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This is to certify that the

dissertation entitled

The Regulation and Localization of Adenosine
Receptors in Renal Epithelial Cells
presented by

David Grant LeVier

has been accepted towards fulfillment
of the requirements for

Ph_n' degree in PhX§iOIng

 

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THE REGULATION AND LOCALIZATION OF ADENOSINE RECEPTORS
IN RENAL EPITHELIAL CELLS
By

David Grant LeVier

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology

1992

to

{78‘ 0

ABSTRACT

THE REGULATION AND LOCALIZATION OF ADENOSINE RECEPTORS
IN RENAL EPITHELIAL CELLS

By

David Grant LeVier

The research presented in this thesis explores the regulation and localization of G-
protein linked adenosine receptors in renal epithelial cells. Desensitization of
adenosine receptors in primary cultures of rabbit cortical collecting tubule (RCCI')
cells involves the altered function of both Al and A2 adenosine receptor subtypes.
Additionally, altered function of the regulatory G—protein, G,, confers a
heterologous component to this desensitization. The adenosine receptor-mediated
pathways were localized by functional criteria to either the apical or basolateral
plasma membrane domains of porcine LLC-PKl renal cells in monolayer culture.
The adenosine receptor mediated pathway that stimulates inositol phosphate
turnover was localized to the basolateral surface of LLC-PKl cells. The inhibitory
adenosine Al receptor pathway was also localized to the basolateral aspect of the
cell monolayer, although a minor apical component could not be ruled out. The
stimulatory adenosine A2 receptor pathway was found on both sides of the
monolayer, although the major component was apical with a smaller, albeit
significant, component residing on the basolateral side of LLC-PK, cells.

Desensitization of stimulatory adenosine A2 receptors on both sides of LLC-PK,

cells, following selective exposure of adenosine to either the apical or basolateral
side of the monolayer, was found to be homologous. This pattern of homologous
desensitization was the same irrespective of the side of the monolayer on which the
receptors were exposed to adenosine. This observation suggested that adenosine
A2 receptors on the side of the monolayer opposite that of adenosine exposure,
which were presumably unoccupied/unactivated, could be desensitized

independently of other unoccupied/unactivated G-protein linked receptors.

This dissertation is dedicated to my parents, Robert and Vivian, my brother, Steven,
and most especially to Sue. Only with their love and constant encouragement was
I able to complete this work.

With all my love, Dave

iv

ACKNOWLEDGEMENTS

I would like to acknowledge my committee members Drs. John E.
Chimoskey, Seth R. Hootman, William L. Smith, William S. Spielman and Robert
B. Stephenson. I would especially like to thank my major advisor, Dr. William S.
Spielman, for his guidance, support and encouragement thoughout my graduate

catrer.

I would also like to recognize my friends and colleagues for their support; Dr. Lois
Arend, Samita Bhattacharya, Dr. Maria Burnatowska-Hledin, Dr. David McCoy,

Dr. Beth Meade, and Barb Olson.

TABLE OF CONTENTS

Bag;
List of Tables .................................................................................................... viii
List of Figm'es ..................................................................................................... ix
1. Introduction ....................................................................................................... 1
II. Literature Review ............................................................................................ 3
A. Adenosine Metabolism ......................................................................... 3
B. Adenosine Receptors ............................................................................ 7
C. Regulation of [LAdrenergic Receptors .............................................. 12
D. Regulation of Adenosine Receptors .................................................. 19
E. Receptor Localization ......................................................................... 23
El . Peptide Hormone Receptor Localization ............................. 24
5.2. Adenosine Receptor Localization ........................................ 26
III. Desensitization of Adenylyl Cyclase Coupled Adenosine
Receptors in Rabbit Cortical Collecting Tubule Cells .............................. 29
A. Introduction ....................................................................................... .29
B. Methods .............................................................................................. 32
C. Results ........................... 36
D. Discussion ........................................................................................... 47
IV. The Functional Localization of Adenosine Receptor
Mediated Pathways in the LLC-PKl Renal Cell Line ............................... 53
A. Introduction ........................................................................................ 53
B. Methods ............................................................................................. .55
C. Results ................................................................................................. 66
D. Discussion ........................................................................................... 79

TABLE OF CONTENTS, CONT’D.

Page
V. Desensitization of Adenosine A2 ReceptOrs in the
LLC-PKl Renal Cell Line: Homologous Desensitization
Occur Independently of Receptor Occupation/Activation ........................... 88
A. Introduction ........................................................................................ 88
B. Methods .............................................................................................. 90
C. Results ................................................................................................. 94
D. Discussion ......................................................................................... 102
VI. Summary and Conclusions ........................................................................ 111
Appendix A. Adenosine Deaminase Activity .................................................. 116
VII. Literature Cited ......................................................................................... 117

LIST OF TABLES

Page
Table 1. Coupling of Adenosine Receptors to Effectors .......................... 10
Table 2. Apical versus basolateral suprarnaximal adenosine
stimulation of CAMP production in LLC-PKl cells ................... 80

viii

Elam

Figure I.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

LIST OF FIGURES

Page

Flow diagram of adenosine sources and srnks ..... 4

Time course of desensitization of adenosine A2
receptors in RCCT cells ............................................................... 37

Effect of four hour NECA pretreatment (10 nM to 10 pM)
on desensitization of adenosine A2 receptor stimulated
cAMP production in RCCT cells ................................................ 38

Effect of four hour 10 pM NECA pretreatment with

and without 10 pM 1,3-diethyl-8-phenylxanthine

(DPX) on desensitization of adenosine A2 receptors

in RCCT cells ............................................................................... 40

Effect of four hour 10 pM NECA pretreatment with and
without 1 pM 8-cyclopentyl-1,3-dipropylxanthine

(DPCPX) or 24 hour pertussis toxin treatment (1 pg/ml)

on desensitization of adenosine A2 receptors in

RCCI‘ cells ................................................................................... 41

10 pM NECA four hour pretreatment of 100 nM
forskolin stimulation of cAMP production
in RCCT cells ............................................................................. 43

Effect of 4 hour 10 IIM NECA pretreatment on
cholera toxin stimulated CAMP production in
RCCT cells ................................................................................... 44

Effect of 4 hour pretreatment with CHA (1 nM to
100 pM) on adenosine A, receptor inhibition of
basal CAMP production in RCCT cells ....................................... 45

Figure

Figure 9.

Figme 10.

Figure 11.

Figure 12.

Figure 13.

Figme 14.

Figure 15.

Figure 16.

Figure 17.

Figtne 18.

LIST OF FIGURES, CONT’D.

l?
0

Effect of 4 hour 10 IIM NECA pretreatment on
arginine vasopressin (AVP) stimulated cAMP
production in RCCT cells ............................................................ 46

Diagram of LLC-PK, cells grown on a
Millicellm-CM insert. ................................................................... 58

Permeability of LLC-PK, cells grown on gelatin
coated Millicellm-CM inserts to 1 pM I‘C-sucrose .................... 67

Spectrofluorometer generated tracing of intracellular
free calcium concentration in LLC-PK, cells in response
to adenosine and AVP ................................................................. 68

Spectrofluorometer generated tracing of intracellular

free calcium concentration stimulated by adenosine

in LLC-PK, cells following chelation of

extracellular calcium with EGTA ................................................ 70

Adenosine stimulation of inositol phosphates in
LLC-PK, cells in suspension ....................................................... 71

Adenosine dose response stimulation of inositol
phosphate (IPI) in LLC-PK, cells with and without
1 pM DPCPX ............................................................................... 72

Apical versus basolateral 10 pM adenosine stimulation
of inositol phosphates in LLC-PK, cells grown on
Millicelln—CM inserts .................................................................. 73

Apical versus basolateral 30 nM adenosine inhibition
of 1 pM forskolin stimulated cAMP production in
LLC-PK, cells grown on Millicellu-CM inserts ........................ 75

Apical versus basolateral 100 pM adenosine
stimulation of cAMP in LLC-PK, cells grown
on Millicelln-CM inserts ............................................................. 76

Figure

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.
Figure 24.
Figure 25.

Figure 26.

LIST OF FIGURES, CONT’D.

Apical versus basolateral time course to
100 pM adenosine stimulation of cAMP in

LLC-PK, cells grown on Millicellm-CM inserts ...........

Model of adenosine receptor functional

localization in LLC-PK, cells .........................................

Selective 100 uM adenosine pretreatment of
apical or basolateral adenosine A2 receptors

in LLC-PK, cells .............................................................

Apical versus basolateral 100 pM adenosine
pretreatment effect on R-PIA inhibition of
1 pM forskolin stimulated cAMP production

in LLC-PK, cells .............................................................

Effect of apical versus basolateral 100 pM
adenosine pretreatment on AVP-stimulated

cAMP production in LLC-PK, cells ..............................

Effect of pretreatment with 1 uM forskolin
on adenosine A, receptor responsiveness

in LLC-PK, cells .............................................................

Effect of pretreatment with 100 IIM adenosine
i. 1 pM staurosporine on adenosine A2 receptor

desensitization in LLC-PK, cells ...................................

Effect of the PKA inhibitor, Rp—cAMPS, on
adenosine A2 receptor desensitization induced
changes in adenylyl cyclase activity in

LLC-PK, cell membranes ...............................................

Page

............. 78

............. 86

............. 96

............. 98

........... 100

........... 101

............ 103

........... 106

I. Introduction

Adenosine is a catabolic product of adenine nucleotides which was first
proposed as a retaliatory metabolic regulator of coronary blood flow by Burne in
1963. Since then, adenosine has been implicated as a physiological regulator of
a number of physiological phenomena including several aspects of renal function
(reviewed by Spielman et al. 1987; Spielman and Arend 1991). During increased
metabolic demand and/or reduced availability of oxygen, catabolism of ATP is
increased leading to an increase in adenosine production. Adenosine then exits the
cell and acts on cell surface receptors. Adenosine is thought to act as a local
hormome, because it is rapidly degraded by adenosine deaminase present in tissues
and blood (Conway and Cooke 1939). Adenosine released into the interstitium is
thought to act in a feedback loop to re-establish the appropriate level of energy
utilization and/or oxygen demand for the ctnrent physiological conditions.

Adenosine receptors that, when activated, stimulate and inhibit adenylyl
cyclase activity have been shown to exist in renal epithelial cells (Arend et al.
1987). Adenosine also has been shown to stimulate the turnover of inositol
phosphates and to mobilize calcium in renal epithelial cells (Arend et al. 1988;

Arend et al. 1989; Weinberg et al. 1989). Despite this characterization, little work

2

has been done to elucidate the regulation or localization of adenosine receptors in
these epithelia.

This dissertation presents research on the regulation and localization of
adenosine receptors in renal epithelial cells grown in culture. The first chapter is
a literature review, the first two sections of which discuss the metabolism of
adenosine and the cell surface receptors through which it acts, while the following
three sections cover receptor regulation and localization as they relate to the
research presented in the thesis. The three chapters that follow the literature review
encompass the three research projects that comprise the thesis. Each consists of
an Introduction, Methods, Results and Discussion section. The first of these
presents research on the desensitization of adenosine receptors in primary cultures
of rabbit cortical collecting tubule cells. The second presents results of
investigation of the spatial localization of adenosine receptors in the porcine renal
cell line, LLC-PK,. The last chapter extends that preceding it with an investigation
of the desensitization of adenosine receptors in the LLC-PK, cell line with regard
to their spatial distribution. The final chapter summarizes the research presented

in this thesis.

 

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II. Literature Review
A. Adenosine Metabolism

Adenosine, a purine nucleoside, is both a percursor and metabolite of the
adenine nucleotides (Figure 1). Adenosine is also coupled to the production of S-
adenosylhomocysteine by SAH hydrolase. The equilibrium of this reaction favors
the production of S-adenosylhomocysteine, although under normoxic conditions the
low intracellular concentrations of adenosine and homocysteine that prevail actually
contribute to the hydrolysis of S-adenosylhomocysteine and the production of
adenosine (Lloyd and Schrader 1987; Porter and Boyd 1992). In spite of this, SAH
hydrolase is inhibited by adenosine and inosine and is assumed not to play an
important role in the production of adenosine under hypoxic conditions (Eloranta
1977; Lloyd and Schrader 1987; Schatz et al. 1977). Therefore, the majority of
adenosine produced both intracellularly and extracellularly in response to increased
oxygen demand and/or hypoxic conditions is thought to arise from the catabolism
of adenine nucleotides.

Adenosine originates from the dephosphorylation of 5’-AMP by 5 ’-
nucleotidase which is found as both a cytosolic enzyme and an ectoenzyme
(Dendorfer et a1. 1987; Frick and Lowenstein 1976; Pearson et al. 1980). Cytosolic
5’-nucleotidase is thought to be responsible for the increase in adenosine observed
under hypoxic conditions (Altschuld et al. 1987; Collinson et al. 1987; Frick and

Lowenstein 1976; Newby et al. 1987; Shryock et al. 1988). Ecto 5’-nucleotidase

adenylyl
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5

is thought to be important in terminating the action of extracellular ATP on P2
purinergic receptors (Gordon 1986). The close association of ecto-ATPase, -
ADPase and -5’-nucleotidase along with the nucleoside transporter has been linked
to the conversion of extracellular ATP to adenosine and the subsequent
translocation of adenosine into the cytosol in the rat heart (Frick and Lowenstein
1978).

Adenosine that is released into or generated in the extracellular compartment
can be removed by two mechanisms; 1) conversion to inosine by adenosine
deaminase and 2) uptake via facilitated diffusion by the nucleoside transporter into
cells and subsequent phosphorylation by adenosine kinase to 5 ’-AMP(Plagemann
and Wohlhueter 1980). The nucleoside transporter is relatively non-selective for
adenosine or inosine and allows the bidirectional passage of these nucleosides down
their respective concentration gradients. Adenosine deaminase is found both intra-
and extracellularly (Baer et al. 1966; Conway and Cooke 1939), and will dearninate
adenosine to inosine. Due to the ubiquitous nature of this enzyme, it is thought
that any adenosine that enters the circulation will be converted quickly to inosine.
Additionally, red blood cells have been shown to take up adenosine and metabolize
it (Meyskens and Williams 1971). Adenosine deaminase (EC 3.5.4.4) has a
relatively high KIn (35 pM), but it also has a high V“, which allows it to
effectively reduce adenosine concentrations to below 1 pM (Manfredi and Holmes

1985; Sparks and Bardenheuer 1986). Adenosine kinase is an intracellular enzyme

6
that phosphorylates adenosine to 5’-AMP. Adenosine kinase has a low Km (0.4

11M) and is inhibited by high concentrations of adenosine (Miller et al. 1979).
Consequently, the majority of adenosine at high concentrations (>50 M), which
occur during increased energy demand and/or hypoxia, is removed by the action
of adenosine deaminase. At low concentrations (< 1 pM) prevalent under normoxic
conditions, adenosine is phosphorylated to 5’-AMP by adenosine kinase.

The kidney has been shown to both take up and release adenosine
(Thompson et al. 1985). It also has been conclusively established that the ischemic
kidney will produce adenosine (Miller et al. 1978; Osswald et al. 1980). However,
the specific sites of uptake and release of adenosine in the kidney have not been
clearly elucidated. Using stop-flow techniques, Miller et al. ( 1978) suggested that
the proximal tubules make a significant contribution to the extracellular adenosine
produced in the ischernic kidney. In regard to the uptake of adenosine and its
conversion into the nucleotide pool, experiments using isolated renal proximal
tubules have shown that exogenous ATP is more effective in increasing
intracellular ATP pools than is exogenous adenosine (Weinberg and Humes 1986;
Weinberg et al. 1988). Adenosine at a concentration of 250 M was preferentially
deaminated to inosine, which is in keeping with substrate inhibition of adenosine
kinase by adenosine. Adenine nucleotides at a concentration of 250 pM lead to
increases in intracellular ATP which could be blocked by S-iodotubercidin, an

adenosine kinase inhibitor. These observations concur with those of Frick and

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Lowenstein ( 197 8) in suggesting that adenosine production by ecto-5’-nucleotidase
in the rat heart is coupled to the uptake of adenosine.

Recent advances in immunohistochemistry have enabled researchers to
localize ecto-5’-nucleotidase in frozen sections of rat kidney. Using a monoclonal
antibody, ecto-5’-nucleotidase was localized to the apical aspect of proximal
tubules, the apical aspect of intercalated cells of the collecting tubule, interstitial
cells of the cortex, and the surrounding cells of the afferent and efferent arterioles
(Gandhi et al. 1990). Whether this ecto-5’-nucleotidase is involved in the
production of extracellular adenosine or the vectorial uptake of adenosine via the
nucleoside transporter is unknown. While it is clear that adenosine is released by
the ischemic kidney, the specific sOurce(s) of this adenosine has yet to be

elucidated.

