Release of cAMP from CCCT Cells. PGEZ was tested
at a variety of concentrations and on either side of CCCT cell
monolayers for its ability to elicit the release of cAMP. Significant
cAMP release was observed with Z 10'8'!_PGE2, and there was no
difference in the dose-reSponse curves for PGEZ added to either the
basolateral or apical surfaces (Figure 26). Thus, PGEZ, unlike AVP,
can act from either side of the CCCT cell to cause cAMP release. As
with AVP induction, the cAMP produced was released into both sides of

the monolayer (Figure 27).

Figure 25.

103

Sidedness of bradykinin effect on the release of iPGEz
from CCCT cells on Millipore filters. Each bar indicates
the amount of iPGE measured on the apical (A) or
basolateral (BL) Slde of cell monolayers following
treatment with no effector or with bradykinin (10'6 M)
added to either the apical (A) or basolateral (BL) side of
the monolayer. The data represent the mean of six
chambers. All treatments were performed at 37° for 60 min.
Radioimmunoassays for PGEZ were as described in MATERIALS
AND METgODS. *Significantly different from control values
P<0.05 .

104

 

l5

i PGEZ (wholes/60 min)
5
I

 

CONTROL

BRADYKININ (A) BRADYKININ

itr-r-

 

\\\\\\\\\\\\V—'

 

 

 

 

(BL)

 

 

7A

 

fig:

A

A BL
SIDE OF MONOLAYER

Figure 25

A BL

 

105

.00 0 000.0000 000000000 00

00 00000 ..0.00 00000> 0000000 5000 000000000 00000000000000 .000500000
00 0005 000 000000000 0000 000 .0000002 oz< m0<0000<z 00 000000000 00

000 0000000 000 0000—0005 000 00 0000 0000 5000 00>0500 00: 500005 .0500 0000000000
000000000 000.0000< .0000000500 ~00 00 0 00000 00m 00 00000000 00 000:

000 00 0

0000000 000 00 005
000 0000000000 030
«000 0>000000000550

00 00000000 000000

0-000 0000000000 00 00000000 000 00 00500 000000000 000 000 000000000 0003

0000\00000 000 x 00 0000000 000 0.000 0000 000 x 0 0003 000000 0000000 0000000000

 

 

 

0.0 0 0.00 0.0 0 0.00 000 00 .0000000000
0.0 0 0.00 N.0 0.0.00 005 00 .0000000000
0.0 0 0.00 0.0 0 0.00 000 0 .0000000000
0.0 0 0.0 0.0 0 0.0 000 N .0000000000
0.0 0 0.0 0.0 0 0.0 000 0 .0000000000
0.0 0 0.0 0.0 H 0.0 000 00 .00000000 oz
0000 00000000000 0000 000000
00005000\00—0500

N000 0>000000000550

000500000

 

 

00000—00 0000000000 00 00000 0000
00 0000 00 0000000 0000000-0000x»000m 00 000000 0500

m 00000

.00 0 000.0v00000000000

00 0000000 ..0.00 00000> 0000000 0000 000000000 3000000000000. .000000000 000 00000000
0000000000 00 0000 000 000000000 0000 000 .00000000000000000 »0 00000000 F0000 000 mooxbm: 020
00<0000<z 00 000000000 00 0000000 003 000000000 000 00 0000 000000 E000 500002 .x200 z -00

00 00000000 000 00 0003 00000000000 __< .0000000000 ~00 an 0 0000: 00m 00 .00000000 m0 003

00 .000000000 000 0o 0000 0000 00000000000 00 000 000000 000 00 00 -000 0>< 00 00 0-000
0000000000 00 00000000 000 00 00s 00 000 000000000 0003 00000000 0% 00000000 00000 00000

 

 

106

 

0.0 0 0.00 0.0 0 0.00 0.0 0 0.00 0.0 0 0.00 0000 0><

0.0 0 0.0 0.0 0 0.0 0.0 0 0.00 0.0 0 0.00 000 0>0

0.0 0 0.0 0.0 0 0.0 0.0 0 0.0 0.0 0 0.0 0000 0000000000

0.0 0 0.0 0.0 0 0.0 0.0 0 0.0. 0.0 0 0.00 000 0000000000

0.0 0 0.~ 0.0 0 m.N 0.0 0 0.0 N.0 0 0.0 00000000 oz
0000 00000000000 000m 00000< 0000 —0000000000 0000 00000< 000000000

00000000\00e 00\00—0000 00000000\00E 00\00_0s00

0z<0 0000—000000x0 N000 0>00000000=EEH

 

 

00000M00 00000000: 00
00000 0000 >0 0z<0 000 000 00 0000000 0000000s00000000

m 0—000

107

.Amo.0vao Amman oo

mucmmnm ..m.pv mmapm> Pogucoo sag» ucmgmwmwu xpocmu_wwcm_m* .mgmnEmsu mumuwpg_gu $0 cams ms»

mucmmmggmg p=_og sumo .moozpmz oz< mo<omop<z cw cmn_gummnnmm az<u Loo vm~apu=m new um_oog

ago; mgwxmpocos mg“ we mmv_m goon sag; ovum: .xzmo z -oH oo mucmmmga mgu :_ cos so

go» cum on vmsgowgwa mgw: mcowumnsoco .mgmxwpocos Fpmu Fooo so we muwm Ago Fmgmumpomon Lo
on Pmu_am an» ngu_m o» mco_umgucmocou vapouwu:_ ago on umucm mm: Noon .mgmu—_$ mgoawppwz
co mgmxmpocoe __mu pooo sag» az<u mo mmmmpwg umuaucwumooa mg“ Low mocmucwamu co_umgucmu:oo

.cN mgsmwu

108

 

 

 

 

 

l l I I l I
0
— O «.1 I
3k at ._
* a.»
I— ‘0 -
* .—
El
l «l
:3 an
p. ( -1-0 0
l -|CL|
N
m
o I
- a.
.1
MN ‘
0 I: ~
0- %i‘ J:-
pn-
.J j _
< o
2 m
0. <I
. <1 m -
o . if
~~ ‘L

Té if—‘é—‘é—‘é :l-To

(momma/um 09/salowd) dwvo W'In'l'IBOVULXB

 

Figure 26

Figure 27.

109

Sidedness of PGE effect on cAMP release from CCCT cell
monolayers on Millipore filters. The bars indicate the
amount of extracellular cAMP measured on the apical (A) or
the basolateral (BL) side of cell monolayers following
treatment with no effector or with 10'6 M PGE added

to the apical (A) or basolateral (BL) side. he data
represent the mean of six chambers. All treatmenta were
performed at 37° for 60 min in the presence of 10‘ M
IBMX. Radioimmunoassays for extracellular cAMP were—
performed as described in MATERIALS AND METHODS.
*Significantly different from control values (i.e., absence
of PGE2) (P<0.05).

110

 

 

 

 

 

 

 

 

 

 

CONTROL PGEZ (A) PGE: (BL)
lCXD- ’6-T_-
r-
3 ..
E 30_
O
‘3
3 +-
3 L
a 60 .9 ~
CL
5, _.
05 *
g 4(-
a 4OL
E _
m
20-
LE J 1 J /
A BL A BL A BL

SIDE OF MONOLAYER

Figure 27

 

DISCUSSION

CCCT Cells on Millipore Filters. The initial goal of the studies
reported here was to test the concept that there is a sidedness to the
release of prostaglandins from CCCT cells and to the effects of
prostaglandins on cAMP metabolism by CCCT cells. This hypothesis
developed from the observations of Stokes and Kokko that PGEZ
inhibited Na+ resorption when added to the basolateral but not the
luminal surface of segments of rabbit cortical collecting tubule (96).
The CCCT cell-Millipore filter system was developed to examine the
sidedness of prostaglandin biosynthesis and function. The criteria
which have qualified this CCCT cell system for use in these studies are
as follows: (a) CCCT cells were morphologically asymmetric as
determined by TEM; (b) CCCT cells exhibited a transcellular potential
difference of the appropriate sign; (c) CCCT cells were impermeable to
both inulin and PGE2 and (d) CCCT cells formed cAMP in response to
AVP added to the basolateral but not the apical surface of the cell
monolayer. Although MDCK cells also exhibit some of these properties
(4,5,143-145), this is the first time that a sidedness to the response
of cells on Millipore filters to a hormone (AVP) has been

demonstrated.