B. Adenosine Receptors
For more than 60 years it has been recognized that adenosine has

physiological effects (Drury and Szent-Gyorgi 1929). To cite just a few, adenosine
has been linked to changes in coronary blood flow (Burne 1963; Bume 1980), renal
function (reviewed by Spielman et al. 1987; Spielman and Arend 1991), release of
neurotransmitters (Fredholm et 'al. 1987), and inhibition of lipolysis (Pain and
Malbon 1979). As discussed earlier, adenosine is metabolized quickly and most

probably does not act as a blood borne hormone, but rather as an autocrine and

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paracrine effector. Adenosine is released into the extracellular space in response
to increased energy demand and/or reduced oxygen supply, and acts on cell surface
receptors activating second messenger systems to bring about functional changes.

The purinergic receptors, P, and P,, were first described by Bumstock
(1978). The P, and P, classifications are based on the potency of either adenosine
or adenine nucleotides at these respective sites; P, receptors show an agonist
potency profile of adenosine > AMP > ADP > ATP, while P, receptors have a
potency profile of ATP > ADP > AMP > adenosine. Moreover, the P, receptors
are antagonized by methylxanthines, such as caffeine and theophylline, while P,
receptors are not. To date there have been no antagonists developed for the P,
class of purinergic receptors.

The P, receptors are further classified as adenosine A, and A, receptors
(Londos et al. 1980). These cell sm'face adenosine receptors have typically been
distinguished on the basis of their selectivity for adenosine analogs. Three of the
adenosine analogs in wide use are, N°-cyclohexyl adenosine (CHA), R(-)N°-(2-
phenylisopropyl) adenosine (R-PIA), and 5 ’-N-ethylcarboxamido adenosine
(NECA). The adenosine A, receptor has an agonist potency profile of CHA > R-
PIA > NECA, while the adenosine A, receptor has the reverse potency profile of
NECA > R-PIA > CHA. Typically, analogs with substitutions at the N‘ position
are selective for the adenosine A, receptor, while analogs with 5’- and 2-

substitutions are selective for the adenosine A, receptor (Daly and Padgett 1992).

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The prototypical adenosine A, receptor is activated by nanomolar
concentrations of agonist and is inhibitory towards adenylyl cyclase. The
adenosine A, receptor is activated by micromolar concentrations of agonist and is
stimulatory towards adenylyl cyclase. The adenosine A, receptor is coupled to
adenylyl cyclase through the regulatory guanine nucleotide binding protein, G, In
contrast, the adenosine A, receptor is coupled to multiple effectors through
pertussis toxin-sensitive regulatory G-proteins, presumably of the G, family. In
addition to inhibiting adenylyl cyclase (Table l), adenosine A, receptors stimulate
(Arend et al. 1989) and inhibit (Delahunty et al. 1988) inositol phosphate turnover,
inhibit chloride transport (Kelley et al. 1990; Schwiebert et al. 1992), activate
potassium channels (Kurachi et al. 1986), and stimulate cGMP production (Kurtz
1987). In further distinguishing the A, receptor from the A, receptor, an antagonist
with a 700 fold selectivity for the adenosine A, receptor, 8-cyclopentyl-l,3-
dipropylxanthine (DPCPX), has been developed and proven to be very useful
(Lohse et al. 1987). Unfortunately, an antagonist with similar selectivity for the
adenosine A, receptor has not yet been developed. The best adenosine A,
antagonist, 3,7-dimethyl-l-propargylxanthine (DMPX), has only a 5-20 fold
selectivity for the adenosine A, receptor (Scale et al. 1988; Sebastiao and Ribeiro
1989).

There is another "adenosine receptor" located on the catalytic subunit of

adenylyl cyclase termed the P site. This intracellular P site inhibits adenylyl

Table l. Coupling of Adenosine Receptors to Effectors

Effect of A, stimulation

Inhibition of adenylyl cyclase

Increased inositol phosphate
Decreased inositol phosphate

Decreased chloride transport

Activated K‘M, channels

Increased cGMP production
Effect of A, stimulation

Stimulation of adenylyl cyclase

Tissue

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28A-RCCI‘ cells
GH3 pituitary cells

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28A—RCCI‘ cells

Atrial cells

smooth muscle cells
Tissue

RCCT cells
28A-RCCI‘ cells
vascular smooth muscle
Platlets

Striatum

11

cyclase and is activated by millimolar concentrations of adenosine. In contrast to
the extracellular adenosine receptors, the P site is not antagonized by
methylxanthines. The physiological function of the P site is still in question,
because adenosine kinase and adenosine deaminase maintain intracellular
concentrations of adenosine well below the micromolar range. Therefore, it
appears unlikely that adenosine plays a role as the endogenous ligand of the P site.
A metabolite of nucleic acids, 3’-AMP, however, is a likely candidate as an
endogenous ligand because it occurs at sufficient concentrations and has sufficient
affinity to activate the P site (Johnson et al. 1989).

Very recently, the structural features of the adenosine receptors have begun
to be elucidated. Photoaffinity and purification studies have demonstrated that the
adenosine A, receptor has an apparent molecular weight of 35,000 daltons (Klotz
et al. 1985; Lohse et al. 1986; Nakata 1990). Similar studies also have established
that the adenosine A, receptor is glycosylated (Klotz and Lohse 1986; Stiles 1986).
Furthermore, the adenosine A, and A, receptors have been cloned and can be
distinguished on the basis of different nucleotide and amino acid sequence (Libert
et al. 1991; Maenhaut et al. 1990). The clones of the adenosine A, and A,
receptors have seven transmembrane-spanning domains that have become a
distinguishing characteristic of G-protein linked receptors. The amino acid
sequences of these two adenosine receptors share a 51% identity with one another

in their transmembrane domains. The cloned adenosine A, receptor has a

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molecular weight of 36,000 daltons, while the cloned adenosine A, receptor has a
molecular weight of 45,000 daltons (Libert et al. 1991; Maenhaut et al. 1990). In
addition, the adenosine A, receptor has a putative glycosylation site on the second
extracellular loop and putative phosphorylation sites for protein kinase A (PKA)
and protein kinase C (PKC) on the third intracellular loop (reviewed by Stiles
1992). The possible significance of phosphorylation sites on these receptors will

be discussed in the section on receptor regulation.

C. Regulation of QAdrenergic Receptors

Quite often, biological systems regulate their responsiveness to stimuli such
that a stimulus does not elicit the same response upon a second or prolonged
exposure. This phenomenon is often termed tachyphylaxis, indicating a reduced
effectiveness of a drug over time. The biochemical basis for this phenomenon has
recently gained considerable attention, with a goal being to control tachyphylaxis
and extend the usefulness of pharmacologic agents. A large number of
pharmacologic agents act through cell surface receptors, of which the B—adrenergic
receptor is a well studied example.

The B-adrenergic receptor, like the rhodopsin receptor, belongs to the family
of cell surface receptors that are characterized by seven transmembrane spanning
domains. These receptors couple to their various effectors through guanine

nucleotide binding proteins (G-proteins). The G-protein linked receptors have been

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observed to undergo tachyphylaxis and this has been termed desensitization of
receptor response. With regard to desensitization, the most extensively studied of
these G-protein linked receptors is the B—adrenergic receptor system (reviewed in
Benovic et al. 1988; Hausdorff et al. 1990). The B—adrenergic receptor is linked
through G, to adenylyl cyclase and thus increases the production of cAMP upon
activation by an agonist. Consequently, there are at least three points at which
altered function could act to bring about desensitization, those being the B-
adrenergic receptor, 6,, and adenylyl cyclase.

There are two forms of desensitization, ”homologous" and ”heterologous".
Homologous desensitization is the reduced responsiveness of a specific receptor
system following exposure of that system to agonist. In contrast, heterologous
desensitization is the reduced responsiveness of multiple distinct receptor systems
following exposure of one of those systems to agonist. Both forms of
desensitization involve the phosphorylation of the receptor by various kinases,
which leads to the rapid uncoupling of the receptor from its G-protein (N ambi et
al. 1985).

Heterologous desensitization of the Sadrenergic receptor occurs at low
(nanomolar) concentrations of agonist and involves the phosphorylation of the
receptor (Clark et al. 1989; Loshe et al. 1990a). This phosphorylation appears to
be responsible for a rightward 'shift of the concentration-response curve but does

not alter the maximum response to agonist (Hausdorff et al. 1989). The rightward

l4
shift of the concentration-response has been speculated to be due to reduced

coupling efficiency of the B—adrenergic receptor to 6,, although the phenomenon
of "spare" receptors allows the system to continue to respond maximally at high
concentrations of agonist. There are two consensus sites, delineated by the amino
acids RRSS, on the B-adrenergic receptor for phosphorylation by cyclic AMP
dependent protein kinase (PKA) (Hausdorff et al. 1990). One site is found in the
third intracellular loop and the other is in the proximal portion of the carboxy-
terminus tail. Using site-directed mutagenesis, the site in the third intracellular
loop has been identified as important in the PKA-induced desensitization of B-
adrenergic receptors (Clark et al. 1989). In contrast, the PKA phosphorylation site
in the proximal portion of the carboxy-terminal tail can be altered without effect
on heterologous desensitization of the B—adrenergic receptor. This is consistent
with the observation that the third intracellular loop of the B~adrenergic receptor is
important in coupling to G, (Strader et al. 1987; O’Dowd et al. 1988). Therefore,
the phosphorylation of [iadrenergic receptors via PKA and subsequent reduction
in receptor-G-protein coupling efficiency appears to be an important component in
the heterologous desensitization of this receptor.

In addition to phosphorylation by PKA, the pmified B‘adrenergic receptor
has been reported to be phosphorylated by the Ca“ phospholipid-dependent,
diacylglycerol-activated kinase, protein kinase C (PKC) in a cell free system

(Bouvier et al. 1987). While agonist occupation of the B-adrenergic receptor

15
increased phosphorylation by PKA, phosphorylation of the receptor by PKC was

not influenced by agonist occupation. This suggests that, while PKC can
phosphorylate B—adrenergic receptors in vitro, it does not participate in the
regulation of adenylyl cyclase-coupled B-adrenergic receptor function in viva.
There are no reports on the functional consequences of PKC phosphorylation of B-
adrenergic receptors.

In contrast to heterologous desensitization, homologous desensitization of the
fl-adrenergic receptor occurs at high agonist concentrations (micromolar) and has
a faster time of onset (minutes as opposed to hours). In addition to a rightward
shift of the agonist dose response curve, as in heterologous desensitization,
homologous desensitization is distinguished by a decrease in the maximal response
to agonist. This decrease in the maximal response is thought to occur through a
complete uncoupling of phosphorylated B-adrenergic receptor from G,.
Homologous desensitization has been characterized by rapid uncoupling of the
receptor and sequestration from the cell surface followed by i a slower down-
regulation of receptor number. In addition to phosphorylation by PKA, a second
receptor—specific kinase termed B—adrenergic receptor kinase (BARK) has been
identified (Benovic et al. 1986). This kinase has been implicated in the rapid
uncoupling of Sadrenergic receptors during homologous desensitization by
phosphorylating only the agonist-occupied form of receptor (Lohse et al. 1989).

BARK is a cytosolic protein that translocates to the plasma membrane upon agonist

16

occupation of a receptor. Strasser et al. (1986) reported that agonist stimulation of
both B«adrenergic and prostaglandin E, receptors was associated with the
translocation of BARK from the cytosol to the plasma membrane. Additionally,
BARK has been implicated in the short term agonist promoted desensitization of
inhibitory a,-adrenergic receptors (Liggett et al. 1992). These studies support the
hypothesis that BARK may be an adenylyl cyclase-coupled receptor kinase that can
regulate the function of many species of both stimulatory and inhibitory adenylyl
cyclase-coupled receptors.

The site(s) that BARK phosphorylates are distinct from those that PKA
phosphorylates (Hausdorff et at 1989). These sites are thought to be located in a
serine, threonine rich area at the distal end of the carboxy-terminal tail. When the
three serines and one threonine in the amino acid sequence 355 to 364 of the
carboxy-terminal tail are replaced with either alanine or glycine, the ability of the
Badrenergic receptor to undergo homologous. desensitization and rapid
sequestration is abolished. Paradoxically, down-regulation of the B-adrenergic
receptor still takes place with prolonged (24 hour) exposure to 10 pM isoproterenol
(Hausdorff et al. 1991). Classically, the rapid sequestration of B-adrenergic
receptors from the cell surface, as defined by decreased binding of hydrophilic
radioligands, following phosphorylation by BARK was thought to be an early step
in the down-regulation of receptors exposed to agonist for long periods of time.

These mutations in the carboxy-terminal tail prevented the phosphorylation and

17

rapid sequestration of the B-adrenergic receptors. However, mutations in the
carboxy-terminal tail did not prevent long term down-regulation of the B-adrenergic
receptor. This suggests that long term agonist-induced down-regulation of B-
adrenergic receptors occurs through a mechanism independent of either
phosphorylation or rapid sequestration.

Purified BARK retained its ability to phosphorylate B—adrenergic receptors
in a cell free system. Surprisingly, this did not lead to desensitization of B-
adrenergic receptor mediated activation of adenylyl cyclase. Nevertheless,
phosphorylation of B-adrenergic receptors with crude extracts of BARK uncoupled
the receptor from 6,. These results led investigators to propose that a cofactor was
involved in the homologous desensitization of B-adrenergic receptors. In an
analogous receptor system, the light sensitive receptor rhodopsin, binding of an
additional cytosolic protein called arrestin following receptor phosphorylation is
required for desensitization to occur. An analogous protein has been cloned called
B-arrestin that regulates B—adrenergic receptors much as arrestin regulates
rhodopsin. B-arrestin is thought to bind to the phosphorylated carboxy-terminal tail
and prevent the interaction of B-adrenergic receptors with G, (Lohse et al. 1990b).
This mechanism is consistent with the diminished maximal response to B-
adrenergic agonists following homologous desensitization.

In addition to desensitization, B-adrenergic receptor function is altered

through a mechanism termed cross-regulation. Cross-regulation refers to the

18

influence of inhibitory receptor activation on subsequent stimulatory receptor
function. Persistent (48 hour) activation of the inhibitory adenosine A, receptor
pathway in smooth muscle DDT,-MF2 cells caused the increased expression of the
stimulatory B-adrenergic receptor and a 50 fold decrease in the ED,,, for
isoproterenol. This cross-regulation was accompanied by an increase in the mRNA
level for B~adrenergic receptors and a decrease in the level of Gin-protein (Hadcock
et al. 1991). In a second study, acute activation (60 min) of the inhibitory
adenosine A, receptor resulted in an increase in the maximal response to B-agonist
(Port et al. 1992). The increase in maximal response was attributed to a decrease
in basal B-adrenergic receptor phosphorylation. Activation of adenosine A,
receptors lowers cAMP levels and reducs PKA activity, thereby reducing the basal
level of B—adrenergic receptor phosphorylation. B—adrenergic receptor cross-
regulation of other receptors also has been observed. Short term stimulation of B-
adrenergic receptors has been shown to increase mRN A levels of the a,-adrenergic
receptor (Morris et al. 1991). This effect of B-adrenergic receptors could be
mimicked by forskolin or cAMP analogs, indicating that this may be a PKA-

mediated event.

D. Regplation of Adenosine Receptors

Far less is known about regulation of adenosine receptors than B—adrenergic

receptors. While desensitization of the stimulatory adenosine A, receptor and the

19

inhibitory adenosine A, receptor has been observed, very little is known about the
biochemical mechanisms that are responsible for these phenomena.

Homologous desensitization of the stimulatory adenosine A, receptor has
been reported in rat brain Striatum (Hawkins et al. 1988; Porter et al. 1988), rat
vascular smooth muscle cells (Anand-Srivastava et al. 1989), hamster DDT, MF-2
smooth muscle cells (Ramkumar et al. 1991), and rat kidney fibroblasts (Newman
and Levitzki 1983). Adenosine A, receptors are found in abundance in the straitum
of the brain. Prolonged exposure (24 hours) to the adenosine analog NECA
induced a homologous pattern of desensitization of adenosine A, receptors in the
Striatum (Porter et al. 1988). This desensitization was accompanied by a reduction
in adenosine A, receptor stimulated adenylyl cyclase activity and a reduction in
receptor number. Although the adenosine analog NECA is not selective for
adenosine A, versus A, receptors, it did not induce a desensitization of adenosine
A, receptors in the Striatum within the 24 hour exposure time. Additionally, the
response of G, to stimulation by NaF was not affected by desensitization of
adenosine A, receptors. This suggests that the homologous desensitization of
adenosine A, receptors in Striatum was due to altered function of the receptor itself
(Porter et al. 1988).

Desensitization of adenosine A, receptors in cultured vascular smooth
muscle cells was found to be both time- and concentration-dependent. Micromolar

concentrations of the adenosine analog NECA were required to induce

20

desensitization and the effect was rapid, with full desensitization occurring within
60 minutes. Adenylyl cyclase activity stimulated by GTP, forskolin, and
isoproterenol was unaffected, indicating a homologous desensitization of adenosine
A, receptors in vascular smooth muscle cells (Anand-Srivastava et al. 1989). As
in vascular smooth muscle cells, the desensitization of adenosine A, receptors in
DDT, MF-2 cells was rapid and homologous. DDT, MF-2 cells are derived from
hamster vas deferens smooth muscle. The t, ,, for desensitization was 45 minutes
and was also rapidly reversible with 80% of control adenylyl cyclase activity
returning four hours after the removal of agonist. In contrast to desensitization of
adenosine A, receptors of rat Striatum, the adenosine A, receptors of DDT, MF-2
cells did not undergo down-regulation (Ramkumar et al. 1991). Consequently, the
desensitization of adenosine A, receptors appears to be rapid and homologous in
most of the tissues and cell systems studied.

In contrast to adenosine A, receptors, the desensitization of inhibitory
adenosine A, receptors has a slower time of onset (days versus hours). Although
the majority of desensitization studies of adenosine A, receptors have been carried
out with adipocytes (Green 1987; Green et al. 1990; Green et al. 1992; Hoffman
et al. 1986; Longabaugh et al. 1989; Parsons and Stiles 1987), studies also have
been carried out with DDT, MF-2 cells (Ramkumar et al. 1991), and embryonic
chicken heart (Shryock et al. 1989).

Desensitization of adenosine A, receptor-mediated inhibition of lipolysis in

21

adipocytes isolated from rats following a six day infusion of the adenosine analog
R-PIA was first described by Hoffman et al. (1986). Upon further investigation
using this in vivo model, desensitization of adenosine A, receptors was found to be
heterologous, involving changes in adenosine A, receptor number and G-protein
levels (Longabaugh et al. 1989; Parsons and Stiles 1987). These investigators
found a 30% to 50% reduction in adenosine A, receptor number. Western blots
of the (1 subunits of G,, G,,, G,,, and GB revealed an increase in 6,, no change in
G,, and a decrease in G,, and 6,. There was no change in the mRNA levels for
any of the a subunits of these G-proteins, suggesting a change in the post-
transcriptional processing of these proteins. Additional work carried outwith
isolated adipocytes in culture has supported the in vivo studies (Green 1987; Green
et al. 1990; and Green et al. 1992). These changes in G-protein levels therefore
constitute the basis, at least in part, for the heterologous desensitization of
inhibitory pathways towards adenylyl cyclase with prolonged exposure of the
adenosine A, receptor to agonist in adipocytes.

Desensitization of adenosine A, receptors also has been reported in DDT,
MF-2 smooth muscle cells. The time of onset of this desensitization (t,,,, 8 hours)
was shorter than that reported for adipocytes and involved a 40% reduction in
adenosine A, receptor number. This is the only study to show an increase in the
phosphorylation state of an adenosine receptor following desensitization. The

ability of NaF, forskolin and isoproterenol to stimulate adenylyl cyclase was

22

unaltered in this study, suggesting a homologous pattern of adenosine A, receptor
desensitization in DDT, MF-Z cells (Ramkumar et al. 1991). Furthermore,
adenosine A, receptors have been shown to become desensitized in cardiac tissue
following 44 hours of exposure to the adenosine analog R-PIA. This desensitzation
was homologous and was characterized by a 60% decrease in receptor number
(Shryock et al. 1989).

Therefore, the desensitization of adenosine A, receptors is characterized by
a long time of onset (days versus hours) and a down-regulation of receptor number.
Additionally, there is at least one report of adenosine A, receptor phosphorylation
following desensitization (Ramkumar et al. 1991). In all tissues other than
adipocytes, desensitization of adenosine A, receptors appears to be homologous.
In contrast, the desensitization of this receptor in adipocytes is heterologous and
involves changes in the levels of G,, G,, and G,,, along with a down-regulation in
receptor number. The levels of G, were increased, leading to an increased
sensitivity to stimulatory agonists, while G,, and 6,, were decreased, leading to a

desensitization of inhibitory inputs.

E. Receptor Localization

The distribution of many hormone receptors in various tissues has been
investigated. For example, adenosine A, receptors in the kidney were found to be

concentrated in the medulla by autoradiographic visualization of mI-I-IPIA' binding

23

sites (Weber et al. 1988). While similar studies of many hormones in many tissues
have been carried out, only recently have researchers begun to investigate the
distribution of hormone receptors on epithelial cells. Epithelial cells, whose major
function is the vectoral transport of water and solutes, can be grown as polarized
monolayers (reviewed by Rodriguez-Bolan and Nelson 1989). That is, epithelial
cell monolayers have an apical and basolateral side separated by tight junctions.
When epithelial cells grow in culture, they orient with their basolateral sides
attached to the substratum and their apical sides up to the media. The tight
junctions function as a barrier, segregating the integral membrane proteins on either
side of the cellular monolayer. Thus, an integral membrane protein targeted to one
or the other side cannot cross the tight junction barrier. Some integral membrane
proteins, such as the Na’lK+ ATPase, which is basolaterally located, are expressed
on only one side of the epithelial cell monolayer (Rodriguez-B clan and Nelson
1989). This polar distribution of transport proteins is the very basis for the vectoral
transport of water. and solutes. With this in mind, researchers have very recently
begun investigating the distribution of hormone receptors on polarized epithelial

cells that may regulate the activity of these transport proteins.

E.l. Peptide Hormone Receptor Localization

It has been known for some time that arginine-vasopressin (AVP) stimulates

cAMP production only when presented to the basolateral side of isolated kidney

24

collecting tubules (Grantham and Burg 1966; Schwartz et al. 1974). This increase
in cAMP is thought to mediate the increase in water permeability of collecting
tubules in response to AVP. For much of this time it has been assumed that there
is no effect of luminal AVP in the collecting tubule. However, recently there have
been reports of AVP initiating responses from the apical side of the cellular
monolayer. Apically applied AVP was shown to be involved in the release of
PGE, (Garcia-Perez and Smith 1984) from primary cultures of canine cortical
collecting tubule (CCCT) cells. Additionally, AVP exposed to the luminal side of
isolated rabbit cortical collecting tubules stimulated the sustained hyperpolarization
of transepithelial potential differance (Ando et al. 1991). Therefore it would appear
that while AVP stimulates cAMP production only from the basolateral side of
isolated collecting tubules, it is also involved in other signal transduction pathways
on the luminal side.

Bradykinin, a nine amino acid peptide of the kinin family, has been shown
to stimulate the release of PGE, only when exposed to the apical surface of CCCI‘
cells (Garcia-Perez and Smith 1984). In primary cultures of canine tracheal
epithelia, bradykinin stimulated arachidonic acid release only from the apical side,
but stimulated inositol phosphate tmnover from both sides (Denning and Welsh
1991). These two studies suggest that the ability of bradykinin to stimulate
arachadonic acid release, and therefore prostaglandin production, from only the

apical surface of epithelial cells may be a general feature of bradykinin receptor

25

organization. However, in one of these studies bradykinin also stimulated the
turnover of inositol phosphates from either side of the epithelium (Denning and
Welsh 1991). This suggests that there may be two types of bradykinin receptors,
or alternatively that there is one type of bradykinin receptor linked to two signal
transduction pathways via distinct G-proteins. That is, there may be a G-protein
involved in the release of arachadonic acid that is isolated to the apical aspect of
epithelia and is responsive to bradykinin receptor stimulation.

Localization of vasoactive intestinal peptide (VIP) receptors has been
investigated in differentiated human colonic adenocarcinoma cells (HT29—D4)
(Fantini et al. 1988). This cell line differentiates when grown in a glucose-free,
galactose-containing media. As HT29-D4 cells differentiated and reached
confluency, the binding of 12’I-VIP applied to the apical surface declined to non-
specific background. Cells grown on permeable supports, however, displayed
sustained binding of 12‘I-VIP to their basolateral aspect. Moreover, VIP could elicit
the stimulation of cAMP production when presented to the basolateral, but not the
apical side of differentiated 141129-134 cells. Therefore it was concluded that the
VIP receptors of differentiated I-IT29-D4 cells are located exclusively to the
basolateral side of the monolayer (Fantini et al. 1988). A

These studies suggest that several hormone receptors have differential
distributions on polarized epithelial cell monolayers. The distribution of a hormone

receptor on a monolayer of epithelial cells may be indicative of the side on which

26

the hormone is most likely to be present in viva, and is most certainly important

in the functional response of a particular epithelium to a hormone signal.

E.2. Adenosine Receptor Localization

The distribution of adenosine A, receptors has been investigated in several
epithelial cell preparations (Barrett et al. 1989; Husted et al. 1990; Pratt et al.
1986). The adenosine analog 2-chloroadenosine was employed to study the
polarity of adenosine A, receptors in canine tracheal epithelium (Pratt et al. 1986).
2-Chloroadenosine was found to stimulate the secretion of Cl' from either side of
the epithelium. The stimulation of Cl' secretion is thought to occur via adenosine
A, receptor mediated increases in cAMP production. The short circuit current (In)
elicited by 2-chloroadenosine stimulation of Cl' secretion was greater on the apical
side than on the basolateral side. Apical addition of 2-chloroadenosine following
basolateral stimulation could further increase the change in I“. In contrast, the
basolateral addition of 2—chloroadenosine following apical stimulation could not
cause any further increase in I“. When the adenosine receptor antagonist 8-
phenyltheophylline was used to antagonize this effect, it only blocked when
presented to the apical surface of the monolayer. This led the authors to suggest
that the adenosine A, receptor is localized to the apical aspect of canine tracheal
epithelium (Pratt et al. 1986).

The adenosine analog NECA was shown to stimulate chloride secretion

27

(measured as I") when applied to either side of monolayers of the colonic cell line
T8,, (Barrett et al. 1989). The rank order of potency of several adenosine analogs
to stimulate I,c was consistent with stimulation of adenosine A, receptors. While
the kinetics of the change in I,c was the same when stimulated from either side,
addition of NECA to the basolateral side elicited a greater change in I,c than did
apical exposure. Furthermore, the ability of NECA to stimulate cAMP production
in T“ cells was markedly greater when presented to the basolateral side. When
cells were preincubated with NECA for 7 days, the cAMP response and the apical
I,c response were completely desensitized. However, the basolateral I,c response
was only reduced by 50%. From this it was suggested that while the adenosine A,
receptor appears to reside on both sides of the T,” cell monolayer, it may be
coupled to different signal pathways. Additionally, there appears to be a subset of
adenosine receptors coupled to the stimulation of I,c on the basolateral side of the
cell that are resistent to desensitization (Barrett et al. 1989).

Adenosine A, receptor stimulated cyclic AMP production was shown to be
greater in response to apically applied NECA than basolaterally applied NECA in
primary cultures of rat inner medullary collecting tubule cells (Husted et al. 1990).
These studies suggest differential distributions of adenosine A, receptors on several
types of epithelia. Although it is still unclear whether this receptor is exclusively
localized to only one side of epithelial monolayers or simply has a higher density

on one side. One caveat to both this study (Husted et al. 1990) and the one

28
preceeding it (Barrett et al. 1989), is that NECA has a lipophilic nature that may

allow it to cross a confluent monolayer of cells and stimulate receptors on the
contralateral side. In regard to the inhibitory adenosine A, receptor, there are as

yet no published reports of the differential distribution of this receptor on epithelia.

III. Desensitization of Adenylyl Cyclase Coupled Adenosine Receptors in

Rabbit Cortical Collecting Tubule Cells

A. Introduction

Adenosine, a product of purine nucleotide metabolism, has been shown to
alter the cellular transport properties of several types of epithelial cells such as
amphibian A6 cells and secretory cells of the shark rectal gland (Forrest et al.
1980; Forrest et al. 1982; Lang et al. 1985). Adenosine also has been shown to
alter renal function by mediating changes in renal hemodynamics, renin release, and
tubule function (Spielman et al. 1980; Spielman and Thompson 1982; Spielman
1984). More recently, Dillingham and Anderson (1985) documented the inhibition
of AVP-stimulated water permeability following administration of adenosine
analogs to isolated rabbit cortical collecting tubules. Previous work from our
laboratory has shown that rabbit cortical collecting tubule (RCCT) cells grown in
culture possess adenylyl cyclase-coupled adenosine A, and A, receptors (Arend et
al. 1987). Activation of the inhibitory adenosine A, receptor has been shown to
attenuate AVP stimulated cAMP production, whereas activation of the stimulatory
adenosine A, receptor increases cAMP production (Arend et al. 1987).

Recently, our attention has focused on the processes of adenosine receptor-
coupled signal transduction in renal tubule cells. Collecting tubule cell adenosine

receptors are coupled to adenylyl cyclase via the guanine nucleotide binding

29

30

proteins, Gi and G,. Adenosine A, receptors, which are coupled to an inhibitory
G, protein, inhibit adenylyl cyclase whereas adenosine A, receptors, which are
coupled to the stimulatory G, protein, stimulate adenylyl cyclase (Londos et a1.
1978; Londos et al. 1980). The two receptor subclasses are further distinguished
by their relative affinites for adenosine. Adenosine A, receptors are activated by
adenosine in nanomolar concentrations whereas adenosine A, receptors are
stimulated by adenosine at micromolar concentrations.

Extensive work has been done to characterize the mechanism(s) of
desensitization in B adrenergic receptor-coupled adenylyl cyclase systems (Benovic
et al. 1988; Nambi et al. 1985; Sibley et al. 1986). Using amphibian erythrocytes
and avian erythrocytes as cell models to investigate B adrenergic receptor
desensitization, Sibley et. al. (1986) concluded that B adrenergic receptor
desensitization in the amphibian erythrocyte occurs by concurrent receptor
phosphorylation and sequestration from the cell surface. In the avian erythrocyte,
however, desensitization is characterized by B adrenergic receptor phosphorylation
and uncoupling from adenylyl cyclase with no concomitant sequestration from the
cell surface (Nambi et al. 1985). In contrast, desensitization of chinese hamster
fibroblast B adrenergic receptors involved receptor phophorylation and regulation
of mRNA levels (Bouvier et al. 1989). These results indicate that while the
mechanisms of desensitization may vary from system to system, altered receptor

function remains the common endpoint.

31

Although considerable information is known about 8 adrenergic receptor
desensitization, much less is known about desensitization of the adenosine receptor-
, coupled adenylyl cyclase systems. Desensitization of the stimulatory adenosine A,
receptor system has been reported in rat kidney fibroblasts and vascular smooth
muscle cells from rat aorta (Anand-Slivastava et al. 1989; Newman and Levitzke
1983). Desensitization of stimulatory adenosine A, receptors in rat brain striatum
also has been reported (Hawkins et al. 1988; Porter et al. 1988). In all cases,
desensitization of adenosine A, receptors was found to be homologous.
Desensitization of inhibitory adenosine A, receptors has been reported in rat
adipocytes, where loss of high affinity binding sites along with decreases in G, and
increases in G, were reported (Hoffman et al. 1986). This desensitization of
adenosine A, receptors in adipocytes was reported to lead to decreased
responsiveness of inhibitory receptors and increased responsiveness of stimulatory
receptors towards adenylyl cyclase (Green et al. 1990; Parsons and Stiles 1987).

The purpose of the present study, therefore, was to investigate
desensitization of adenosine receptors coupled to adenylyl cyclase as a means of
further elucidating the mechanism(s) of adenosine receptor signal transduction in

rabbit cortical collecting tubule (RCCT) cells.

32
B. Methods

Materials: Cell culture reagents were obtained from Gibco BRL, (Grand
Island, NY). Adenosine analogs, NECA and CHA, and the adenosine receptor
antagonist 1,3-diethyl-8-phenylxanthine (DPX) were gifts from Warner Lambert
Co.,(Ann Arbor, MI). The adenosine receptor antagonist 8-cyclopentyl-1,3-
diphenylxanthine (DPCPX) was purchased from Research Biochemicals Inc.,
(N atick, MA). Pertussis toxin was purchased from Peninsula Laboratories, Inc.,
(Belmont, CA). The phosphodiesterase inhibitor RO 20-1724 was obtained from
Biomol Research Labs, Inc., (Plymouth Meeting, PA). All other agents were of
reagent grade and obtained from standard sources.