Functional Asymmetry of CCCT Cells. The major findings on aspects

of asymmetry are: (a) that PGEZ is released from both the

111

112

basolateral and apical surfaces of CCCT cells, (b) that PGEZ

functions to elevate cAMP in CCCT cells when added to either the
basolateral or apical side, (c) that bradykinin acts only from the
apical surface of CCCT cells to elicit prostaglandin formation, and (d)
that AVP can act from either the apical or basolateral surface to cause
prostaglandin production by CCCT cells.

Our studies on the biosynthesis of prostaglandins by CCCT cell
monolayers indicate that PGEZ is the major if not the exclusive
prostaglandin product formed both under basal conditions and in
response to hormonal stimuli. PGEZ was found on both sides of the
cell monolayer in similar amounts. This result cannot be ascribed to
simple transcellular diffusion since the flux of PGE2 across the
monolayer is quite slow. Thus, PGEZ is released on both sides of the
CCCT cell monolayer suggesting, in turn, that PGEZ is released on
both the blood and urine sides of the canine collecting tubule in 1119.
A previous report had indicated that the major site of entry of primary
prostaglandins into urine occurs at the level of Henle's loop (146).
The experiments reported were performed in rat kidneys perfused with
angiotensin II, which appears to cause prostaglandin synthesis in
glomerular epithelial cells, certain portion of the renal vasculature
and in renomedullary interstitial cells (17). Angiotensin II does not
elicit prostaglandin formation by collecting tubules (17) or by CCCT
cells. In addition, recent studies show that AVP, but not DD-AVP,
stimulate PGEZ synthesis by rat renomedullary interstitial cells in
culture (85); yet DD-AVP as well as AVP stimulates renal PGEZ
excretion in the rat in vivo (147-149). These data imply that the

increase in urinary PGEZ excretion in response to AVP or DD-AVP is

113

due to increased PGEZ synthesis not by renomedullary cells but rather
by the vasopressin-sensitive collecting tubule (149). Our results
further suggest that, at least in the dog, PGEZ can enter the urine
at the level of the collecting tubule under conditions where renal
bradykinin or AVP concentrations are elevated (150). Our results also
indicate that if PGE2 functions extracellularly to influence
collecting tubule metabolism, PGEZ presumably can affect events from
both the apical and basolateral cell surfaces. As will be discussed in
more detail in Chapter IV, this concept is consistent with the lack of
sidedness to the effects of PGE2 on cAMP metabolism in CCCT cells.

Although there was no asymmetry to PGEZ release, there was a
sidedness to the effect of bradykinin on prostaglandin synthesis.
Bradykinin acted only from the apical surface to cause PGEZ release
(Figure 28). This sidedness of bradykinin action on the prostaglandin
biosynthetic system was conceptualized by McGiff and coworkers (151),
who proposed that only urinary and not blood-borne kinins are involved
in eliciting prostaglandin synthesis by collecting tubules. The
sidedness of bradykinin action observed in CCCT cells is also
consistent with studies showing that kallikrein may be located on the
apical side of the distal tubule (152,153). It should be noted,
however, that bradykinin appears to act only from the basolateral
surface of rabbit collecting tubules to antagonize the hydroosmotic
effect of AVP and that this antagonism appears to be prostaglandin
mediated (154).

The lack of a sidedness to the effect of AVP on PGEZ formation
was unexpected (Figure 28). AVP caused the release of PGE2 when

added to either side of CCCT cells even though AVP elicited cAMP

114

Figure 28. Apical-basolateral asymmetry of effector actions in CCCT
cells.

115

BASOLATERAL

APICAL

 

F‘SEEz: ‘9""

 

 

BK

Pol:2 ——> cAMP <—— PGEZ

 

AVP ——> PGEZ e— AVP

AVP -—> cAMP

 

Figure 28

116

release only when added to the basolateral side of the cells. These
data suggest that AVP interacts with different receptor systems in the

cases of prostaglandin and cAMP formation.

CHAPTER IV

AVP-PGE? INTERACTIONS IN CANINE
CORTICAL COLLECTING TUBULE (CCCT) CELLS

There is strong circumstantial evidence that prostaglandins
inhibit the hydroosmotic effect of AVP 1n_vivg as well as jg_vitrg
(99,117). For example, indomethacin treatment and essential fatty acid
deficiency, two regimens which inhibit renal prostaglandin production,
increase urine osmolality (103,104,155). Thus, PGEZ appears to have
a physiological as opposed to simply a pharmacological action on the
collecting tubule.

Recently, PGEZ has been observed to inhibit AVP-induced cAMP
accumulation in the renal cortical collecting tubule. This action of
PGE? accounts for inhibition of the hydroosmotic effect of AVP (156).
However, the molecular mechanisms underlying this inhibition have yet
to be determined.

In performing the studies on the sidedness of hormonal responses
in CCCT cells described in Chapter 111, it was noted (a) that extremely
low concentrations of PGE2 (10‘12 fl) would inhibit the release
of cAMP that normally occurred in response to AVP and (b) that this
inhibitory effect occurred with PGE2 added to either side of the CCCT
cell monolayer. In this chapter, experiments on the inhibition by

PGEZ of AVP-induced cAMP formation by CCCT cells are described.

117

MATERIALS AND METHODS

Materials. All materials were as described in Chapter III.

Isolation and Growth of CCCT Cells on Millipore Filters. CCCT

 

cells were isolated by immunodissection as described in Chapter II.
The cells were seeded on Millipore filters and their asymmetrical
functions and permeability characteristics were monitored as described
in Chapter III. Alternatively, CCCT cells were seeded as described in
Chapter II, in multi-well culture dishes for intracellular cAMP

measurements.

Effector-induced cAMP Release. For CCCT cells grown on Millipore
filters, cAMP released into the surrounding media was measured.
Effector dose-response curves and experiments involving more than one
effector (e.g., AVP plus PGEZ) were performed using one treatment per
chamber. All other procedures were as described in Chapter III and

detailed in figure legends and table captions in this chapter.

Effector-induced Intracellular cAMP Formation. Treatment of
monolayer cultures of nonconfluent CCCT cells with effectors (i.e.,
AVP, PGEZ, or both) was done in triplicate using 24-well culture
dishes seeded at a density of 5 x 104 cells/well. Cells were rinsed

free of media with Krebs buffer, pH 7.4 containing 10-4 fl IBMX; 0.3

118

119

ml of buffer alone or buffer containing 10’11 N_PGE2 was then

added for a preincubation period of 60 min at 37°. Following this
preincubation, the solutions were removed from the wells and 0.3 ml of
buffer alone or buffer containing an effector was added to the cells.
The monolayers were incubated for the desired time at 37°. The
solution in each well was removed and 500 pl of cold 6% TCA was added.
The wells were incubated at -80° for 20 min, thawed at 24° for 25 min
and incubated for 2 hrs at 0-4°. The liquid in each well was then
transferred to a test tube and extracted four times with 10 volumes of
diethyl ether. Any remaining ether was evaporated in a 60° water-bath
for 10 min. The remaining aqueous phase was lyophilized. The
lyophilized samples were resuspended in 0.125-0.2 ml of buffer and

assayed for cAMP by radioimmunoassay as described previously (13).

Statistical Analysis. All experiments were done using a minimum

 

of three replicates per treatment. A completely random analysis of
variance was used to test for differences between sample means at
P<0.05 (124). Dunnett's test was used for comparing differences
between treatment means with the control mean (124). When it was
desired to compare all the treatment means to each other and not only
to the control mean, Student-Newman-Keuls' test for all possible

comparisons was used (124). Error bars on the figures are t SE.

RESULTS

Inhibition of AVP-induced cAMP Release by PGEZ° At concentra-

 

tions of 10-10 )1 or less, PGEZ had no significant effect on
cAMP release (Chapter III, Figure 26). Therefore, we used these con-
centrations to test the effects of PGE2 on AVP-induced cAMP release
from CCCT cells. In the first experiment, PGEZ was added to both the
apical and basolateral side of the cell monolayer at a concentration of
IO'IQN on both sides one hour prior to the addition of AVP.
Monolayers used as no effector controls or monolayers treated with AVP
alone were preincubated for 60 min with buffer that did not contain
PGEZ. The preincubation medium was removed after 60 min; then,
following a further one hour incubation in the presence of no effector,
AVP (lo-8 [1) alone or both AVP (10-8 )1) and P352 (10'10
N), the medium was assayed for cAMP. Under these conditions, PGEZ
completely blocked AVP-induced cAMP release (Figure 29).

Figure 30 shows the sidedness and the concentration dependence for
PGEZ acting as an antagonist of the release of cAMP occurring in
response to AVP (10"8 )1). Significant inhibition of AVP-induced
cAMP release was noted at concentrations of PGE2 as low as 10.12 5,
Interestingly, the dose-response curves for inhibition of AVP-induced
cAMP release were the same for PGEZ added to either side of the CCCT

cell monolayer. One cannot ascribe the inhibition obtained with

10'1?g PGEZ to diffusion of PGE2 across the monolayer since a

120

Figure 29.

121

Inhibition by PGEZ of DD-AVP-induced cAMP release from

CCCT cell monolayers on Millipore filters. Each bar
represents the amount of cAMP measured on the apical (A) or
basolateral (BL side 08f the CCCT cell monolayer following
treatment with (a) M DD-AVP added to the

basolateral side, (b) 10:8 M DD-AVP added to the
basolateral side) plus 10-10 M PGEZ added to both

sides) or (c) no effector. ATl samples were preincubated
for 60 min at 37°. PGEZ was added for the duration of the
preincubation period to samples that were to be treated with
PGEZ. All other samples were preincubated with buffer
alone. At the end of the preincubation period the media was
removed and the monolayers were incubated for an additional
60 min at 37° with the effectors indicated. Following this
incubation period, the media from the two sides were removed
and assayed for cAMP as described in MATERIALS AND METHODS.
All treatments were performed with triplicate chambers in
the presence of 10'4 M IBMX. *Significantly different

from control values (FK0.05).

122

 

DD-AVP (BL) DD-AVP (BL)

 

CONTROL
4o

 

 

 

 

 

 

 

 

 

BL

A

BL

A
SIDE OF MONOLAYER

BL

 

 

 

m s
Ar
IV////////////////////////////////////
L
E jA

2.5 028.25. “=23 «5348455

Figure 29

123

Figure 30. Concentration dependence for the inhibition by PGE of
DD-AVP-induced release of cAMP. CCCT cells on Mil ipore
filters were preincubated for 60 min at 37° with the
indicated concentrations of PGE2 on the apical or
basolateral side of the monolayers. The chambers were then
incubated for a second 60 min period at 37° with the same
concentrations of PGE2 plus DD-AVP (10'8 M). DD-AVP
was added only to the basolateral side. Media from both
sides of the monolayer were pooled and assayed for cAMP as
described in MATERIALS AND METHODS. All treatments were
performed in the presence of 10'4 M IBMX. Each point
represents the mean of duplicate chambers.

*Significantly different from control values (i.e., no
effectors; dotted line); *Significantly different from
values of samples treated with DD-AVP alone) (P<0.05).

l2

.5

II 7.11!
fix
1

DD-AVP-INDUCED EXTRACELLULAR cAMP
(pmolos/SO min/chamber)

l2
* d
8 .x. * '1
.8222‘3’2--_-_. ................ ..
or PGEZ

4.. ..
b d
H94 1 _ J. L:

o IO no"2 no" io"°

124

’ l I I T

APICAL Poe2 ‘

 

-L.
d)—

.—
u)—

BASOLATERAL Poe, *

 

 

 

@653 (Ml

Figure 30

125

maximum of 10% of the PGEZ could have crossed the monolayer during

the 60 min preincubation period (Chapter III, Figure 18). Thus, PGE2
apparently can act from either the apical or basolateral surface of
CCCT cells to inhibit AVP-induced cAMP release, even though AVP appears
to act only from the basolateral surface to cause cAMP formation
(Chapter III, Figure 20).

The concentration of PGE2 surrounding the CCCT cell monolayers
after a 60 min preincubation (Figures 23-25) ranges from 10'9 to
10'10‘M_due to endogenous synthesis of the prostaglandin. When
endogenous synthesis was blocked with either Ibuprofen or aspirin, a
potentiation of the AVP response was observed (Table 8). Nevertheless,
exogenous PGEZ (10'11 M) completely suppressed AVP-induced cAMP
release in the presence or absence of cyclooxygenase inhibitors. Thus,
the effect of newly formed prostaglandin, while measureable, does not
appear to have an overriding influence on this system. This appears to
be due to two factors. First, the inhibitory effect of PGE2 requires
a prolonged incubation with this prostaglandin (see Table 9), and the
PGEZ arising from the cells themselves is not present at elevated
concentrations during the entire preincubation period. And second,
after the preincubation period, the medium surrounding the cells is
removed and replaced with fresh medium to begin the 60 min treatment

(e.g., with AVP).

Inhibition of AVP-induced Accumulation of Intracellular cAMP by
PGEZ. In studies on CCCT cells seeded on Millipore filters, only
cAMP release was monitored. To determine if PGEZ inhibits

AVP-induced accumulation of intracellular cAMP, experiments were

.Amo.o v av oeo_o a>< new: oeoanoco Lo o=_os eoee oeocoeeeo apeeooee_emeaa

”Acouommem o: ..m._v m=~o> .oeucou soc» “cosmewwu a—ucmu_m_=mwm# .mconEozo muou_Pq:m eo come we»
mocmmmcams mapo> zoom .uo_sma :_E oma ago uaosmsocgu mcmasosu Ppo :_ ucmmmca mo: xzmo z wiofi .mcou
iwawgcp scam uaocuwz memQEogu cw ms_p msom on» Loo umm: mo: cmwwzm .Acws omH Lo —ouou o ..m _o mvowcmq
“cosuomcu can :o_ooa=ucpwea uaosmaoegu cusp awn newcma cowuonaymwmca as» o» Lowca mwuchs om opvme

we» :_ ucmmmcq mo: u_ .umm: mo: Acmmocaano z ioH Lo =_cpqmo z uofiv couwnw;c_ mwmmgwcxm =_u=opm
noumoea o mem;3.mcmnEoco ms» cm .om_m poewoowomon maple» »_:o mauve mo: ¢>< .mmoa z HaioH mapa

¢>< 2 tea so mac—o mom 2 ioH .mcopm o>< z -oH .Louumemm 0: new: okm um uopcmq :_5

oo Pocowuwuco so so; owuonzucw mew: mcmxopomwe mew vco um>oeme mo: o_uwe as» .uopcma copumaau:_mea we»
eo ucm on» u< .Lmampoeme mg» we mwuwm soon :0 AN vcm H muewsuomehv mcopm Lemon; now: go fie uco m mucwe
-uomehv Noon 2 Hfiuofi ;p_z cum on :_2 co com cmoonzuc_mea mew: memupwe usage—Fez co m__mu hoooo

 

 

126

 

vaumge mduwga mauege Qfiufioa 32+a2 é
Confie HOHWN wanes Roufio 22(32 a
H.H.u «.moe m.o.H m.HH# H.H.u m.efie m.fl H w.oHn oeo.< a>< .N
flfiufio floudk douqo doueo csgtwg A
e_c_am< coo_a_eeo oz eoeocaaao cooea_;e_ oz oeoEoeoce

Aemnsogo\:_e oo\mw—o5qv
az<u copap—muocuxm

 