RCCT Cell Immunodissection: Irnmunodissection of cortical collecting
tubule cells was carried out as described previously (Smith and Garcia-Perez 1985;
Spielman et al. 1986). In brief, six polystyrene culture dishes were treated with 0.2
mg each of a rabbit collecting tubule cell-specific antibody (Rct-30) in 3 ml of
phosphate buffered-saline (PBS) for two to three hours. The dishes were then
washed three times with 3 ml of 1% bovine serum albumin (BSA) in PBS to block
any non-specific protein binding sites.

A six week old New Zealand White rabbit was euthanized by administering
an intraperitoneal overdose of sodium pentobarbitol. The kidneys were excised
using aseptic technique, the renal capsules were removed, and the kidneys were

placed in ice cold PBS. The renal cortex was then removed and minced into 1min3

33

cubes using a sterile razor blade. The resulting cortical "mash" was incubated in
0.1% collagenase in Krebs buffer [mM: 118 NaCl, 25 NaHCO,, 14 glucose, 10 N-
2-hydroxyethylpiperazine-N ’-2-ethanesulfonic acid (HEPES), 4.7 KCl, 2.5 CaCl,,
1.8 K,HPO,, and 1.8 MgSO,, pH 7.4] at 37°C for 40 minutes. At 10 minute
intervals the cortical rrrixture was pipetted up and down (2—3 times) in a 25 ml
pipet to aid in the break up of cortical tissue. At the end of the 40 minute
collagenase incubation, the tissue was centrifuged (500 x g) for five minutes. The
supernatant was aspirated and the pellet resuspended in 10 ml of 0.2% NaCl for 30
seconds to lyse red blood cells. An equal volume of 1.6% NaCl was then added,
followed by an equal volume of PBS in order to bring the solution to isotonicity.
The tissue mixture was then filtered through 250 pm Gelrnan filters to remove any
large tissue pieces. The resulting suspension of single cells and tubule fragments
was centrifuged (500 x g), resuspended in 10% BSA in PBS, and recentrifuged to
remove cellular debris. The final pellet, consisting of single cells and tubule
fragments, was resuspended in 6 ml of PBS. '

Following cortical cell preparation, 1 ml aliquots of the cell suspension were
added to each of six Rct-30 antibody treated-culture dishes. The cellular
suspension was allowed to remain on the culture dishes for two to three minutes
to allow Rct-30 antibody to bind to collecting tubule cells. The culture dishes were
then washed three to five times with 3 ml of PBS to remove any unbound cells and

tubule fragments. Media containing Dulbecco’s modified eagle’s medium

34
(DMEM), 10% decomplemented fetal bovine serum (FBS), 2mM glutamine, and

50 pg/mI of penicillin and streptomycin was added to the dishes (8 ml per dish)
and the RCCT cells were grown to confluency (4 to 5 days).

At confluency, RCCT cells were removed from the culture dishes with 0.1 %
trypsin + 0.05% (Ethylenedininilo)tetraacetic acid (EDTA) in PBS and reseeded
onto 24 well culture plates in 1 ml aliquots per well. Typically, one 100 mm
culture dish of confluent RCCT cells was resuspended into 50 ml of culture media.
This RCCT cell suspension was then added to the wells of two 24 well plates in
1 ml aliquots. The cells were again allowed to grow to confluency (4 to 5 days)
before undergoing any experimental procedure.

Desensitization Procedure: All experimental pretreatrnents and treatments
were done in triplicate (i.e. three culture wells per treatment regimen). For cells
receiving 5 ’-N-ethylcarboxamidoadenosine (NECA), N°-cyclohexyladenosine
(CHA), 8-cyclopentyl-l,3-dipropylxanthine (DPCPX), or 1,3-diethyI-8-
phenylxanthine (DPX) pretreatment, culture media was aspirated and replaced with
300 pl of DMEM containing the specific effector. Control wells had their culture
media removed and replaced with 300 pl of DMEM. Following three hours of
incubation at 37°C in a 7% CO, atmosphere, all cells (pretreated and control) were
washed with 1 ml of Krebs buffer. Cells in control wells were incubated for one
hour at 37°C in 300 pl of Krebs buffer containing 100 pM RO 204724, a

phosphodiesterase inhibitor that is not an adenosine receptor antagonist. Cells in

35
pretreatment wells also were incubated for one hour at 37°C in 300 pl of Krebs

buffer containing RO 20-1724 along with the same concentration of pretreatment
effector used in the previous three hour incubation.

Following the four hour desensitization period described above, all cells
were washed once with 1 ml of Krebs buffer. Specific adenosine receptors were
then stimulated with the appropriate agent, nanomolar concentrations of CHA for
A, stimulation and micromolar concentrations of NECA for A, stimulation, at
37°C. In addition, cholera toxin was used to directly stimulate adenylyl cyclase to
assess the effect of pretreatment on G,. CHA, NECA or cholera toxin were added
in 300 pl of Krebs buffer containing 100 pM RO 20-1724. At the end of a 30
minute stimulation period, all cells were treated with 75 pl of 0.2 N HCl to
terminate cellular processes. Cells in four control wells received 300 pl of 8%
TCA and were subsequently used for protein determination. Culture plates were
then frozen at -80°C to lyse the cells, thawed, and 200 pl of 0.5 M Na,HPO, was
added to each well to neutralize the acidic supernatant. A radioimmunoassay was
performed immediately to determine cellular cAMP content (Frandsen and Krishna
1976). Cyclic AMP values were expressed as pmol cAMP/mg protein. Protein
content was determined by a modified Lawry’s assay (Markwell et al. 1978).

Statistics: Data were analyzed by analysis of variance (ANOVA) for dose
response of desensitization, effects of DPX, DPCPX, and pertussis toxin. In

addition, the effect of a NECA pretreatment on arginine-vasopressin (AVP)

36
response in RCCT cells were also analyzed by AN OVA. If a difference among the

means was indicated, Scheffe’s multiple comparisons test was used to determine
which means were statistically different. The CHA inhibition of basal cAMP
production, time course to desensitization, and cholera toxin pretreatment effects
were analyzed by Student’s t-test. Statistical significance was ascribed for p < 0.05
and all values are expressed as mean :1: SEM. Statistical analysis was performed ,
using Apple MacIntosh software (StatView"" 512+ from BrainPower, Inc.,

Calabasas, CA).

C- 8.6%

Figure 2 illustrates the time course of desensitization for adenosine A,
receptor-stimulated cAMP production. After 30, 60 and 120 minutes of
pretreatment with 10 pM NECA, 67%, 33%, and 0% of control stimulated cAMP
production was observed. N 0 further alteration of the adenylyl cyclase-coupled
adenosine A, receptor system was evident following three and four hours of
pretreatment (data not shown). The T,,, for this desensitization response was 45
minutes.

The four hour desensitization dose response relationship using 10 nM to 10
pM NECA is shown in Figure 3. Pretreatment with 10 nM NECA had no effect
on the subsequent ability of 10 pM NECA to stimulate cAMP production in RCCT

cells. Following 40 nM NECA pretreatment, 72% of control stimulated cAMP was

96 OF CONTROL NECA-STIMULATED
cAMP PRODUCTION

Figure 2.

37

 

 

 

125 -
. —o— BASALcANPPRooucmN
100 - * —o— touMNEGAPRETREATMENT
ANDRESTWIJLATION
75 -
so I-
#
25 - :=—--.
L
o I L j l _l l I l j

 

 

0 20 40 60 80 100 120 140 160 180
PRETREATMENT TIME (min)

Time course of desensitization of adenosine A, receptors in RCCT
cells. Cells were restimulated with 10 pM NECA following
pretreatment for the indicated times with 10 pM NECA. Values are
expressed as the mean 3; SEM of two experiments in triplicate. The
mean basal and stimulated cAMP values without pretreatment were
19.6 i: 5.9 and 75.6 i 23 pmol cAMP/mg protein, respectively.

* p < 0.05 (Student’s t-test; two tailed) indicates a significant
difference from basal. # p < 0.05 indicates a significant difference

from 100%.

Figure 3.

100 q,
80 -- ,

x OF NECA-SITMULATED cAMP PRODUCTION

38

120-,

60 -~

40.

 

 

 

NECA PRETREATMENT CONCENTRATION (Log M)

Effect of four hour NECA pretreatment (10 nM to 10 pM) on
desensitization of adenosine A, receptor stimulated cAMP production
in RCCT cells. Values are expressed as mean 1 SEM of two
experiments in triplicate. * p < 0.05 (ANOVA) indicates a significant
difference from control NECA-stimulated cAMP production.

39
recovered. After 100 nM NECA pretreatment, 20% of control (10 pM NECA)

stimulated cAMP production could be regained. Following pretreatment with 1 pM
NECA, only 12% of 10 pM NECA stimulated cAMP production could be regained,
whereas pretreatment with 10 pM NECA resulted in subsequent 10 pM NECA
stimulated cAMP production below basal. The IC,0 for this response was 80 nM
NECA.

Figure 4 shows the effect of adenosine receptor antagonism on adenosine A,
receptor desensitization. When 10 pM DPX , an adenosine receptor antagonist,
was added concurrently with 10 pM NECA during the pretreatment period, 54%
of control (10 pM NECA) stimulated cAMP production was retained. This is in
contrast to only 11% retention of control-stimulated cAMP production following
adenosine A, receptor desensitization with 10 pM NECA alone, which is not above
basal production. DPX at concentrations lower than 10 pM had no effect on A,
receptor desensitization (data not shown).

When 1 pM DPCPX , a specific A, adenosine receptor antagonist, was
added concurrently with 10 pM NECA, NECA-stimulated cAMP production was
increased above that of non-DPCPX treated cells, yet adenosine A, receptor
desensitization was still complete (Figure 5). Basal cAMP levels in DPCPX treated
RCCT cells were significantly higher than in non-DPCPX treated cells. Figure 5
also shows the effect of 24 hour of pertussis toxin (1 pg/ml) treatment on adenosine

A, receptor desensitization. Although basal cAMP production was increased after

El NOTNEGAPREIREATED
iouMNECAPREmEATED o

i

. l

120-

8

 

at or cournol. uses snuuureo
cAMP Pnooucnou
8
T

 

 

 

 

 

 

     

T

BASAL 10 UM NECA BASAL 10 UM NECA
10 uM DPX PRETREATED

 

Figure 4. Effect of four hour 10 pM NECA pretreatment with and without 10
pM 1,3-diethyl-8-phenylxanthine (DPX) on desensitization of
adenosine A, receptors in RCCT cells. Values are expressed as mean
i SEM of two experiments in triplicate. * p < 0.05 (AN OVA)
indicates a significant difference from basal cAMP production.

cAMP (pmol/mg protoln)

Figure 5.

41

. * c
45° El NOTPRETREATED
400 IOMNEGAPRETREATED l
350 -
3oo- * b
I r:
250 -
b * a
zoo - l
150 -

 

 

 

 

 

 

 

 

 

1 UM DPCPX 1 uglml PERTUSSIS

Effect of four hour 10 pM NECA pretreatment with and without 1
pM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or 24 hour pertussis
toxin treatment (1 pg/ml) on desensitization of adenosine A, receptors
in RCCT cells. Values are expressed as mean 1 SEM of two
experiments in triplicate. * p < 0.05 (AN OVA) indicates a significant
difference from basal cAMP production of the respective treatment
group. Letters are significantly different at a p < 0.05.

42
24 hours of pertussis toxin exposure and 10 pM NECA-stimulated cAMP

production was significantly higher than that of control cells, 10 pM NECA-
induced adenosine A, receptor desensitization was still complete. Pretreatment with
10 pM NECA for four hours did not alter forskolin—stimulated cAMP production
in RCCT cells (Figure 6).

A possible role for G, in adenosine A, receptor desensitization was
investigated by stimulating G, with cholera toxin following preueatment with 10
pM NECA (Figure 7). At both concentrations investigated cholera toxin stimulated
cAMP production, albeit to levels that were significantly less than control.

Four hour pretreatment with CHA, an adenosine A, receptor specific agonist,
at concentrations of 100 nM or less did not diminish adenosine A, receptor
mediated inhibition of basal cAMP production (Figure 8). However, pretreatment
with CHA at concentrations above 100 nM, known to occupy the adenosine A,
receptor, eliminated the ability of CHA (SOnM) to inhibit basal cAMP production.

The effect of adenosine A, receptor desensitization on AVP-stimulated
cAMP production is shown in Figure 9. Following a four hour pretreatment with
10 pM NECA, the response to 1 pM AVP was significantly reduced to 47% of
control AVP-stimulated cAMP production, although NECA pretreated cells could

still significantly increase AVP stimulated cAMP production above basal levels.

43

140 P

E] comm.
12° - IOuMNECA PRETREATMENT

   

cAMP (pmol/mg protein)

 

 

BASAL 100 nM FORSKOLIN
TREATMENT

Figure 6. 10 pM NECA four hour pretreatment of 100 nM forskolin stimulation
of cAMP production in RCCT cells. Values are expressed as mean
1: SEM of two experiments in triplicate. * p < 0.05 (ANOVA)
indicates a significant difference from basal cAMP production.

cAMP (pmol/mg protein)

Figure 7.

El NOTPPErnEATED
totMNECAPRETREATED

 

150'-

100-

 

 

 

 

 

 

 

0.3 uglml 0.5 uglmi
CHOLERA TOXIN CONCENTRATION

Effect of four hour 10 pM NECA pretreatment on cholera toxin
stimulated cAMP production in RCCT cells. Values are reported as
mean 1 SEM of two experiments in triplicate. * p < 0.05 (Student’s
t-test; two tailed) indicates a significant difference from non-pretreated
cholera toxin stimulated cAMP production.

45

96 INHIBITION OF BASAL
cAMP PRODUCTION

 

 

-100 . . . . . a
0 -9 -8 -7 -6 -5

CHA PRETREATMENT CONCENTRATION (Log M)

 

Figure 8. Effect of four hour pretreatment with CHA (1 nM to 100 pM) on
adenosine A, receptor inhibition of basal cAMP production in RCCT
cells. Following pretreatment with CHA, basal production of cAMP
was inhibited with 50 nM CHA. Values are expressed as mean 1
SEM of one experiment in triplicate. The 0 CHA pretreament group
is the mean i SEM of six treatment groups (n-l8). * p < 0.05
(Student’s t-test; one tailed) indicates a significant decrease from basal
cAMP production.

cAMP (pmol/mg protein)

Figure 9.

El NOTPRETREATED .
towNECAPRETREATED

100'-

 

 

 

 

 

 

 

BASAL 1 uM AVP

Effect of four hour 10 pM NECA pretreatment an arginine
vasopressin (AVP) stimulated cAMP production in RCCT cells.
Values are expressed as mean 1 SEM of three experiments in
triplicate. * p < 0.05 (AN OVA) indicates a significant difference
from basal cAMP production. # p < 0.05 indicates a significant
difference from non-pretreated AVP-stimulated cAMP production.

47

D. Discussion

Our laboratory has shown that primary cultures of RCCT cells possess both
inhibitory adenosine A, and stimulatory adenosine A, receptors coupled to adenylyl
cyclase (Arend et al. 1987). The present study sought to determine whether
adenosine receptors in RCCT cells are always fully functional or, if they are
regulated by prior exposure to agonist. The desensitization of hormone-stimulated
adenylyl cyclase systems, such as the B—adrenergic receptor system, has been well
documented (Benovic et al. 1988; Bouvier et al. 1989; Nambi et al. 1985; Sibley
et al. 1986). Moreover, desensitization of adenosine A, receptors in rat kidney
fibroblasts (Newman and Levitzke 1982), vascular smooth muscle (Anand-
Srivastava et al. 1989), vas deferens smooth muscle (Ramkumar et al. 1991) and
Striatum (Hawkins et al. 1988; Porter et al. 1988) have been reported. In all cases,
the desensitization of adenosine A, receptors was reported to be homologous.
Desensitization of adenosine A, receptors in rat adipocytes has also been described
(Green et al. 1990; Hoffman et al. 1986; Parsons and Stiles 1987). The
desensitization of adenosine A, receptor has also been reported in smooth muscle
cells (Ramkumar et al. 1991). The present report, however, is the first to document
concurrent adenosine A, and A, receptor desensitization in primary cultures of
cortical collecting tubule cells.

Desensitization of adenosine A, receptors in RCCT cells grown in culture

was both time and concentration dependent. A significant reduction in 10 pM

48

NECA-stimulated cAMP production occurred within 30 minutes, with complete
desensitization of adenosine A, receptor stimulated cAMP production occurring
within two hours. Adenosine A, receptor desensitization occurred only when
pretreatment concentrations of NECA approached micromolar concentrations.
These results are consistent with the hypothesis that adenosine A, receptor
occupation and activation are necessary to initiate desensitization. These results
also suggest that adenosine A, receptor occupation and activation, which
presumably occurs at lower concentrations of NECA, does not produce adenosine
A, receptor system desensitization within the four hour exposure time frame.