 

mnemozucam :_u:opmoumoea eo
meouwn_zco eo mucmmweo ego cw mcmupwu «can? —P2
:0 msmxopocoz .pmo pooo soc» mmompmm az<u umuzucwuo>< eo won an coeu_n_;co

w m—nwh

127

.zmo.o v oo ozooo o>< zoez ozoEoooco eo oozes soce ooocoec_o

ozooooee_ooeoa mtooooto oz ..o.eo o=_o> .ocoooo eoce oeocoeeoo »_oooo_eeooomo .moozemz oz< mo<oeme<z
cw vmoweummv mo zommooc=EErmwvoc an coezmome mo: oz<u Lo_=~pmuosp=~ .mppmz muoowpapeu we come ms»
mpcmmmcomc mapo> gamut .xzmo z -oH monoucmmmca we» co umscomcmo mew: meowumm=u=_ uco mcopuonzucwmeo
.mooo z -oH mama o>< z onoH so mco_o mooo ioH .mcopo o>< z -oH .couummwm o: ;p_:

cps m Lo :_5 N so» owuonzomm mew: m—pmo mg» can um>osmc mo: momma ms» .uowema cowmonao:_wea F—ae mg» mo

ucm wcu u< .Amooo mo wucmmwca mg» no :o_uon:o:kmcq we c_s m An ooze—pom mac—o cmeean cw cooponaocomca eo

cos mm omuapucw e ucmsuomc» ..m.mv Nooo z HioH zoo: copuonaocwmea as» x5 vmzo__ow moo—o cmwwan cw
umuonzuc_mcn mom: mp_mu mg» .Akio mpcwEuomchv co? cm can» mmm_ coo vascooewa mo: «moo goo: cmouonauc_meo
mgu meme: macmEpomcu co .Amifi mucmEHomehv mcopo emewan ;u_: so Amie mucmEuomepv Noon 2 wmiofi zoo:

 

 

 

cum um floor—ma 0:5.» vmuwowbcw mg“ bony vouwnzucwwga 0L9» mwcmwv whoa—.30 co Uwvmwm 2.me #000 a: choucozw
o.o.w N.~e m.o H.m.~e Nooo + o>< oo o .o
m.o.H ~.~a N.o.w H.~a Nmoo + o>< oo om .e
H.o H.L.Ha o.o H.o.~e Nooo + o>< oN oo .o
z.o H.~.oo m.z H.H.~o Nooo + ozz oz om .m
o.o.w m.m# m.o.w H.oo Nooo + o>< m mm .o
m.H.H m.oo o.o H.w.oo Nuoo + o>< o oo .m
z.o.w o.mo ~.o H.o.oo ooo_< o>< o oo .N
~.o + ~.z ~.o + o.~ cooooeeo oz o oo .z
are m cos N «cosummgh .I mcpev Acweo
zoooooeo .zoo oo\mo_oeeo A: Hz-ozo moo zoo: oe_e ooozo coeeoo zo_z oeee
az<u copap—muocuco cowuoaaucwmco

 

 

ooozopo ocoo_oo eo o__oo eooo zo oz<o eo oo_ooeLoa
oooooe_-o>< co Nooo zo oooo_o_zoo ozo eo oeoe oooooooooeoco Lo oooeeo

o ozooe

128

performed using CCCT cells grown as nonconfluent monolayers on plastic
culture dishes. As shown in Table 10, 10"11 M_PGE2 suppressed

the AVP-induced accumulation of intracellular cAMP at 2 and 3 min time
points. This inhibition also occurred when endogenous PGEZ formation
was blocked (Table 11). Curiously, inhibition was only observed after
a 20 min preincubation of PGE2 with the CCCT cells (Table 9). A
similar time dependence was observed in the inhibition of AVP-induced
cAMP release from CCCT cell monolayers on Millipore filters. This time
dependence suggests that PGE2 is causing a chain of metabolic events

to occur which prevents AVP-induced cAMP accumulation.

Torikai and Kurokawa suggested that PGEZ may exert its
inhibitory effect on AVP-induced cAMP formation by activating cAMP
phosphodiesterase activity (156). Here this true in the CCCT cell
system, one would expect 10'12-10'10 M_PGE2 to decrease
basal levels of intracellular cAMP. However, these concentrations of
PGE2 had no effect on intracellular cAMP levels in the presence of
10-4 M IBMX (Table 12).

A dual receptor mechanism for prostaglandin action was conceived
to accomodate the biphasic nature of the effects of PGE2 on cAMP
metabolism in CCCT cells. We propose that one class of receptors,
displaying a low affinity for the ligand (i.e., PGEZ), is involved in
the activation of adenylate cyclase. We suggest that another class of
receptors, having a high affinity for the ligand, is involved in the
suppression of AVP-induced adenylate cyclase activation. In support of
this concept were the observations that PGFZa inhibited AVP-induced

cAMP formation but PGFZQ did not increase cellular cAMP levels.

129

.Hmo.ovoo wooHo ozz zoo: oooEHooeo eo ooHos eoee

acmewewHU AHHchHFHcmHme HHeouuweew o: ..m.HV maHm> Hoeucou Eoee acmeweewu xHucouHeH:mHm#
.moozpoz oz< mo<Hao»<: :_ uwaHeomwu mm HommmocasEHoHume A: cweamome mo: nz<w eoHaHHwomewcH
.mHHw: wuouH—quw mo cows mew mucwmweowe wsHo> sumo .xzmH : oioH we momwmweq

me» =_ queoeemoiwew: mcoppmnsucw nmm cowumaaocpweo .Nooo z HHioH mzHa o>< z ioH

eo mac—m Noon 2 HHioH .wcon o>< z -oH .eouoweew o: cup: vmuouHucH mcoHewo mew»

mew eoe wwwooaucH mew: mHku we“ vem nw>oswe mm: mHuwe we» .uoHewo coouonzucwweo weyieo ocw
mew H< .HN vco H mwcwewoweho wee—m ewewan guH: eo He uco m mocmsumwepv Nooo z HHioH

sue: on um cos oo eoe omuoozucHweo mew: mwgmHu mesa—so co cwuwwm mHwa Hooo pcwzHecoocoz

a

 

 

 

H.o H N.m H.o A m.m« H.o A N.m« m.o H o.e# Now; + o>< .c
m.o A e.¢ N.o A N.m N.o u o.¢ H.o « N.m mcoH< Noon .m
w.o A N.@ o.H w N.w# H.o A o.m# m.o w N.¢# mcoH< a>< .N
m.o u o.v H.o « m.e v.0 w H.v m.o w w.N eouumemu oz .H
.cwe o .cHE m .cwa N .:_E H ucmsumwe»

H:_wuoea Hku ma\meosmv
oz<w eoHaHkuweucH

 

 

ooozoeo weooHoo oo WHHoo Hooo zo
oz<o eo ooeooEeoe ooooooe-o>< eo Nmoo zo ooeoeoezoo

oH mHnmh

130

Table 11

Inhibition by PGEZ of AVP-induced Formation of cAMP by
CCCT Cells in the Presence of Flurbiprofena

 

 

Intracellular cAMP
(fmoles/ug cell protein)

 

Treatment No Inhibitor Flurbiprofen

No Effector 1.9 t 0.3 4.9 i 0.3
2. AVP Alone 13.5 1 0.3 $8.5 t 0.2
3. AVP + PGEZ *1.4 t 0.3 *4.6 i 0.4

 

 

aNonconfluent CCCT cells seeded on culture dishes were preincubated
for so min at 37° with 10-11 M 9652 (Treatment 3) or with

buffer alone (Treatments 1 and 2). At the end of the preincubation
period, the media was removed and the cellg were incubated for 3 min
with no effector, 10‘8 M AVP alone, or 10' M AVP plus

10'11 M PGEZ. In the wElls where FlurbiprofEn (10"5 M)

was used, it was present in the media 30 min prior to the preincuba-
tion period and then throughout preincubation and treatment periods
(i.e., a total of 93 min). Buffer was used for the same time in
wells without inhibitor. 10'4 M IBMX was present in all wells
throughout the 93 min period. Each value represents the mean of
triplicate wells. Intracellular cAMP was measured by radioimmuno-
assay as described in MATERIALS AND METHODS. *Signicicantly
different from control value (i.e., no effector); *Significantly
different from value of treatment with AVP alone (P<0.05).

131

Table 12

Effect of Low Concentrations of PGE on Basal Levels
of Intracellular cAMP in CC T Cellsa

 

 

Intracellular cAMP

 

Treatment (fmoleslug cell protein)
1. No Effetor 8.6 i 0.1
2. 10"12 M PGEZ 8.5 s 0.3
3. 10'11 n PGEZ 8.7 s 0.1
4. 10'10 )1 PGEZ 8.8 e 0.2

 

 

aNonconfluent CCCT cells seeded on culture dishes were preincubated
for 60 min with the indicated concentrations of PGE (Treatments
2-4) or with buffer alone (Treatment 1). At the en of the preincu-
bation period, the media was removed and the cells were incubated for
3 min with no effector or the indicated concentrations of PGE2.
Preincubations and incubations were performed in the presence of
10’ M IBMX. Each value represents the mean of triplicate wells.
Intracellular cAMP was measured by radioimmunoassay as described in
MATERIALS AND METHODS. No significant difference was observed
between treatments (P<0.05).