To further probe the involvement of adenosine A, receptors in adenosine A,
receptor desensitization, the specific adenosine A, receptor antagonist DPCPX was
administered during adenosine A, receptor desensitization. When 1 pM DPCPX
was present, adenosine A, receptor desensitization remained intact. This finding
is consistent with the dose dependency of adenosine A, receptor desensitization, in
that agonist occupation/activation of the adenosine A, receptor is not necessary for
desensitization. Moreover, addition of DPCPX significantly raised basal cAMP
production suggesting the removal of a tonic inhibitory input by adenosine A,
receptors to adenylyl cyclase.

Pertussis toxin, which inactivates the G, subfamily of G-proteins, did not
prevent adenosine A, receptor desensitization, suggesting that G, is not primarily

involved. However, cells treated with pertussis toxin had significantly higher

49
NECA-stimulated cAMP production than non-pertussis toxin treated cells.

Pertussis toxin also raised basal cAMP production, which again suggests the
removal of a tonic inhibition on adenylyl cyclase. Not surprisingly, the DPCPX
and pertussis toxin results appear to be qualitatively similar. Both interventions
appear to remove a tonic inhibitory influence on the adenylyl cyclase system, but
do not alter adenosine A, receptor desensitization. The blockade of adenosine A,
receptors with DPCPX allowed for a significant increase in 10 pM NECA-
stimulated cAMP production over control. Pertussis toxin inactivation of G,
resulted in an even larger significant increase of control 10 pM NECA-stimulated
cAMP production. These two observations suggest that the adenosine A, receptor
system is only one of many that may be involved in the tonic inhibition of adenylyl
cyclase in RCCT cells. Desensitization of adenosine A, receptors did not
significantly alter the ability of forskolin to stimulate cAMP production. Together,
the forskolin and pertussis toxin experiments suggest that neither adenylyl cyclase
nor G, are directly involved in adenosine A, receptor desensitization. Because
neither DPCPX nor pertussis toxin administration significantly altered the pattern
of adenosine A, receptor desensitization, it would appear that the mechanism
responsible for this phenomenon resides within the stimulatory pathway of the
adenosine receptor-adenylyl cyclase system.

Ten micromolar DPX, an adenosine A, and adenosine A, receptor antagonist

at this concentration, presented concurrently with 10 pM NECA pretreatment

50

significantly attenuated adenosine A, receptor desensitization. With 10 pM DPX
present, 54% of control NECA-stimulated cAMP production was regained as
opposed to only 11% without DPX present. Basal cAMP production was 11% of
control 10 pM NECA stimulated cAMP production. Pretreatment with DPX alone
at concentrations higher than 10 pM produced adenosine A, receptor desensitization
(data not shown). The desensitization of adenosine A, receptors by DPX at
concentrations higher than 10 pM may be due to inhibition of phosphodiesterases
and, therefore, increased cellular cAMP levels. Alternatively, DPX may act as a
partial adenosine A, receptor agonist at concentrations higher than 10 pM.
Nevertheless, agonist occupation/activation of adenosine A, receptors appears to be
necessary for adenosine A, receptor desensitization.

Following pretreatment of RCCT cells with 10 pM NECA, cholera toxin, a
potent activator of G,, retained its ability to stimulate cAMP production, albeit, to
levels that were significantly attenuated as compared to non-pretreated cells.
Cholera toxin at concentrations of 0.3 pg/ml and 0.5 pg/ml stimulated cAMP
production to 37% and 62%, respectively, of those levels achieved in the absence
of NECA pretreatment. A reduction in functional G, for cholera toxin induced
ADP-ribosylation may explain the attenuation in cAMP production following
pretreatment with 10 pM NECA. Indeed, Edwards et al. (1987) observed decreased
ADP-ribosylation of the or-subunit of G,-protein following prolonged exposure of

platelets to the prostacyclin analog iloprost.

51

The effect of pretreatment with CHA on adenosine A, receptor
responsiveness in RCCT cells also was investigated. While desensitization of the
adenosine A, receptor system’s ability to inhibit basal cAMP production was
observed, adenosine A, receptor desensitization did nOt occur until pretreatment
concentrations of CHA reached concentrations (1 pM) known to occupy/activate
the A, adenosine receptor. This provides further support for the hypothesis that
adenosine A, receptor occupation/activation is needed to initiate desensitization of
both adenosine A, and adenosine A, receptors in RCCT cells.

The ability of AVP to increase cAMP production was attenuated following
desensitization of the adenosine receptor system with 10 pM NECA. In this
experiment, 1 pM AVP-stimulated cAMP production was 47% of control.
However, pretreatment of RCCT cells with NECA at nanomolar concentrations,
which activates only adenosine A, receptors, did not alter adenylyl cyclase
responsiveness to 1 pM AVP. This is not surprising because adenosine A, receptor
stimulation by CHA at these concentrations did not desensitize the adenosine
receptor system. Taken together, these results suggest that adenosine A, receptor-
induced heterologous desensitization of AVP responsiveness in RCCT cells had
occurred. .

In summary, it appears that neither adenylyl cyclase nor G, are directly

involved in adenosine receptor-coupled adenylyl cyclase system desensitization in

RCCT cells. Desensitization of this system appears to require. agonist

52

occupation/activation of the adenosine A, receptor subclass and results in a reduced
availability of functional G,. The desensitization of adenosine receptor-coupled
adenylyl cyclase is additionally characterized by a reduced responsiveness to AVP.
This reduced responsiveness of AVP receptor-coupled adenylyl cyclase following
adenosine receptor desensitization may, in part, be due to altered function of G,
and constitutes a heterologous desensitization. Desensitization of this system is
further characterized by the inability of adenosine A, receptor stimulation to inhibit
basal cAMP production following prolonged occupation/activation of adenosine A,
receptors. Because pertussis toxin inactivation of G, does not affect the pattern of
adenosine A, receptor desensitization, and adenylyl cyclase appears not to be
affected by desensitization, it seems reasonable to speculate that altered function
of the adenosine A, receptor has occurred.

In all of the experiments where adenosine receptor desensitization occurred,
the stimulatory adenosine A, receptor-coupled adenylyl cyclase system was
occupied/activated. Prolonged occupation/activation of the adenosine A, receptor
system lead to the reduced ability of cholera toxin to stimulate the regulatory G,
protein. Moreover, the heterologous desensitization of AVP receptor-coupled
adenylyl cyclase is supportive of altered function of G,. In conclusion,
desensitization of adenosine receptor-coupled adenylyl cyclase in RCCT cells is
characterized by alterations in the function of adenosine A, and adenosine A,

receptors and the regulatory protein G,.

IV. The Functional Localization of Adenosine Receptor Mediated Pathways

in the LLC-PK, Renal Cell Line

A. Introduction

The purine nucleoside, adenosine, is known to regulate a large number of
physiological functions including A the regulation of neurotransmitter release,
vasodilation, and inhibition of lipolysis in adipose tissue (Daly et al. 1981; Londos
et al. 1978). In the kidney, adenosine is known to alter renal function through
changes in hemodynamics, renin release and tubule function (Dillingham and
' Anderson 1985; Spielman 1984; Spielman et al. 1980; Spielman and Thompson
1982). . .

These physiological effects of adenosine are thought to be mediated
primarily by its interaction with extracellular adenOsine receptors. Adenosine
receptors are coupled to adenylyl cyclase through the guanine nucleotide regulatory
proteins G, and G,.(Arend et al. 1987; Londos et al. 1978; Londos et al. 1980)
These receptors are divided into two subclasses based on the following criteria: 1)
ligand/receptor pharmacology, 2) coupling to G-proteins, 3) second messenger
systems, and 4) structures deduced from molecular cloning (Libert et al. 1991;
Londos et al. 1978; Londos et al. 1980). The adenosine A, receptor is known to
act through an inhibitory G-protein (G,) to inhibit adenylyl cyclase activity with the

following adenosine analog potency profile: CHA > PIA > NECA. The adenosine

53

54

A, receptor has a high affinity for adenosine analogs and is stimulated at
nanomolar concentrations. The adenosine A, receptor acts through a stimulatory
G-protein (G,) to stimulate adenylyl cyclase activity. This subclass of adenosine
receptor has an adenosine analog potency profile of NECA > PIA > CHA, and is
of lower affinity, responding to micromolar concentrations. A third adenosine
receptor mediated pathway found in renal epithelial cells stimulates inositol
phosphate turnover with a subsequent release of calcium from intracellular stores
(Arend et al. 1988; Arend et al. 1989; Weinberg et al. 1989). This pathway is
blocked by the specific adenosine A, receptor antagonist DPCPX and is pertussis
toxin sensitive. However, unlike the adenosine A, and A, receptors, this receptor
mediated pathway has an adenosine analog potency profile of CHA = PIA =
NECA. Therefore, this receptor mediated pathway has been suggested to be a
subtype of the adenosine A, receptor (Arend et al. 1988; Arend et al. 1989).
Transporting epithelial cells separated by tight junctions are known to grow
in culture as a polarized monolayer, displaying both an apical and a basolateral side
(Pfaller et al. 1990; Rodriguez-Bolan and Nelson 1989). These epithelia express
transport proteins such as Na+ * ATPase at their cell surface in a polarized
manner. This polarity is the basis for the vectoral transport of solutes and water.
Recently, studies have focused on the spatial localization of receptors that regulate
transport in several types of epithelial cells. For example, the vasoactive intestinal

peptide (VIP) receptor has been localized to the basolateral side of the colonic cell

55
line HT29-D4 (Fantini et al. 1988). In addition, the majority of adenosine

receptors responsible for changes in short circuit current in the T8, colonic cell line
were also found to be basolaterally located (Barrett et al. 1989). However, in
canine tracheal epithelium, adenosine applied to the mucosal surface was found to
stimulate chloride secretion through an apically located adenosine receptor (Pratt
et al. 1986).

Renal tubule cells are exposed to two spatially separate fluid compartments;
the interstitial fluid (basolateral) compartment, and the tubular lumen or urine
compartment (apical). Adenosine in these two extracellular compartments
represents separate pools that may act differentially to regulate tubular epithelial
transport via spatially distinct receptors. Therefore, the purpose of these studies
was to localize the adenosine receptor mediated pathways in the LLC-PK, renal

cell line.

B- M

Cell Culture: LLC-PK, cells (passage number 194) were obtained from
American Type Culture Collection, Rockville Maryland. Cell culture reagents were
obtained from Gibco BRL, Grand Island NY. The cells were cultured in a media
consisting of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum (FBS), 2 mM glutamine and 50 pg/ml each of penicillin and

streptomycin at 37°C in a 7% CO, atmosphere. Cells were routinely passaged

56
every 7 days.

Gelatin Substrate Coating of Culture Inserts: Cell culture inserts (12 mm
diameter, Millicell'm-CM; obtained from Millipore Corp. Bedford MA.) were coated
with gelatin (Millipore technical brief: Lit. No. TB017 copyright 1989) as an
extracellular matrix upon which LLC-PK, cells adhere and grow. Using aseptic
techniques in a laminar flow hood, inserts were placed in a sterile 24 well plate and -
wetted with 0.5 ml of phosphate buffered saline (PBS mM; 138 NaCl, 3 mM KCI,
8.1 mM Na,I-IPO,, 1.5 mM KH,PO,). A solution consisting of 5% w/v gelatin and
5% w/v sucrose in dH,O was made. The resulting slurry was heated in a boiling
water bath to allow the gelatin and sucrose to go into solution. The PBS was
aspirated and the inserts were coated with 200 pl of warm gelatin solution. If the
inserts were not pre-wetted with PBS, the gelatin solution would form as a droplet
and not coat the insert evenly. The gelatin solution was immediately aspirated and
the remaining gelatin film was allowed to air dry on the culture inserts in a laminar
flow hood. After drying, the gelatin film was rehydrated with 0.5 ml PBS per
insert for 10 minutes. The PBS was aspirated and the gelatin film was fixed with
0.5 ml of 1% glutaraldehyde in PBS for 30 minutes. This fixation procedure also
had the advantage of sterilizing the gelatin film. For reasons that are unclear, LLC-
PK, cells do not grow well on unfixed gelatin. Inserts were washed twice with 0.5
ml of PBS to remove the glutaraldehyde and the fixed gelatin film was treated with

0.5 ml of 0.1 M glycine in PBS for 30 minutes to quench reactive groups

57
introduced by the glutaraldehyde. Inserts then were washed twice with 0.5 ml PBS

and placed in a sterile 24 well plate containing 0.5 ml of culture media per well.
This media served as the basolateral culture media for the cells. Following two
washes with 5 ml of PBS, a confluent monlayer of LLC-PK, cells was removed
from a 100 mm culture plate by a 3 minute incubation at 37°C with 4 ml of 0.2%
trypsin, 0.05% (Ethylenedinitrilo)tetraacetic acid (EDTA) in PBS. The resulting
suspension of cells was sedimented by centrifugation (500 x g) for five minutes.
The supernatant was discarded and the cells were diluted 1:20 (~105 cells/ml) with
culture media. Three hundred microliters (300 pl) of the cell suspension was then
transfered to the inside of each insert. Epithelial cells seeded in this manner will
orient apical side up and basolateral side down on the gelatin substrate in the insert
(Figure 10). Cells were allowed to grow to confluency for 5 to 7 days before each
experiment.

Permeability of a LLC-PK Cell Monolayer tol‘C-Spcrose: Once confluent,
the permeability of the LLC-PK, cell monolayer to l"C-sucrose was assessed.
Inserts with and without cells were carefully washed with Krebs buffer and then
placed in a new 24 well plate containing 0.5 ml of Krebs buffer (basolateral side).
Three hundred microliters (300 pl) of Krebs buffer containing 1 pM l‘C-sucrose
was added to the inside (apical side) of the inserts. The 24 well plate was then
placed on an orbital shaker at 37°C and shaken gently. At timed intervals (0 to 60

minutes at ten minute intervals), 50 pl aliquots were removed from buffer

58

cell monolayer

 

 

 

 

 

 

 

Figure 10. Diagram of LLC—PK, cells grown on a Millicellm-CM insert.

59

surrounding the inserts (basolateral side) and placed in glass scintillation vials
containing 5 ml of scintillation cocktail. The volume that was removed from the
outside of the inserts was immediately replaced with 50 pl of Krebs buffer to
maintain the outside volume at 0.5 ml. The radioactivity in these samples was
counted for three minutes by a LKB 1209 Rackbeta beta counter. The
concentration of l’C-sucrose that passed through the inserts was calculated and
expressed as percent of the equilibrium concentration, the concentration that would
result from free mixing of the apical and basolateral volumes. The converse
experiment of measuring the flux of 1’C-sucrose from outside (basolateral) the
insert to inside (apical) was performed as described above.

Measurement of the mtential difference and electrical resistance of a LLC-
Emell monolaypg: Once confluent, the potential difference and electrical
resistance of the LLC-PK, cell monolayer was measured to assess the monolayer’s
integrity. All potential difference and electrical resistance measurements were
made immediately before any experimental procedure was undertaken. For this
purpose, an Epithelial Voltohmmeter (EVOM; World Precision Instruments, Inc.,
New Haven CT.) was used. In a laminar flow hood, EVOM electrodes were
sterilized in 95% ethanol and rinsed with culture media. Each of the electrodes
contains a pellet of Ag/AgCl for the measurement of voltage and silver wires for
the passage of current to measure electrical resistance. The potential difference and

electrical resistance of each LLC-PK, monolayer was measured by placing the

60
anode in the media on the inside of the insert (apical) and the cathode in the media

on the outside of the insert (basolateral). Because the Millicell-CM inserts have
a surface area of 1.13 cmz, electrical resistances were multiplied by 1.13 to express
resistance as ohm x cmz. Typical potential differences ranged from 1.0 to 2.0 mV
(apical side negative), while the electrical resistances were approximately 250 ohm

x cmz.

Adenosine A, and A, Receptor cAMP Pathway Localization: Following 5

to 7 days of culture on Millicell‘m-CM filter inserts, LLC-PK, cells were exposed
to adenosine (30 nM or 100 pM) in an attempt to study the sidedness of inhibitory
adenosine A, and stimulatory A, receptor mediated pathways respectively. For the
localization of adenosine A, receptors, 100 pM adenosine was used to stimulate
cAMP production in LLC-PK, cells. Inserts were washed with Krebs buffer and
incubated for 30 minutes at 37°C in Krebs buffer (300 pl apical, 500 pl basolateral)
containing 100 pM RO 20-1724, a phosphodiesterase inhibitor that is not an
adenosine receptor antagonist. This buffer was aspirated from the inserts and
specific cell surfaces were stimulated with 100 pM adenosine in Krebs buffer
containing 100 pM RO 201724 for an additional 30 minutes at 37°C. Krebs
buffer containing 100 pM R0 20-1724 and 1.5 U/ml adenosine deaminase (ADA)
was placed on the contralateral side to deaminate any adenosine that might cross
the monolayer. The stimulation of adenylyl cyclase by A, adenosine receptors was

terminated by adding 75 pl of 0.2 N HCl to the inside (apical) of each insert. The

61

inserts along with the upper volume of buffer were then frozen at -80°C.

The sidedness experiments for the inhibitory adenosine A, receptor were
performed essentially as described above for the adenosine A, receptor, except that
30 nM adenosine was used to inhibit forskolin-stimulated cAMP production.
Forskolin was used to directly stimulate adenylyl cyclase, because inhibition of
stimulated cAMP levels can be more clearly resolved than inhibition of basal
cAMP levels. Confluent LLC-PK, cells on inserts were washed with Krebs buffer
and incubated with Krebs buffer containing RO 201724 for 30 minutes at 37°C
as described above. The specific cell surfaces (apical. or basolateral) were then
stimulated with Krebs buffer containing RO 20-1724 and 1 pM forskolin with or
without 30 nM adenosine for 30 minutes at 37°C. Forskolin was present on both
sides of the monolayer. Krebs buffer containing RO 20-1724 and 1.5 U/ml ADA
was placed on the contralateral side. The inhibition of forskolin stimulated cAMP
was terminated by adding 75 pl of 0.2 N HCl to the inside of the insert. The
inserts were frozen along with the inner volume of buffer at -80°C.

cAMP Determination: Samples from sidedness experiments were thawed
at room temperature and neutralized by adding 200 pl of 0.5 M Na,HPO,. The
cAMP levels in the supernatant of each sample were measured via
radioimmunoassay as described by Frandsen and Krishna (1976). Cyclic AMP
levels were expressed per mass of cell protein as measured by a modified Lowry’s

assay (Markwell et al. 1978).

62

Adenosine Sti_m_u_l_a_ltion of Intracellul_ar Calcium Release: Adenosine
stimulation of free intracellular calcium [Ca‘z], release by LLC-PK, cells in
suspension was performed as described by Grynkiewicz et al. (1985). A confluent
monolayer of cells was removed from a culture dish with a 3 minute incubation
with 0.2% trypsin and 0.05% EDTA in PBS at 37°C. The cells were centrifuged
(500 x g) for five minutes and the cellular pellet resuspended in 300 pl of
simplified saline (SSS mM; 145 NaCl, 5 KCl, 1 Na,I-IPO,, 1 CaCl,, 0.5 MgCl,, 5
glucose, 10 HEPES, pH 7.4). The cells were loaded with the fluorescent calcium
chelator Fura—ZAM, at a concentration of 10 pM with 0.02% pluronic acid in SSS
for 30 minutes at 37°C in a shaking water bath. The pluronic acid was utilized to
aid in the loading of Fara-2AM into the cells. After loading, the cells were washed
twice with 10 ml SSS and resuspended in 400 pl of SSS. Samples of this cell
suspension used for calcium measurements were diluted 1:100 into a 1.5 ml volume
of SSS in a quartz cuvette. The cuvette was therrnostated at 37°C on a magnetic
stirrer in a dual excitation wavelength spectrofluorometer (SPEX Industries, Inc.,
Metuchen, NJ). The cell suspension in the cuvette was continuously stirred and
I effectors were added at a 1:100 dilution to their final concentrations. Cells were
excited at 340 nm and 380 nm, while emmision was monitored at 505 nm. The
ratio of fluorescence (R) emitted by exciting at 340 nm/380 nm was used to

calculate [Ca‘q], as described by Grynkiewicz et al. (1985):

63
[C3+2]i = Kd X (R'Ro/Rs-R) X (Fo/Fs)380

The effective K, of Fura-ZAM for calcium, 224 nM, as determined by Grynkiewicz
et al. (1985) was used. R is the experimental fluorescence ratio, R,, is the
fluorescence ratio at zero free calcium and R, is the fluorescence ratio at saturating
free calcium. F0 is the fluorescence emitted by 380 nm excitation at zero free
calcium and F, the fluorescence at saturating free calcium. Digitonin (50 pM) was
used to measure the fluorescence to saturating concentrations of calcium, while 5
mM Ethylene glycol-bis(B«aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA)
was used to measure the fluorescence at zero free calcium.

Menosine Receptor Mediated Inositol Phosphate Pathway Localization: A
confluent 100 mm plate of LLC-PK, cells was incubated for 6 hours in 5 ml of
Medium 199 supplemented with 2 mM glutamine, 1 pM dexamethasone, 5 pM
tliiodothyronine, and 5 nM insulin with 6 pCi/ml of myo-[2-3H(N)]inositol. This
incubation was utilized to load the phosphotidylinositol pools with myo-[2-
3H(N)]inositol. Cells were removed from the plate by a 3 minute incubation with
0.2% trypsin, 0.05% EDTA in PBS. The cells were washed from the plate with
5 ml PBS and centrifuged at 500 x g for 5 minutes. The resulting cellular pellet
was resuspended in 10 ml of lithium buffer (mM; 58 NaCl, 60 LiCl, 4.7 KCl, 1.5
CaCl,, 1.2 MgSO,, 10 glucose, and 20 I-IEPES. pH 7.4), and incubated for 20

minutes at 37°C in a water bath with constant shaking. Following two washes in

64
10 ml of lithium buffer, the cells were resuspended in 15 ml of lithium buffer and

400 pl aliquots were added to microcentrifuge tubes. Hormones (adenosine or
AVP) at 5x their final concentration were added to the microcentrifuge tubes in 100
pl aliquots (500 pl final volume). The stimulation of inositol phosphates was
allowed to occur for 10 minutes at 37°C, followed by termination of the reaction
with 500 pl of 10% trichloroacetic acid (TCA). The reaction mixture was then
frozen at -80°C. After thawing at room temperature, individual reaction mixtures
were centrifuged and the supernatant applied to anion exchange columns (Bio-Rad,
AG- 1X8, 100- to ZOO-mesh formate form). The inositol phosphates were then
separated as described by Berridge et. al. (1983). Following three washes with 3
ml distilled water and two washes with 3 ml 50 mM ammonium formate plus 5
mM sodium borate, increasing concentrations of ammonium formate (200 mM, 400
mM, 1 M) containing 100 mM formic acid (2x 1 ml) were used to elute the
inositol phosphates; 200 mM ammonium formate for IP,, 400 mM ammonium
formate for IP,, and 1 M ammonium formate for IP,,. The lipids were extracted
from the TCA induced‘precipitate with methanol and chloroform. The radioactivity
in the inositol phosphate pools were normalized to the radioactivty in the lipid
fraction and expressed as percent of control (unstimulated cells).

Experiments for the sidedness of the inositol phosphate response were
carried out as described above with slight modifications. Inserts were placed in

supplemented Medium 199 containing 6 pCi/ml of myo-[2-3H(N)]inositol (300 pl

65

apical, 500 pl basolateral) for 6 hours. The inserts were carefully washed once
with lithium buffer and incubated in lithium buffer at 37°C for 20 minutes and
again washed carefully with lithium buffer. Lithium buffer containing hormone (10
pM adenosine or 1 pM AVP) was then applied to specific cell surfaces (apical or
basolateral). Adenosine deaminase (1.5 U/ml) in lithium buffer was introduced on
the contralateral side as described previously. At the end of the 10 minute
stimulation period, 300 pl of 10% TCA was introduced to the inside of each insert.
The inserts were removed from the 24 well plate and the monolayers along with
the upper (apical) volume of buffer were frozen at -80°C. After thawing the cells
and supernatant, the supernatant was run over an anion exchange column as
described above. Filters with the lysed cells attached were cut from their supports
and the lipids were extracted with methanol and chloroform. Again the
radioactivity of the inositol phosphate fractions were normalized to the radioactivity
in the lipid fraction. The level of inositol phosphate was expressed as percent of
control.

Statistics: Data were analyzed by analysis of variance (ANOVA) for
sidedness effects, time course effects, and agonist concentration effects. If a
difference among the means was indicated, Scheffe’s multiple comparisons test was
used to determine which means were statistically different. Analyses of inositol
phosphate data (sidedness) was performed by Student’s t-test. Statistical

significance was ascribed for p < 0.05 and all values are expressed as~ mean i

66

SEM. Statistics were performed using Apple MacIntosh software (StatView‘m

512+, BrainPower, Inc., Calabasas, CA).

G mt;

The permeability of gelatin-coated Millicell'm-CM filter inserts to l‘C-sucrose
was determined (Figure 11). In the absence of a LLC-PK, cell monolayer, l‘C-
sucrose placed in either the upper or lower chamber reached steady state with the
contralateral chamber within 30 minutes. Approximately 11% of the total
radioactivity added at the beginning of the experiment was removed in the course
of measuring l"C-sucrose flux across an insert without cells. However, in the
presence of a confluent monolayer of LLC-PK, cells l‘C-sucrose reached less than
10% of the steady state concentration on the contralateral side at the end of a 60
minute time course.

Adenosine (10 pM) was found to elevate intracellular calcium in LLC-PK,
cells in a dose dependent manner (Figure 12) as previously reported by Weinberg
et. al. (1989). The response consisted of two phases. The increase in calcium in
the initial phase was 100% over baseline (94 i 9.3 nM), followed by a sustained
phase with calcium elevated by 55% over basal when cells in suspension were
exposed to 10 pM adenosine. Additionally, 1 pM AVP stimulated calcium release
to 200% over baseline. Chelating extracellular calcium with 5 mM EGTA did not

prevent the adenosine-induced peak increase in intracellular calcium but did abolish

67

 

 

 

 

 

 

100 -
'—'O'— W
5 ' —o— LLC-PKCELLS
E so -
r:
E
Ill
‘2"
o 60 "
o
2
2
E
2 40 ’-
a
a
III
II.
O 20 .
a!
r ‘ _ ;_ 3’ %
0 VT? 1 r r r J
0 10 20 30 40 50 60
TIME (min)

Figure 11. Permeability of LLC-PK, cells grown on gelatin coated Millicellm-
CM inserts to 1 pM l‘C-sucrose. Inserts with (open circles) or
without (closed circles) a confluent monolayer of LLC-PK, cells had
1 pM l‘C-sucrose placed on the outside (basolateral) of the insert and
50 pl aliqout samples were taken at the indicated time points from the
inside (apical) of the inserts. Representative graph of two experiments
in duplicate.

68

300 — CAM .
:3: Wm i le

o — I g I
50 pM digitonin

tOpMadenosine 1pMAVP I

 

 

 

SmMEGTA

I I l l I l I I I l l -l J
0 50 100 150 200 250 300

TIME (see)

Figure 12. Spectrofluorometer generated tracing of intracellular free calcium
concentration in LLC-PK, cells in response to adenosine and AVP.
The ordinate is expressed as intracellular free calcium concentration
in nM.

 

 

69

the sustained phase (Figure 13), thereby suggesting that calcium is released from
an intracellular store. Moreover, Weinberg et al. (1989) have shown that
mobilization of calcium in LLC-PK, cells by adenosine is blocked by the adenosine
receptor antagonist, DPCPX, and is pertussis toxin sensitive. Because inositol
phosphate stimulation is known to increase the cytosolic free calcium concentration,
it may be that the adenosine-induced calcium release in LLC—PK, cells is mediated
by inositol triphosphate stimulation. A time course for the stimulation of inositol
phosphates was determined for LLC-PK, cells in suspension (Figure 14). Inositol
phosphate levels reached peak values at 10 minutes following stimulation by 10 pM
adenosine and returned to the same level as the control by 20 minutes. All
remaining inositol phosphate measurements were made using a 10 minute time
course. DPCPX blockade of adenosine stimulated inositol phosphate turnover in
cells in suspension is shown in Figure 15. Adenosine (10 pM) stimulated inositol
phosphate production in a dose dependent manner. However, 1 pM DPCPX
blocked adenosine stimulated inositol phoshpate production. Figure 16 shows
results of inositol phosphate stimulation by 10 pM adenosine applied to either the
apical or basolateral side of cells grown on Millicell'm-CM inserts. None of the
inositol phosphate species was increased above control when cells were exposed
to adenosine on the apical side. However, all three inositol phosphate species were
significantly elevated by 10 pM adenosine applied to the basolateral side of the

monolayer.

70

 

3 mM CaCl
5 mM EGTA
100 uM ADENOSINE
200

E 160 ‘
to . ' .

I I l I I ' I

0 30 60 l 90 120 150

TIME (see)

Figure 13. Spectrofluorometer generated tracing of intracellular free calcium
’ concentration stimulated by adenosine in LLC-PK, cells following
chelation of extracellular calcium with EGTA.

% STIMULATION OVER BASAL

Figure 14.

71

180 P

160

140

120

 

 

100

 

 

80 I I I I
0 5 10 15 20

TIME (min)

 

Adenosine stimulation of inositol phosphates in LLC-PK, cells in
suspension. LLC-PK, cells were incubated in the presence of 10 pM
adenosine for the indicated times. Inositol phosphates were collected
and fractionated as described in "Methods". Values are expressed as
mean i SEM of three experiments in triplicate. * p < 0.05 (ANOVA)
indicates a significant difference from one minute time point.

96 OF BASAL INOSITOL PHOSPHATE

Figure 15.

72

 

130 r-

' —.— mm
125 - “—0— ADO+DPCPX

e

120 l-

i' e
115 l-
110 '-
105 '-

r I I
100 ---<- .............

I /

95 P

L
m —J—/ I I I I

 

 

/
BASAL 10-6 3X10-6 10-5 3X10-5 10-4
ADENOSINE CONCENTRATION (Log M)

Adenosine dose response stimulation of inositol phosphate (1P1) in
LLC-PK, cells with and without 1 pM DPCPX. LLC—PK, cells were
incubated with increasing concentrations of adenosine in the presence
or absence of 1 pM DPCPX for ten minutes. Inositol phosphate (IPl)
was collected as described in "Methods". Values are expressed as
mean -_I-_ SEM of three experiments in triplicate. * p < 0.05 (AN OVA)
indicates a significant difference from 1 pM adenosine stimulated
cAMP production.

.73

           
     

 

 

 

 

 

 

 

 

180 a
. El lP1 ,
t
E 192
g 160 - a ":3
Q
In
2 ‘
at * yep;
J 140 "' E; \:\:\’\
O \’\’\:V
5 . :I:I‘I‘
‘ I [‘1‘
;: \ s s \
g \:\:\:\
2: 12° “ ‘1:
m ‘I\I\I:
( 1 I I I
° 351113:
u, \’\’\’\
° ‘°°‘ 33:32:
i ‘I‘I‘I‘
r §§§ :Izlti:
\I\I\I~
1: I‘I‘Q
so _ L A ‘
BASOLATERAL
TREATMENT
Figure 16. Apical versus basolateral 10 pM adenosine stimulation of inositol

phosphates in LLC-PK, cells grown on Millicell'm-CM inserts. LLC-
PK, cells were exposed to 10 pM adenosine on either the apical or
basolateral side for ten minutes. Inositol phosphates were collected
and fractionated as described in "Methods". Values are expressed as
mean i SEM of three experiments in triplicate. * p < 0.05 (Student’s
t-test; two tailed) indicates a significant difference from the
corresponding apical exposure.

74

LLC-PK, cells were stimulated with 1 pM forskolin and concurrent 30 nM
adenosine was applied to either the apical or basolateral side of the monolayer
(Figure 17). Forskolin stimulated cAMP levels were unchanged by apically applied
adenosine (30 nM). Cells exposed to 30 nM adenosine on the basolateral side
exhibited a significant 23% inhibition of forskolin stimulated cAMP production.

The results of adenosine A, receptor localization in LLC-PK, cells
stimulated with 100 pM adenosine on the apical, basolateral or apical and
basolateral sides are shown in Figure 18. Stimulation of either the apical (129 :l:
14 pmol cAMP/mg protein) or basolateral side (58 i 6 pmol cAMP/mg protein)
evoked significant increases in cAMP levels over baseline (13.5 i 1.8 pmol
cAMP/mg protein). Stimulation from the apical side elicited significantly higher
levels of cAMP than stimulation from the basolateral side. In addition, concurrent
stimulation of apical and basolateral sides produced cAMP levels (189 i 15 pmol
cAMP/mg protein) that were the numerical sum of each of the sides stimulated
independently.