132

Figure 31 illustrates the effect of a wide range of PGan concen-
trations (10"12-10“7 M) on AVP-induced intracellular cAMP
accumulation. All concentrations of PGan tested were effective in
completely inhibiting the AVP-induced response. In addition and
contrary to PGEZ, PGFZG does not induce cAMP increases in CCCT

cells or renal papillary collecting tubule cells (14) even at very high
concentrations (10'7-10'4 M). In this vane, it is interesting

to consider a report on experiments performed in isolated, perfused
cortical collecting tubules. It was shown in these studies that PGEZ
inhibited both Na+ resorption and AVP-induced water resorption.
PGFZa, however, caused only inhibition of AVP-induced water
resorption. PGFZo had no apparent effect on Na+ resorption

(138).

133

.eme.ovoo mooHo o>< ewe: wooEwooew eo ooHos eoee wooeoeeHo szeooweeooeoa

"HmewH ewwwee Hmeewemeem e: ..m.wv mweHe> Heeweew seem weweweewe aneeewewemHmw

.moozeoz oz< me<Hmoh<z ew eweweemme we xemmeeeeeeweweee He emeemems me: ez<u eeHeHHmoeeweH
.mHHm: wweeHHewew ee come mew mwewmmeewe wewee eeem hXZmH -oH we meemmmee

mew ew emeeeeeme mew: meewwmeeuew eee meewweeeuewmee .Hz mion e>< meHe eNeoe

we meewweewemeeee wEem mew eww: ewe m eee ewweeeeew mew: mHHme mew eem em>eeme we: ewewe
mew .eeweme eewweeeeewwee mew we eem mew w< .eNooo we meewweewemeeee ewweuweew mew

eww: on we ewe oo eee emweeee:_mee mew: mmemwe meewHee ee emewwm mHHmw pooo wemeHeeeweez
.eewwmeeee ez<w emeeeewie>< we eNooe xe eewwweweew mew eew meemeememe eewweewemeeeo

.Hm oeooee

134

 

IO'7

i0'°

I
I0’9

 

I
'0'10
[PGFZJ (M)

l
10""

 

'0‘12

 

 

N
(ugatond ueo fid/sagoum
dWVO HV‘ln'l'TBOVHLNI OBOOONl-d/W

Figure 31

DISCUSSION

PGE;:AVP Interactions. The hypothesis that prostaglandins
inhibit AVP-induced cAMP formation in the collecting tubule evolved
from the classic study of Grantham and Orloff (11). Clear evidence
supporting this concept in the collecting tubule system itself has been
difficult to obtain (14,112,113,131,144,147). However, there have been
two recent reports that, in the absence of phosphodiesterase inhibi-
tors, PGEZ causes partial but significant inhibition of AVP-induced
cAMP accumulation in rabbit collecting tubule segments (156,157). We
have found that PGEZ inhibits both the release of cAMP normally
occurring in response to treatment of confluent CCCT cell monolayers
with AVP and the accumulation of intracellular cAMP in nonconfluent
CCCT cell monolayers treated with AVP. The effects observed in the
CCCT cell system have slightly different characteristics than those
reported by Torikai and Kurokawa (156) and Edwards gt_al. (157) in that
inhibition by PGEZ of AVP-induced cAMP release in the CCCT cell
system: (a) is quantitative, (b) is time-dependent and (c) involves
concentrations of PGE2 which are 5-7 orders of magnitude lower than
those reported for the rabbit collecting tubule.

The biochemical mechanism by which PGEZ inhibits AVP-induced
cAMP formation is not yet clear. It appears that the effect occurs
only with intact cells. For example, the phenomenon is apparent in

slices (113), in intact collecting tubules (156,157) and in CCCT cells,

135

136

but PGEZ fails either to activate cAMP phosphodiesterase (157) or to
inhibit AVP-independent adenylate cyclase in permeabilized collecting
tubule segments (131). This suggests that PGEZ may be causing
production, mobilization or sequestration of an intermediate factor(s)
(denoted by an X in Figure 32) which, in turn, modulates cAMP levels in
the collecting tubule. Based on the 20 min time requirement (Table 9),
this intermediate could be a protein (158). Locher gt_gl, (111) have
demonstrated that in human phagocytes there is also a time dependence
to the inhibition of AVP-induced cAMP synthesis by PGEZ.

It seems unlikely that PGEZ is causing its inhibitory effect at
the level of cAMP phosphodiesterase because PGEZ does not decrease
basal levels of intracellular cAMP in CCCT cells. More reasonable is
an inhibitory effect on the AVP-inducible adenylate cyclase system.
PGEZ does not affect the affinity of binding of AVP to human phago-
cytes under conditions in which PGEZ inhibits AVP-induced cAMP
formation (111). Thus, the effect of PGE2 is probably expressed at a
post receptor step. The most applicable precedent for inhibition by
PGEZ of AVP-induced cAMP formation is seen in the heterologous
desensitization of adenylate cyclase in human fibroblasts (159,160).

In this situation PGEZ attenuates the coupling of the hormone--
receptor complex to the catalytic subunit of adenylate cyclase probably
by modifying a GTP binding subunit. Hopefully, the availability of
large numbers of CCCT cells in culture will permit the examination of

this hypothesis at the biochemical level.

Dual Receptor Concept of Prostaglandin Action. The single

experiment performed with PGan (Figure 31) provides suggestive

137

Figure 32. Model for AVP, bradykinin (BK), PGE interrelationships in
canine cortical collecting tubule ( CCT) cells.

138

CCCT CELL

APICALL j BASOLATERAL
5'-AMP

GE: \/ PGE:

H 20

c— o- c- cAHP: AVP
RESORPTION I1",

 

 

 

 

 

PGE __/ ®\ PGE;

J

/'

AVP —t ——-» PGE2+—— 4— AVP

ex/
H   4%

Figure 32

 

 

 

 

139

but incomplete evidence for a dual prostaglandin receptor system.
However, the existence of dual PGEZ receptors (Figure 32) would
explain the observation that PGEZ has two actions in the collecting
tubule. Binding of PGE2 to one type of receptor, exhibiting high
affinity for the ligand, could initiate desensitization of adenylate
cyclase to circulating antidiuretic hormone (ADH). Through this
receptor, PGEZ would act as a negative feedback modulator of the
action of ADH on the collecting tubule. The second type of receptor,
displaying low affinity for the ligand, would be involved in the
activation of adenylate cyclase. This raises the question of the
physiological significance for this latter effect. It is pertinent to
note that only cells which synthesize prostaglandins exhibit
prostaglandin-induced adenylate cyclase activation. This demonstrated
tendency, taken together with the recent observation that cAMP inhibits
prostaglandin formation in platelets (161,162) and MDCK cells (163),
leads to the idea of a feedback mechanism for the regulation of
prostaglandin synthesis with cAMP acting as the negative modulator (not
shown in Figure 32). Prostaglandin derivatives that elicit activation
of adenylate cyclase but not inhibition of AVP-induced cAMP increases
could provide additional suggestive evidence for the dual receptor
model. Furthermore, such prostaglandins and PGFZG could be used to
study the two distinct responses independently.

The identification of heterogeneous classes of PGE2 receptors in
CCCT cells should be pursued. Direct measurements of radiolabeled
PGEZ-binding to CCCT cell membranes could provide more direct
evidence. The biphasic nature of PGE2 effects indicates that binding

dissociation constants should be detectably different. Analysis of the

140

data could be complicated by the presence of heterogeneous receptor
molecules within a class; a possibility suggested by experiments on
RPCT cells (14) and rat hepatocytes (164) for prostaglandin receptors
coupled to adenylate cyclase activation. However, prostaglandins with
affinity for only one class of receptor (e.g., PGFZG) could be used

to facilitate distinction between receptor populations in ligand-recep-
tor binding experiments as well as in the ultimate solubilization and

characterization of the receptor molecules involved.