The time course of 100 pM adenosine stimulation of cAMP production by
apical or basolateral adenosine A, receptors is shown in Figure 19. Both apical and
basolateral stimulation of cAMP production reached a plateau at 30 minutes. The
absolute level of cAMP produced by apical and basolateral stimulation was
A significantly different at all times (Figure 19A). However, when cAMP levels were

expressed as percent of cAMP present at 30 minutes, there was no difference

cAMP (pmol/mg protein)

Figure 17.

75

400 '-

 

 

FOFBKOLN APICAL BASOLATERAL
TR EATME NT

Apical versus basolateral 30 nM adenosine inhibition of 1 pM
forskolin stimulated cAMP production in LLC—PK, cells grown on
Millicell'm-CM inserts. LLC-PK, cells were Heated with 1 pM
forskolin i 30 nM adenosine on either the apcial or basolateral side
for 30 minutes. Cyclic AMP was measured as described in
"Methods". Basal cAMP production was 7.0 i 0.4 pmol/mg protein.
Values are expressed as mean :t SEM of three experiments in
triplicate. * p < 0.05 (ANOVA) indicates a significant difference
from forskolin stimulated cAMP levels.

200-

1

1

1

1

cAMP (pmol/mg protein)

Figure 18.

76

75-
50-1
25-

mat

 

 

BASAL APICAL BASOLATERAL API + BASO
TREATMENT

Apical versus basolateral 100 pM adenosine stimulation of cAMP in
LLC-PK, cells grown on Millicell'm-CM inserts. LLC-PK, cells were
stimulated with 100 pM adenosine on either the apical, basolateral or
both sides for 30 minutes. Cyclic AMP was measured as described
in "Methods". Values are expressed as mean i SEM of four
experiments in duplicate. * p < 0.05 (ANOVA) indicates a significant
difference of each Heatrnent from the others.

Figure 19.

77

Apical versus basolateral time course to 100 pM adenosine
stimulation of cAMP in LLC-PK, cells grown on Millicell'm-CM
inserts. LLC-PK, cells were stimulated with 100 pM adenosine on
either the apical or basolateral side for the indicated times.

A: Absolute cAMP production. B: Relative cAMP production.
Representative graph of two experiments in duplicate."

78

200 i-

—O— APICAL

—o—- BASOLATERAL

 

 

 

20 30

TIME (min)

10

 

m

.539... uE=oE& a2<e

. —e— APICAL

 

 

 

mmSz... on ._.<
ZOF(r_DE:OO( a3<o “—0 i.

TIME (min)

79
between the responsiveness of the apical and basolateral sides (Figure 19B). A 30

minute stimulation with supramaximal doses (100, 300 and 1000 pM) of adenosine
showed a marked difference in the absolute levels of cAMP produced via apical
and basolateral stimulation (Table 2). Neither 100 nM DPCPX nor 24 hour
pretreatment with 0.1 pg/ml of pertussis toxin altered the pattern of responses

elicited by adenosine A, receptor stimulation in LLC-PK, cells (data not shown).

D. Discussion

Transporting epithelia display a polarity consisting of an apical and a
basolateral side separated by tight junctions (Pfaller et al. 1990). The integral
membrane proteins involved in the transport of solutes and water are expressed
asymmetrically in these epithelia (Rodriguez-Bolan and Nelson 1989). This
asymmetric distribution of transport proteins is the basis for the vectoral Hansport
of solutes and water. There is increasing evidence that the receptor systems
regulating Hansport are also distributed in a polarized manner (Barrett et al. 1989;
Fantini et al. 1988; Pratt et al. 1986). While there is growing data on the actions
of adenosine receptor systems in renal and other Hansporting epithelia, very little
is known about the spatial location of adenosine receptors on these epithelia.

The permeability of a confluent monolayer of LLC-PK, cells was assessed
using l"C-sucrose (1 pM). While l‘C-sucrose freely crossed a gelatin Heated filter

insert without cells and reached steady state within 30 rrrinutes, it did not cross a

80

Table 2. Apical versus basolateral supramaximal adenosine stimulation of
cAMP production in LLC-PK, cells.

Adenosine Apical Basolateral
ConcenHation
(Log M) (pmol/mg promin)
10" 104 :13 51.8 i 4.7'
3x10" 130 i 15 54.2 i 6.4‘
10'3 141 j; 17 57.2 i 7.4'

The mean i SEM of cAMP levels of two experiments in duplicate.
* Significantly different from apical cAMP levels at a p < 0.05.

81

confluent monolayer of cells to any significant degree (< 10% of the equilibrium
concentration of 370 nM). Sucrose is thought to be neither transported nor
metabolized by these cells and has a molecular weight similar to adenosine. Both
uptake and metabolism of adenosine occurs in these cells, therefore, the crossing
of l"C—sucrose should represent an over-estimate of the permeability of the
monolayer to adenosine. In addition, adenosine deaminase was present on the
conHalateral side of LLC—PK, monolayers to further ensure that any adenosine
crossing the monolayer was deaminated to inosine. Although the K“' of adenosine
deaminase is 35 pM, it has a relatively high Vm, 1 unit = l pmole/minute at pH
7.4. The ratio of enzyme activity/subsHate flux gave a 40 fold higher adenosine
deaminase activity than the predicted flux rate of adenosine to the conHalateral side
(Appendix A). Therefore, we feel that any adenosine that may have crossed the
monolayer was quickly deaminated to the inactive compound, inosine. Moreover,
non-metabolizable analogs of ‘ adenosine were not used in this study. Those
adenosine analogs possess varying degrees of lipophilicity and, therefore, may
partition into the plasma membrane and cross the monolayer.

Adenosine was found to stimulate the turnover of inositol phosphates in the
LLC-PK, cell in a dose dependent manner. This effect was blocked by the
adenosine receptor antagonist, DPCPX, indicating that adenosine acts through a
receptor mediated mechanism to stimulate inositol phosphate production. When

cells were exposed to adenosine on the apical or basolateral side independently,

82

only exposure on the basolateral side elicited a stimulation of inositol phosphates.
It may be argued that the signal Hansduction machinery for the stimulation of
inositol phosphates is not present in the apical membrane of this cell line.
However, 1 pM vasopressin (AVP) was found to stimulate inositol phosphate
turnover when presented to either the basolateral or the apical side (data not
shown). This finding suggests that the adenosine receptor that mediates inositol
phosphate turnover, and thus calcium release from inHacellular stores, is located
in the basolateral membrane of LLC-PK, cells.

The adenosine A, receptor is known to mediate inhibition of adenylyl
cyclase concurrently activated by stimulatory receptors, such as the AVP receptor
(Arend et al. 1987; Dillingham and Anderson 1985; Roy 1984). However, when
assessing the functional location of the adenOsine A, receptor, we chose to look at
the inhibition of forskolin stimulated cAMP levels to avoid the further complication
of the possible polarity of a stimulatory receptor system. When applied to the
basolateral side, 30 nM adenosine inhibited forskolin stimulated cAMP levels by
23%. In conHast, exposure of 30 nM adenosine to the apical side caused a non-
significant change (~ 8% decrease) in forskolin stimulated cAMP levels. This
finding suggests that inhibitory adenosine A, receptors are located primarily
basolateral, but that the presence of a minor component on the apical side cannot
be completely eliminated.

The functional localization of the stimulatory adenosine A, receptor was

83
assessed by spatially stimulating cAMP production with 100 pM adenosine.

Exposure of adenosine to either side of the monolayer elicited a significant
elevation in cAMP levels above conHol. Stimulation of the apical side produced
significantly higher levels of cAMP than stimulation of the basolateral side.
Concurrent stimulation of both apical and basolateral sides produced cAMP levels
that were the numerical sum of either side stimulated independently.

Because the inhibitory adenosine A, receptor is located basolaterally, we
explored the possibility that the lesser degree of increased cAMP production by
basolateral adenosine A, receptor stimulation may be due to concurrent inhibition
by adenosine A, receptors. The specific adenosine A, receptor antagonist, DPCPX
(100 nM), was used to block adenosine A, receptors while adenosine A, receptors
were stimulated with 100 pM adenosine. Blockade of the adenosine A, receptors
did not alter the pattern of adenosine A, receptor-stimulated cAMP production.
Moreover, when cells were pretreated for 24 hours with 0.1 pg/ml pertussis toxin
to block any other inhibitory influence that may occur via G,. the pattern of
adenosine A, receptor stimulated cAMP production did not change. These findings
suggest that the lower cAMP levels produced by basolateral adenosine A, receptor
stimulation are not due to inhibitory influences that may predominate on that side.

The disparity between cAMP production induced by apical versus basolateral
receptor stimulation may be explained in part by the inability of adenosine to

access the basolateral cell surface because of the mechanical hinderance of the filter

84

insert and gelatin subsHate required for cell growth. When a time course to
stimulate cAMP production was performed, the absolute levels of cAMP were
different between apical and basolateral sides at all time periods. However, the
levels of cAMP reached a plateau at 30 minutes in both cases. When cAMP levels
were expressed as a percent of levels reached at 30 minutes, the rate of stimulation
from both sides was identical. In addition, supramaximal concenHations of
adenosine continued to stimulate cAMP production to different degrees when
applied to the apical and basolateral sides. These data suggest that the lower
cAMP levels resulting from basolateral adenosine receptor stimulation are not due
to a significant hinderance of adenosine access to the basolateral side. Further
support for the primary functional localization of adenosine A, receptors to the
apical cell surface and adenosine A, receptors to the basolateral cell surface can be
seen from the results of inositol phosphate turnover experiments. Using a 10
minute time course, adenosine stimulated inositol phosphate turnover when applied
to the basolateral side only. Interestingly, there was a significant difference in
apical verses basolateral stimulation of cAMP production at the 10 minute time
point in experiments examining the time course of adenosine A, receptor stimulated
cAMP production. Taken together, these results suggest that the majority of
adenosine A, receptors reside in the apical membrane, whereas only a minor
complement of adenosine A, receptors reside in the basolateral membrane of LLC-

PK, cells.

85

From the results discussed above, we have developed a model of the
functional localization of adenosine receptors in the LLC-PK, cell (Figure 20).
This model suggests that the majority of stimulatory adenosine A, receptors are
exposed to the apical surface, while the majority of inhibitory adenosine A,
receptors are exposed to the basolateral surface of the cell. In addition, the
adenosine receptor mediated pathway that stimulates inositol phosphate turnover
and presumably Ca“ release from inHacellular stores is exposed to the basolateral
cell surface. If the adenosine receptors have a similar distribution in the renal
tubule, adenosine A, receptors would be exposed to the tubular lumen, while the
adenosine receptor mediated pathway that stimulates inositol phosphate turnover
and adenosine A, receptors would face the exHacellular space on the vascular side
of the tubule. However, it should be cautioned that the functional distribution of
adenosine receptors on the LLC-PK, cell may be reflective of aberrant targeting of
these receptors by cells in culture. It remains to be determined if a functional
separation of adenosine receptors is observed in viva.

The tubular epithelium of the kidney is bathed by two distinct exHacellular
fluid compartments, one being interstitial fluid (basolateral) and the other being
tubular fluid (apical). There is evidence to suggest that the concenHation of
adenosine in renal blood and urine is different under normal resting conditions
(Spielman and Thompson 1982). Those investigators reported that the adenosine

concenHation of arterial and venous renal blood was ~50 nM, whereas the

 

Figure 20.

 

 

86

APICAL

1P3

 

 

 

Model of adenosine receptor functional localization in LLC-PK, cells.
This figure shows a model of the spatial localization of the adenosine
receptor mediated pathways in the LLC-PK, cell. The relative size
of each "receptor" indicates the predominance of that receptor
pathway on either the apical or basolateral side.

87

concenHation in the urine was ~300 nM. If adenosine receptors are globally
distributed about the tubule cell, then adenosine in the tubular lumen would have
an overriding influence on the resulting signal Hansduction. However, we report
here that adenosine receptors have a polarized distribution in the LLC-PK, renal
cell line and that this distribution may be reflective of the adenosine concenHations

that are present on either side of the epithelium.

V. Desensitization of Adenosine A, Receptors in the LLC-PK, Renal Cell
Line: Homologous Desensitization Can Occur Independently of Receptor

Occupation/Activation

A. InHoduction

The prolonged exposure of cell types or tissues that predominantly express
the stimulatory adenosine A, receptor have shown a homologous pattern of
desensitization. Homologous desensitization of adenosine A, receptors has been
seen in vascular smooth muscle from rat aorta (Anand-Srivastava et al. 1989), rat
kidney fibroblasts (Newman and Levitzki 1983), and rat brain Striatum (Hawkins
et al. 1988; Porter et al. 1988). Ramkumar et al. (1991) observed the
desensitization of both adenosine A, and A, receptors in the DDT, MF-2 smooth
muscle cell line to specific agonists for each of the two receptors. However, the
time course for desensitization of the adenosine A, receptor had a t,,, of 8 hours,
while the t,,, for the adenosine A, receptor was 45 minutes. In conHast, the
adenosine A, and adenosine A, receptors in RCCT cells in culture desensitized at
essentially the same concenHation and time course of exposure to agonist (Arend,
personal communication; LeVier et al. submitted).

Recently the idea of spatial separation of cell surface receptors on a
polarized monolayer of epithelial cells has received attention. Polarized epithelia

are structured such that tight junctions between the apical and basolateral sides

88

89

separate the membrane bound proteins on one side from the other (Rodriquez—Bolan
and Nelson 1989). This polarization of epithelial monolayers and therefore the
proteins responsible for solute Hansport is the basis for vectoral transport of solute
and water across the monolayer. Several researchers have demonsHated the polar
distribution of membrane bound receptors on epithelial cells. These include the
bradykinin (Denning and Welsh 1991), VIP (Fantini et al. 1988), AVP (Ando et
al. 1991), and adenosine receptors (Barrett et al. 1989; Husted et al. 1990; Pratt et
al. 1986) which have been found to have a polarized distribution on several
epithelial cell types. This laboratory has recently established the functional
localization of adenosine receptor pathways in the LLC-PK, renal cell line (LeVier
et al. in press). The inhibitory adenosine A, receptor pathway was found to be
basolaterally located, although a minor component located on the apical side could
not be discounted. In conHast, the stimulatory adenosine A, receptor pathway was
localized to both the apical and basolateral side of the LLC-PK, monolayer with
the major component residing on the apical side. The present study investigated
the effect of selective exposure of adenosine to either the apical or basolateral side
of a monolayer of LLC-PK, cells on the subsequent responsiveness of adenylyl

cyclase-coupled adenosine receptors.

90
B. Methods

Cell Culture: LLC—PK, cells (passage number 194) were obtained from
American Type Culture Collection, (Rockville Maryland). Cell culture reagents
were obtained from Gibco BRL, (Grand Island NY). The cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum
(FBS), 2 mM glutamine and 50 pg/ml each of penicillin and sHeptomycin at 37°C
in a 7% CO, atmosphere. Cells were routinely passaged every 7 days. Cells for
experiments in 24 well plates were lightly Hypsinized from one 100 mm culture
dish with a solution of 0.2% Hypsin and 0.05% EDTA in PBS and sedimented by
centrifugation (500 x g) for five minutes. The resulting pellet was then
resuspended in 25 ml of culture media. This cell suspension was then distributed
to one 24 well plate at 1 ml per well. The cells were then allowed to grow to
confluency before an experiment was undertaken.

Gelatin Substrate Coating of Culture Inserts: Cell culture inserts (12 mm
diameter, Millicell‘m-CM; obtained from Millipore Corp., Bedford MA.) were
coated with gelatin (Millipore technical brief: Lit. No. T3017 copyright 1989) as
an exHacellular matrix upon which LLC-PK, cells adhere and grow. The inserts
were then prepared for cell culture as described in Methods of chapter IV (Figure
9).

Measurement of the Potential Difference and Electrical Resistance of a LLC-

PI_(_, Cell Monolayer: Once confluent, the potential difference and electrical

91

resistance of the LLC-PK, cell monolayer was measured to assess the monolayer’s
integrity. All potential difference and electrical resistance measurements were
made before any experimental procedure was undertaken. The measurement of
potential difference and electrical resistance was carried out as described in
Methods of chapter IV.