SUMMARY

The renal collecting tubule is an attractive system for studying
the physiological function and mechanism of action of PGE2. The
collecting tubule is the part of the renal tubule which exhibits the
highest cyclooxygenase activity and along with the vasculature, the
medullary interstitial cells and the glomeruli constitutes one of the
four major sites of prostaglandin synthesis in the kidney. In
addition, prostaglandins have been shown to have two potent effects on
the transport properties of isolated collecting tubule segments. One
effect is to inhibit water resorption occurring in response to arginine
vasopressin (AVP), and the second is to inhibit Na+ resorption.

To facilitate the study of the mechanisms of actions of
prostaglandins on the collecting tubule, a culture system of canine
cortical collecting tubule (CCCT) cells was developed. These cells
were isolated by immunodissection using culture plates coated with a
monoclonal antibody which specifically reacts with an ecto-antigen on
the canine collecting tubule. CCCT cells, which exhibit many of the
morphological and biochemical properties of collecting tubule cells 13_
situ, can be grown and maintained in culture for several months.
Confluent monolayers of CCCT cells seeded on Millipore filters, showing
characteristics of asymmetry seen with intact collecting tubules, were
utilized to examine aspects of apical-basolateral asymmetry related to

PGEZ metabolism and function. PGEZ is the major prostaglandin

141

142

derivative synthesized by CCCT cells. Figure 32 incorporates the main
concepts progressively developed through the research described in this
dissertation. Although AVP caused cAMP release only when added to the
basolateral side of CCCT cells, AVP caused the release of PGE2 when
added to either the apical or basolateral surface. This result implies
that there are at least two AVP receptor systems, one coupled to cAMP
synthesis and one to PGEZ formation. In contrast to the results
observed with AVP, bradykinin caused PGEZ release only when added to
the apical surface of CCCT cells suggesting that urinary but not
blood-borne kinins elicit PGEZ formation by the canine collecting
tubule. PGEZ was released in comparable amounts on each side of the
monolayer in response both to AVP and to bradykinin.

High concentrations (310'8 M) of PGE2 added to either side
of the monolayer caused the release of cAMP. However, at concentra-
tions (10'10-10'12 M) at which PGEZ had no independent
effect on cAMP release, PGEZ inhibited the release of cAMP normally
occurring in response to AVP. This inhibition occurred with PGEZ
added to either the apical or basolateral surface of the CCCT cell
monolayer. PGEZ (10‘11 11) also inhibited the AVP-induced
accumulation of intracellular cAMP by CCCT cells seeded on culture
dishes. This inhibition was only observed when the cells were prein-
cubated with PGE2 for Z 20 min. The results are consistent with the
concept that inhibition by prostaglandins of the hydroosmotic effect of
AVP is due to inhibition of AVP-induced cAMP production. This inhibi-
tion does not appear to involve a direct physical interaction of PGE2
with the AVP receptor which is coupled to adenylate cyclase since (a)

CCCT cells must be preincubated with PGEZ for 20 min for the

143

inhibition to be observed and (b) PGEZ added to the apical surface of
CCCT cells inhibits cAMP release in response to AVP acting from the
basolateral surface. A dual prostaglandin receptor concept, considered
in light of the biphasic nature of PGE2 effects on cAMP metabolism in

the collecting tubule, is also briefly discussed in this dissertation.

11.

12.

13.

14.

15.

16.

BIBLIOGRAPHY
Morel, F. and DeRouffignac, C. (1973) Ann. Rev. Physiol. 99,
17-540

Grantham, J.J., Irish, J.M. III, and Hall, D.A. (1978) Ann. Rev.
Physiol . _4_g, 249-277.

Burg, M., Grantham, J.J., Abramow, M., and Orloff, J. (1966) Am.
J. Physiol. 299, 1293-1298.

Misfeldt, D.S., Hamamoto, S.T., and Pitelka, D.R. (1976) Proc.
Natl. Acad. Sci. USA 19, 1212-1216.

Rindler, M.J., Churman, LM., Shaffer, L., and Saier, M.H. Jr.
(1979) J. Cell Biol. 99, 635-648.

Saier, M.H. Jr. (1981) Am. J. Physiol. 299, C106-C109.

Misfeldt, 0.5. and Sanders, M.J. (1981) Am. J. Physiol. 299,
C92-C95.

Mullin, J.M., Cha, C.-J.M., and Kleinzeller, A. (1982) Am. J.
Physiol . Q2, C41-C45.

Smith, N.L. and Nilkin, G.P. (1977) Prostaglandins 19, 873-892.

Smith, N.L. and Bell, T.G. (1978) Am. J. Physiol. 299,
F451-F457.

Grantham, J.J. and Orloff, J. (1968) J. Clin. Invest. 91,
1154-1161.

Grenier, F.C. and Smith, H.L. (1978) Prostaglandins 99,
759-772.

Grenier, F.C., Rollins, T.E., and Smith, N.L. (1981) Am. J.
Physiol . 24_1, F94-F104.

Grenier, F.C., Allen, M.L., and Smith, W.L. (1982)
Prostaglandins 29, 547-565.

Moncada, S. and Vane, J.R. (1979) New England J. Med. 999,
1142-1147.

Lands, H.E.M. and Samuelsson, B. (1968) Biochim. Bi0phys. Acta
169, 426-429.

144

17.
18.

19.
20.

21.

22.

23.

24.
25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

145

Smith, N.L. (1981) Mineral Electrolyte Metab. 9, 10-26.

Pace-Asciak, C.R. and Smith, N.L. (1983) In The Enzymes (Boyer,
P.D., Ed.), Vol. 16, pp. 543-603.

Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 99, 580-587.

Broekman, M.J., Hard, J.M., and Marcus, A.J. (1980) J. Clin.
Invest. 99, 275-283.

Mauco, 6., Chap, H., and Douste-Blazy, L. (1979) FEBS Lett. 199,
367-370.

Broekman, M.J., Hard, J.M., and Marcus, A.J. (1981) J. Biol.
Chem. 2_5§, 8271.

Bell, R.L., Kennerly, D.A., Stanford, N., and Majerus, S.M.
(1979) Proc. Natl. Acad. Sci. USA 19, 3238-3241.

Majerus, P.”. and Prescott, S.M. (1982) Meth. Enz. 99, 11-17.

Marcus, A.J., Ullman, H.L., and Safier, L.B. (1969) J. Lipid
Res. 29, 108-114.

Bills, T.K., Smith, J.B., and Silver, M.J. (1977) J. Clin.
Invest. 99, 1-6.

Dennis, E.A. (1983) In The Enzymes (Boyer, P.D., Ed.), Vol. 16,
pp. 307-353.

 

Smith, N.L. (1984) In Biochemistry of Lipids and Membranes
(Vance, D., and Vance, J., Eds.), Benjamin/Cummings Publishing
Co., in press.

Neufeld, E.J. and Majerus, P.w. (1983) J. Biol. Chem. 299,
2461-2467.

Miyamoto, T., Ogino, M., Yamamoto, S., and Hayaishi, 0. (1976)
J. Biol. Chem. 29;, 2629-2636.

Hemler, M., Lands, H.E.M., and Smith, N.L. (1976) J. Biol. Chem.
.299, 5575-5578.

Ohki, S., Ogino, N., Yamamoto, S., and Hayaishi, 0. (1979) J.
Biol. Chem. 299, 829.

VanderOuderaa, F.J., Buytenhek, M., Nugteren, D.H., and VanDorp,
D.A. (1977) Biochim. Biophys. Acta 391, 315.

Pagels, N.R., Marnett, L.J., DeWitt, D.L., and Smith, H.L.
(1983) J. Biol. Chem. 299, 6517.

Ogino, N., Miyamoto, T., Yamamoto, S., and Hayaishi, 0. (1977)
J. 3101. Chem. 152-, 890-8950

36.

37.

38.

39.

40.

41.

42.

43.
44.

45.

46.
47.

48.

49.

50.

51.

52.
53.

146

Nugteren, D.H. (1982) in Progress in Lipid Research (Holman,
R.T., Ed.), Vol. 20, pp. 169-172.

 

Christ-Hazelhof, E. and Nugteren, D.H. (1979) Biochim. Biophys.
Acta 912, 43-51.

Shimizu, T., Yamamoto, S., and Hayaishi, O. (1979) J. Biol.
Chem. Zéfl, 5222-5228.

Salmon, J.A., Smith, D.R., Flower, R.J., Moncada, S., and Vane,
J.R. (1978) Biochim. Biophys. Acta 929, 250-262.

Yoshimoto, T., Yamamoto, S., Okuma, M., and Hayaishi, O. (1977)
J. Biol. Chem. 292, 5871-5874.

Pace-Asciak, C. and Nashat, M. (1975) Biochim. Biophys. Acta
33.8. 243.

Hlodawer, P., Kindahl, H., and Hamberg, M. (1976) Biochim.
Biophys. Acta 999, 603.

wong, P.Y.-K. (1982) Meth. Enz. 99, 117-125.

Hatanabe, K., Shimizu, T., and Hayaishi, O. (1981) Biochem. Int.
2, 603.

Reingold, D.F., Kawasaki, A., and Needleman, P. (1981) Biochem.
Biophys. Acta 999, 179.

Stone, K.J. and Hart, M. (1975) Prostaglandins 99, 273.

Christ-Hazelhof, E. and Nugteren, D.H. (1982) Meth. Enz. 99,
77-840

Shimizu, T., Yamamoto, S., and Hayaishi, O. (1982) Meth. Enz.
99, 73-77.

Roth, G.J., Machuga, E.T., and Strittmatter, P. (1981) J. Biol.
Chem. 299, 10018.

Moonen, P., Buytenhek, M., and Nugteren, D.H. (1982) Meth. Enz.
99, 84-91.

Smith, H.L., Dewitt, D.L., and Day, J.S. (1983) In Advances in

 

Prostaglandin, Thromboxane and Leukotriene Research, Vol. 11,
87.

Dewitt, D.L. and Smith, N.L. (1983) J. Biol. Chem. 299, 3285.
Graf, H., Castle, L., and Ullrich, V. (1983) In Advances in

Prostaglandin, Thromboxane and Leukotriene Research, Vol. 11,
105.

 

54.
55.

56.
57.
58.
59.

60.
61.
62.

63.

64.

65.

66.

67.

68.

69.

70.
71.

72.

73.
74.

147

Stehle, R.G. (1982) Meth. Enz. 99, 436-458.

Hamberg, M., Svensson, J., and Samuelsson, B. (1975) Proc. Natl.
Acad. Sci. USA 12, 2994.

Anggard, E. and Samuelsson, B. (1964) J. Biol. Chem. 299, 4097.
Lee, 5.0. and Levine, L. (1975) J. Biol. Chem. 259, 548.
Ferreira, S. and Vane, J.R. (1967) Nature (London) 299, 868.

Granstrom, E. and Samuelsson, B. (1971) J. Biol. Chem. 299,
7470.

Hamberg, M. and Samuelsson, B. (1971) J. Biol. Chem. 299, 6713.
Hansen, H.S. (1979) Biochim. Bi0phys. Acta 919, 136.

Edwards, N.S. and Pace-Asciak, C.R. (1982) J. Biol. Chem. 292,
6339.

Pace-Asciak, C.R. and Domazet, Z. (1975) Biochim. Biophys. Acta
E), 338.

Yuan, 8., Tai, C.L., and Tai, H.-H. (1980) J. Biol. Chem. 299,
7439.

Dunn, M.J., Liard, J.F., and Dray, F. (1978) Kidney Int. 99,
136-143.

Christ, E.J. and VanDorp, D.A. (1972) Biochim. Biophys. Acta
2_79, 537-545.

Smith, N.L. (1984) In CRC Handbook of Prostaglandins and Related

 

Ligids, CRC Press, in press.

Jouvenaz, G.H., Nugteren, D.H., Beerthuis, R.K., and VanDorp,
D.A. (1970) Biochim. Biophys. Acta 292, 231-234.

Levine, L., Hinkle, P.M., Voelkel, E.F., and Tashjian, A.H.
(1972) Biochem. Biophys. Res. Comm. 91, 888-896.

Bito, L.Z. (1975) Prostaglandins 9, 851-855.

Bito, L.Z. and Baroody, R.A. (1974) Am. J. Physiol. 229,
1580-1584.

Siegl, A.M., Smith, J.B., Silver, M.J., Nicolau, K.C., and
Ahern, D. (1979) J. Clin. Invest. 99, 215.

Jacobson, H.R. (1981) Am. J. Physiol. 24_1, F203-F218.
Knepper, M. and Burg, M. (1983) Am. J. Physiol. 299, F579-F589.

75.

76.
77.

78.

79.
80.

81.

82.

83.

84.

85.

86.

87.
88.

89.

90.

91.

92.
93.

148

Lee, J.B., Covino, B.G., Takman, B.H., and Smith, E.R. (1965)
Circulation Res. 91, 57.

Hamberg, M. (1969) FEBS Lett. 9, 127.

Zusman, R.M. and Keiser, H.R. (1977) J. Biol. Chem. 292,
2069-2071.

Janszen, F.H.A. and Nugteren, D.H. (1973) Adv. Biosci. 1973,
287-292.

Bohman, S.O. (1977) Prostaglandins 99, 729-744.

McGiff, J.C. and Wong, P.Y.-K. (1979) Federation Proc. 99,
89-930

Sraer, J., Foidart, J., Chansel, D., Mahieu, P., Kouznetzova,
B., and Ardaillou, R. (1979) FEBS Lett. 999, 420-424.

Hassid, A., Koniecskowski, M., and Dunn, M.J. (1979) Proc. Natl.
Acad. Sci. USA 19, 1155-1159.

Jackson, B.A., Edwards, R.M., and Dousa, T.P. (1980) J. Lab
Clin. Med. 99, 119-128.

Kirschenbaum, M.A., Lowe, A.G., Trizna, W., and Fine, L.G.
(1982) J. Clin. Invest. 19, 1193-1204.

Beck, T.R., Hassid, A., and Dunn, M.J. (1980) J. Pharmacol. Exp.
Ther. 299, 15-19.

Zusman, R.M. and Keiser, H.R. (1977) J. Clin. Invest. 99,
215-223.

Hassid, A. and Dunn, M.J. (1980) J. Biol. Chem. 299, 2472-2475.

Needleman, P., Bronson, S.D., Wyche, A., Sivakoff, M., and
Nicolaou, K.C. (1978) J. Clin. Invest. 99, 839-849.

Ally, A.I. and Horrobin, D.F. (1980) Prostaglandins Med. 9,
431-438.

Morrison, A.R., Nishikawa, K., and Needleman, P. (1977) Nature
(London) 292, 259-260.

Lee, S.C., Pong, S.S., Katzen, 0., Wu, K.Y., and Levine, L.
(1975) Biochemistry 99, 142-145.

Nissen, H.M. and Andersen, H. (1968) Histichemie 99, 189-194.
Frolich, J.C., Hollifield, J.W., Michelakis, A.M., Vesper, B.S.,

Wilson, J.R., Shand, D.G., Seyberth, H.J., Frolich, W.H., and
Dates, J.A. (1979) Circulation Res. 99, 781-787.

94.

95.

96.

97.

98.
99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

149

Gerber, J.C., Keller, R.T., and Nies, A.S. (1979) Circulation
Res. 99, 796-799.

Dunn, M.J. and Hood, V.L. (1977) Am. J. Physiol. 299,
F169-F184.

Stokes, J.B. and Kokko, J.P. (1977) J. Clin. Invest. 99,
1099-1104.

Jamison, R.L., Sonnenberg, H., and Stein, J.H. (1979) Am. J.
Physiol . gfl, F247-F261.

Rocha, A.S. and Kokko, J.P. (1974) Kidney Int. 9, 379-387.

Handler, J.S. and Orloff, J. (1981) Ann. Rev. Physiol. 99,
611-624.

Nimmo, H.G. and Cohen, P. (1977) Adv. Cyclic Nucleotide Res. 9,
145-266.

Strewler, G.J. and Orloff, J. (1977) Adv. Cyclic Nucleotide Res.
9, 331-361.

Zambraski, E. and Dunn, M.J. (1979) Am. J. Physiol. 299,
F552-F558.

Fejes-Toth, G., Magyar, A., and Walter, J. (1977) Am. J.
Physiol. 292, F416-F423.

Anderson, R., Berl, T., McDonald, K., and Schrier, R. (1975) J.
Clin. Invest. 99, 420-426.

Kramer, H., Backer, A., Hinzen, S., and Dusing, R. (1978)
Prostaglandins Med. 9, 341-349.

Berl, T., Ray, A., Wald, H., Horowitz, J., and Czaczkes, W.
(1977) Am. J. Physiol. 292, F529-F537.

Levison, S. and Levison, M. (1978) J. Lab Clin. Med. 92,
570-576.

Lum, G.H., Aisenberg, G.A., Dunn, M.J., Berl, T., Schrier, R.W.,
and McDonald, K.M. (1977) J. Clin. Invest. 99, 8-13.

02er, A. and Sharp, G. (1972) Am. J. Physiol. 222, 674-680.
Block, L.H., Locher, R., Tenschert, W., Siegenthaler, W.,
Hofmann, T., Mettler, R., and Vetter, W. (1981) J. Clin. Invest.
99, 374-381.

Locher, R., Vetter, W., and Block, L.H. (1983) J. Clin. Invest.
_7_1_, 884-891.

112.

113.

114.

115.

116.
117.

118.

119.

120.

121.

122.

123.

124.

125.

126.
127.

128.

129.

150

Marumo, F. and Edelman, I. (1971) J. Clin. Invest. 99,
1613-1620.

Beck, N., Kaneko, T., Zor, V., Field, J., and Davis, 8. (1971)
J. Clin. Invest. 99, 2461-2465.

Herman, C., Zenser, T., and Davis, B. (1979) Biochim. Bi0phys.
Acta 992, 496-503.

Birnbaumer, L. and Yang, P. (1974) J. Biol. Chem. 299,
7848-7856.

Zenser, T. and Davis, B. (1977) Prostaglandins 99, 437-447.

Beck, T.R. and Dunn, M.J. (1981) Mineral Electrolyte Metab. 9,
46-59.

Galfre, G., Howe, S.C., Milstein, C., Butcher, G.W., and Howard,
J.C. (1977) Nature (London) 299, 550-552.

Shulman, M., Wilde, C.D., and Kohler, G. (1978) Nature (London)
279, 269-270.

Cotton, R., Secher, D., and Milstein, C. (1973) Eur. J. Immunol.
9, 135-140.

DeWitt, D.L., Rollins, T.E., Day, J.S., Gauger, J.A., and Smith,
W.L. (1981) J. Biol. Chem. Zéfi, 10375-10382.

Medgyesi, G.A., Fust, G., Gergely, J., and Bazin, H. (1978)
Immunochemistry 99, 125-129.

Lowry, D.H., Rosebrough, N.H., Farr, A.L., and Randall, R.J.
(1951) J. Biol. Chem. 12;, 265-275.

Steel, R.G.D. and Torrie, J.H. (1980) Principles and Procedures
of Statistics, New York, McGraw-Hill.

 

Bancroft, J.D. (1975) Histochemical Techniques, London,
Butterworth, pp. 278-290.

Kriz, W. (1981) Am. J. Physiol. 299, R3-R16.

Spargo, B.H. (1966) The Kidney (Mostofi, F.K. and Smith, D.E.,
Eds.), Baltimore, Williams and Wilkins, pp. 17-59.

 

Forster, R.P. (1961) In The Cell: Specialized Cells (Brackett,
J. and Mirsky, A.E., Eds.), New York, Academic Press, Vol. 5,
Part 2, pp. 89-161.

 

Jefferson, D.M., Brown, J.A. Jr., Zadumaisky, J.A., and Scott,
W.N. (1982; Abstract). Federation Proc. 99, 1266.

130.
131.
132.

133.
134.

135.

136.

137.

138.
139.
140.
141.
142.
143.

144.

145.

146.

147.

151

Morel, F. (1981) Am. J. Physiol. 299, F159-F164.
Torikai, S. and Kurokawa, K. (1981) Prostaglandins 29, 427-438.

Wysocki, L.J. and Sato, V.L. (1978) Proc. Natl. Acad. Sci. USA
19, 2844-2848.

Chan, 5. and Silverman, M. (1977) Kidney Int. 99, 348-356.

Herzlinger, D.A., Easton, T.G., and Ojakian, G.K. (1982) J. Cell
Biol. 99, 269-277.

Allen, M.L., Garcia-Perez, A., and Smith, W.L. (1982; Abstract).
Federation Proc. 99, 1693.

Savoy-Moore, R.T., Carretero, D.A., and Scicli, A.C. (1984;
Abstract). Federation Proc. 99, 359.

Holt, W.F. and Lechene, c. (1981) Am. J. Physiol. 299,
F452-F460.

Stokes, J.B. (1979; Abstract). Kidney Int. 99, 839.

Hassid, A. (1981) Prostaglandins 29, 985-1001.

Iino, Y. and Imai, M. (1978) Pflugers Arch. 999, 125-132.
Iino, Y. and Brenner, B.M. (1981) Prostaglandins 22, 715-721.
Powell, W.S. (1982) Meth. Enz. 99, 467-477.

Adler, E.M., Fluk, L.J., Mullin, J.M., and Kleinzeller, A.
(1982) Science_29z, 851-853.

Pugliese, F., Sato, M., Williams, S., Aikawa, M., Hassid, A.,
and Dunn, M.J. (1983) In Advances in Prostaglandin, Thromboxane

 

and Leukotriene Research (Samuelsson, B., Paoletti, R., and
Ramwell, P., Eds.), Vol. 11, New York, Raven Press, pp.
517-5230

 

Cereijido, M., Robbins, E.S., Dolan, W.J., Rotrinno, C.A., and
Sabatini, D.D. (1978) J. Cell Biol. 99, 853-880.

Frolich, J.C., Williams, W.M., Sweetman, B.J., Smigel, M., Carr,
K., Hollifield, J.W., Fleischer, S., Nies, A.S., Frisk-Holmberg,
M., and Dates, J.A. (1976) In Advances in Prostaglandin and
Thromboxane Research (Samuelsson, B. and Paoletti, R., Eds.),
Vol. 1, New York, Raven Press, pp. 65-80.

 

 

Dunn, M.J., Greely, H.R., Valtin, H., Kinter, L.B., and
Beeuwkes, R. III (1978) Am. J. Physiol. 299, E624-E627.

152

148. Dunn, M.J., Kinter, L.B., Shier, D., and Beeuwkes, R. 111 (1980;
Abstract). Clin. Res. 22, 496A.

149. Zusman, R.M. (1981) Ann Rev. Med. 92, 359-374.

150. Kirschenbaum, M.A. and Serros, E.R. (1980) Am. J. Physiol. 299,
F107-F111.

151. McGiff, J.C., Itskovitz, H.D., Terragno, A., and Wong, P.Y.-K.
(1976) Federation Proc. 99, 175-180.

152. Chao, J. and Margolius, H.S. (1979) Biochim. Bi0phys. Acta 929,
330'3400

153. Carretero, O.A. and Scicli, A.G. (1980) Am. J. Physiol. 299,
F247-F255.

154. Schuster, V.L., Kokko, J.P., and Jacobson, H.R. (!983;
Abstract). Proc. Am. Soc. Nephrology, 178A.

155. Hansen, H.S. (1981) Lipids 99, 849-854.

156. Torikai, S. and Kurokawa, K. (1983) Am. J. Physiol. 299,
F58-F66.

157. Edwards, R.M., Jackson, B.A., and Dousa, T.P. (1981) Am. J.

158. Anderson, W.B., Johnson, G.S., and Pastan, I. (1973) Proc. Natl.
Acad. Sci. USA 99, 1055-1059.

159. Clark, R.b. and Butcher, R.W. (1979) J. Biol. Chem. 299,
9373-9378.

160. Kassis, s. and Fishman, P.H. (1982) J. Biol. Chem. 291,
5312-5318.

161. Malmsten, C., Granstrom, E., and Samuelsson, B. (1976) Biochem.
Biophys. Res. Commun. 99, 569-576.

162. Minkes, M., Stanford, N., Chi, M.M.Y., Roth, G.J., Raz, A.,
Needleman, P., and Majerus, P.W. (1977) J. Clin. Invest. 99,
449-464.

163. Hassid, A. (1983) Am. J. Physiol. 299, C369-C376.
164. Garrity, M.J., Westcott, K.R., Eggerman, T.L., Andersen, N.H.,

Storm, D.R., and Robertson, R.P. (1983) Am. J. Physiol. 299,
E367-E372.