Desensitization Protocol: LLC-PK, cells were grown to confluency on either
Millicell'm-CM inserts or 24 well plates. All cells were washed with Krebs buffer.
Cells grown in 24 well plates were pretreated for 1 hour with 100 pM adenosine
in Krebs buffer (500 pl). Cells grown on filter inserts were pmHeated for 1 hour
with 100 pM adenosine in Krebs buffer on either the apical or basolateral side of
the monolayer (300 pl apical, 500 pl basolateral). Krebs buffer with 1.5 U/ml of
adenosine deaminase (ADA) was placed on the conHalateral side of preHeated
inserts. CronHol groups were preHeated for 1 hour with Krebs buffer without
adenosine. Following the pretreatment time period, the monolayers were washed
with Krebs buffer. Each monolayer was then stimulated for 30 minutes with 100
pM adenosine in Krebs buffer (300 pl in wells; 300 pl apical, 500 pl basolateral in
inserts) in the presence of 100 pM RO 20-1724, a phosphodiesterase inhibitor that
is not an adenosine receptor antagonist. ‘ The protein kinase C inhibitor,
staurosporine (1 pM), was included in the preHeatment regime of cells on a 24 well
plate to investigate the possible involvement of PKC in the signal pathway for

desensitization. The stimulation of cAMP production was halted by adding 75 pl

92

of 0.2 N HCl to each well or upper side of inserts. The cells were then frozen at -
80°C to lyse cells and release cellular contents into the supernatant for subsequent
measurement of cAMP production.

_c_A_MP_I,)etermination: Samples were thawed at room temperature and the
pH was neutralized by adding 200 pl of 0.5 M Na,HPO,. The cAMP levels in the
supernatant of each sample were measured via radioimmunoassay as described by
Frandsen and Krishna (1976). Cyclic AMP levels were expressed per mass of cell
protein as measured by a modified Lowry’s assay (Markwell et al. 1978).

Adenylyl Cyclase Activity: A highly specific competitive protein kinase A
(PKA) inhibitor, Rp—cAMPS (from BioLog life science institute in LaJolla CA.),
was used to specifically inhibit PKA and investigate its role in the desensitization
of adenosine A, receptors. LLC-PK, cells grown to confluency on 60 mm culture
dishes were incubated in the presence or absence of 100 pM adenosine :1: 10 pM
or 100 pM Rp-cAMPS in Krebs buffer at 37°C for 1 hour. The cells were
preincubated for 10 minutes with Rp-cAMPS prior to the inHoduction of 100 pM
adenosine. The cells were washed twice with 5 ml PBS and removed from the
culture dishes by incubation at 37°C for 3 minutes with 0.2% Hypsin, 0.05% EDTA
in PBS. The cells were collected by washing the dish with 5 ml PBS and
centrifuged (500 x g) for 5 minutes. The resulting cellular pellet was resuspended
in 2 ml of cyclase buffer (mM: 50 Tris-HCl, 5 MgCl,, pH 7.4) and gently

homogenized with a small glass-teflon homogenizer. The ensuing homogenate was

93
centrifuged at 12,000 RPM in a Beckman JA-20 rotor for 15 minutes at 4°C. The

resulting crude membrane pellet was resuspended in 0.5 ml cyclase buffer so as to
yield a protein concenHation of ~ 1 mg/ml. Adenylyl cyclase activity was initiated
by adding ten microliters (~20 pg membrane protein) of the crude membrane
preparation to microcentrifuge tubes in a final reaction volume of 250 pl. The final
concenHation of reaction mixture components were: 0.31 mg/ml creatine kinase,
1.68 mg/ml creatine phosphate, 0.3 mg/ml BSA, 5 units/ml adenosine deaminase,
0.25 mM ATP, 0.25 mM cAMP, 0.5 mM EGTA, 25 pM GTP, 0.25 mM R0 20-
1724, and 10 pCi/ml oer-ATP in cyclase buffer. Adenosine A, receptor
stimulated adenylyl cyclase activity was allowed to run for 30 minutes at 30°C in
the presence or absense of 10 pM NECA and terminated by adding 250 pl 6.7%
TCA. Adenylyl cyclase activity in the membranes was determined as described by
Salomon et al. (1974).

Statistics: All data sets were analyzed by analysis of variance (ANOVA).
If a difference among means was indicated, Scheffe’s multiple comparisons test
was used to determine which means were statistically different. Statistical
significance was ascribed for a p < 0.05. All values are expessed as mean :1: SEM.
Statistics were performed using Apple MacIntosh software StatView'm 512+ from

BrainPower, Inc., Calabasas, CA.

94

C- M

As has been previously demonsHated (LeVier et al. in press) the functional
response of adenosine A, receptors in LLC-PK, cells shows a marked polarity.
Adenosine applied to either side of the monolayer stimulates cAMP production via
the adenosine A, receptor with the apical side responding to a greater degree than
the basolateral side. Moreover, the response to adenosine presented to both sides
simultaneously is the numerical sum of either side stimulated independently. A
preHeatment time of one hour was chosen to minimize the possible flux of
adenosine across the cellular monolayer. Following a one hour preHeatment with
adenosine on either the apical or basolateral side of a LLC-PK, cell monolayer, the
ability of selectively applied 100 pM adenosine to restimulate cAMP production
was significantly blunted as compared to conHol on either side of the monolayer
(Figure 21 A & B). Apical preHeatment lead to a 51% decrease of apical and a
42% decrease of the combined apical and basolateral adenosine A, receptor
stimulated cAMP production. Furthermore, apical preHeatment induced a complete
desensitization of the adenosine A, receptor response on the basolateral side.
Basolateral pretreatment also lead to a complete desensitization of adenosine A,
receptor stimulated cAMP production on the basolateral side. In addition,
basolateral pretreatment lead to a 53% decrease of apical and 66% decrease of
combined apical and basolateral adenosine A, receptor stimulated cAMP

production.

Figure 21.

95

Selective 100 pM adenosine preHeatment of apical or basolateral
adenosine A, receptors in LLC-PK, cells. Cells grown to confluency
on Millicell‘m-CM inserts were preHeated with 100 pM adenosine for
one hour on either the (A) apical or (B) basolateral side. The cells
were then rechallenged with 100 pM adenosine for 30 minutes on
either the apical, basolateral or both sides. Cyclic AMP was
measured as described in "Methods". Values are reported as mean
i SEM of two experiments in duplicate. * p < 0.05 (ANOVA)
indicates a significant difference from the corresponding conHol
value.

 

 

 

 

 

cAMP LEVEL (pmol/mg protein)

cAMP LEVEL (pmol/mg protein)

 

96

 

 

 

 

 

 

 

 

 

 

 

    

 

 

 

 

    

 

 

 

 

A
250 -
. '3 “m I.
ONEHOURPRETREA‘INENT
zoo -
150 -
100 -
b
so I-
o
B
zoo -
El comm.
. CNEI-IOIRPFEI'REATNEINIT
150 -
100 - l
n * i
so -
BASAL APICAL BASOLATERAL API + aaso

97

The adenosine A, receptor selective agonist, R-PIA, was used to inhibit
cAMP production stimulated by forskolin following preHeatment with adenosine.
A one hour preHeatment with 100 pM adenosine on either the apical or basolateral
side did not blunt the ability of 30 nM R—PIA to inhibit 1 pM forskolin stimulated
cAMP production via the adenosine A, receptor (Figure 22). ConHol, apically and
basolaterally preHeated cells all responded to 30 nM R-PIA by inhibiting 1 pM
forskolin stimulated cAMP production by 40%.

A one hour pretreatment with 100 pM adenosine on either the apical or
basolateral side of the cell monolayer did not alter the pattern of 1 nM arginine
vasopressin (AVP) stimulated cAMP production (Figure 23 A & B). LLC-PK,
cells were equally responsive to 1 nM AVP added to either the apical or basolateral
side of the monolayer. In addition, 1 nM AVP added to both sides concurrently
stimulated cAMP production that was the numerical sum of the two sides
stimulated independently.

Forskolin was used to explore the effect of elevated cAMP levels on
adenosine A, receptor function in the LLC-PK, cell (Figure 24). Forskolin (1 pM)
increased cAMP production to levels that were similar to those stimulated by 100
pM adenosine. Following preHeatment for one hour with 1 pM forskolin, 100 pM
adenosine stimulation of cAMP production on the apical, basolateral or both sides
of LLC-PK, cells was unaltered. The PKC inhibitor, staurosporine, was used to

investigate the possible role of PKC in the desensitization of adenosine A,

cAMP (pmol/mg protein)

Figure 22.

98

El BASAL
1 uM FORSKOUN
500 . 1 uM FORSKOLIN + so nM R-PIA

   

             
        

 

 

 

 

.
400-
.
300-
' II I
xx xx
II II I
xx xx xx
II II I
xx xx xx
200.- II II I
xx xx xx
II II I
xx xx xx
II II I
xx xx xx
II II I
’ xx xx xx
II II I
xx xx xx
II II I
xx xx xx
II II I
100,. xx xx xx
II II I
xx xx xx
II II I
xx xx xx
II II II
xx xx xx
> II II I
xx xx xx
II II I
xx xx xx
II II II
xx xx xx
0 II II II
o a

WIT-"K1 APICAL BASOLATERAL
100 uM ADENOSINE PRETREATMENT

Apical versus basolateral 100 pM adenosine preHeatment effect on
'R-PIA inhibition of 1 pM forskolin stimulated cAMP production in
LLC-PK, cells. Following pretreatment with 100 pM adenosine on
either the apical or basolateral side for one hour, LLC-PK, cells were
Heated with 1 pM forskolin i 30 nM R-PIA for 30 minutes. Cyclic
AMP was measured as described in "Methods". Values are expressed
as mean i SEM of four experiments in duplicate. * p < 0.05
(ANOVA) indicates a significant difference from forskolin stimulated
cAMP levels.

Figure 23.

99

Effect of apical versus basolateral 100 pM adenosine preHeaHnent on
AVP-stimulated cAMP production in LLC-PK, cells. Cells grown to
confluency on Millicell‘m-CM inserts were preHeated for one hour
with 100 pM adenosine on the (A) apical or (B) basolateral side.
Cyclic AMP production was stimulated with 1 nM AVP for 30
minutes on the apical, basolateral or both sides. Values are expressed
as the mean i SEM of two experiments in duplicate. Cyclic AMP
was measured as described in "Methods". An ANOVA was
performed as described.

 

 

cAMP LEVEL (pmol/mg protein)

cAMP LEVEL (pmol/mg protein)

5500
5000
4500
4000
3500
3000
2500
2000
1 500
1 000

500

5000

4500

3500

3000

2500

1 500

1 000

500

 

 

 

 

 

 
 

BASAL APICAL BASOLATERAL API 4» BASO

 

BASAL APICAL BASOLATERAL API + BASO

cAMP LEVEL (pmol/mg protein)

Figure 24.

101

El comm
ONEHOURPRETREATIENT
250 -

200 '-

100 -

50'-

  

  

 

 

 

 

 

 

BASAL APICAL BASOLATERAL API + BASO

Effect of preHeatment with 1 pM forskolin on adenosine A, receptor
responsiveness in LLC-PK, cells. Cells grown to confluency on
Millicell'm-CM inserts were preHeated with 1 pM forskolin on both
sides for one hour. Cyclic AMP production was stimulated with 100
pM adenosine for 30 minutes on the apical, basolateral or both sides.
Values are reported as mean 1': SEM of two experiments in duplicate.
Cyclic AMP was measured as described in "Methods". All ANOVA
as performed as described.

102
receptors (Figure 25). When 1 pM staurosporine was added concurrently with 100

pM adenosine during a one hour preHeatment of LLC-PK, cells grown on a 24
well culture plate, the pattern of adenosine A, receptor desensitization was not
altered.

The cell permeant cAMP analog Rp-cAMPS was used to assess the
contribution of protein kinase A in the homologous desensitization of adenosine A,
receptors (Figure 26). Rp-cAMPS is an analog of cAMP that is a competitive
inhibitor of PKA with an 1C50 of approximately 10 pM (Botelho et al. 1988).
Adenylyl cyclase activity was increased by 85 i 3% when conHol membranes were
stimulated with 10 pM NECA. Following a one hour preHeatment with 100 pM
adenosine, adenylyl cyclase activity could only be stimulated by 30 i 3.7%.
NECA stimulated adenylyl cyclase activity following inclusion of Rp-cAMPS at
10 pM or 100 pM in the one hour preHeatment with 100 pM adenosine was

increased by 34 i: 4.7% or 34 i 2.6% respectively.

D. Discussion

While desensitization of receptor coupled-adenylyl cyclase systems has been
thoroughly researched, the idea of spatial effects on desensitization is something
new. Virtually none of the studies in polarized epithelia have tested the effects of
the polar distribution of a receptor its desensitization. The adenosine A, receptor

has been reported to be desensitized in a number of cell and tissue types, however

103

120 . [j NOTPFETREATED
IOOUMADENOSINEPRETREATED

:1; _T_

8

8

8

 

96 OF CONTROL 100 UM ADENOSINE
STIMULATED cAMP ACCUMULATION
#1
o
I

8

     

 

 

 

 

 

.
_ _ .I:e:e:"l.e'-'I. :e: ”I...
.; . ,5. . .e 5... c .‘ '___'
E- : ”3"“? :35:

BASAL 100 UM ADENOSINE BASAL 100 UM ADENOSINE

 

 

 

1 uM STAUROSPORINE

Figure 25 . Effect of preHeatment with 100 pM adenosine -_i-_ 1 pM staurosporine
on adenosine A, receptor desensitization in LLC-PK, cells. The PKC
inhibitor, staurosporine (1 pM), was added concurrently with 100 pM
adenosine in a one hour preHeatment of cells grown on a 24 well
plate. The cells were then rechallenged with 100 pM adenosine for
30 minutes. Values are reported as mean i SEM of two experiments
in triplicate. Cyclic AMP levels were measured as described in
"Methods". * p < 0.05 (ANOVA) indicates a significant difference
from conHol stimulated cAMP production.

96 STIMULATION OF ADENYLYL CYCLASE
ACTIVITY ABOVE BASAL

Figure 26.

104

 
   

1o uM NECA STIMULATION

 

OCNIFO. ADEIDSINE WNE ADBICBIE
10 uM Ftp-cAMPS 100 uM Rp-cAMPS

 

PRETREATMENT

Effect of the PKA inhibitor, Rp-cAMPS, on adenosine A, receptor
desensitization induced changes in adenylyl cyclase activity in LLC-
PK, cell membranes. Cells grown to confluency on a culture dish
were concurrently preHeated with 100 pM adenosine and the
competitive PKA inhibitor Rp—cAMPs at concenHations of 10 pM or
100 pM for one hour. Membranes were prepared and adenylyl
cyclase activity measured as described in "Methods". Values are
expressed as mean i SEM of two experiments in triplicate. * p <
0.05 (ANOVA) indicates a significant difference from conHol
stimulated adenylyl cyclase activity.

105

none of these cells or tissues are epithelial in nature (Anand-Srivastava et al. 1989;
Hawkins et al. 1988; Newman and Levitzki 1983; Porter et al. 1988; Ramkumar
et al. 1991). In conHast, this laboratory has reported the desensitization of
adenosine A, and adenosine A, receptors in renal cortical collecting tubule cells
(Arend personal communication; LeVier et al. submitted). The afore mentioned
work was carried out using adenosine analogs that are sufficiently lipophilic to
access the basolateral side of cells grown to confluency on a culture dish. The
present work was performed using the hydrophilic ligand adenosine exposed
selectively to either the apical or basolateral aspect of cells grown on Millicell“-
CM filter inserts.

Recently, adenosine A, receptor function was determined to be present on
both apical and basolateral sides of LLC-PK, cells (LeVier et al. in press). The
major component of adenosine A, receptors was found to be apical with a minor
component on the basolateral side. In addition, concurrent stimulation of adenosine
A, receptors on both sides resulted in an increase in cAMP production that was the
numerical sum of either side stimulated independently. Nonetheless, adenosine
placed on either side evoked a significant increase in cAMP production over basal.
This differential distribution of adenosine A, receptors provided an opportunity to
selectively occupy and activate a subpopulation of receptors on the same cell.

In preliminary experiments on the desensitization of adenosine receptors

performed on cells grown to confluency on a solid plastic support (24 well plate),

106

a significant reduction in adenosine A, receptor responsiveness (50%) had occurred
within 30 minutes of exposure to 100 pM adenosine and continued to decline for
up to four hours (data not shown). Following selective exposure of either the
apical or basolateral side to 100 pM adenosine for 1 hour, the adenosine A,
receptor response on the basolateral side was abolished. However, the response on
the apical side was reduced by 50%. The 50% reduction in response on the apical
side is strikingly similar to that seen with desensitization of LLC-PK, cells groWn
to confluency on a plate. It would appear that a subpopulation of adenosine A,
receptors on the apical side of LLC-PK, cells is refractory towards desensitization
over the time course studied. Surprisingly, selective exposure of either side of the
monolayer to 100 pM adenosine resulted in the same pattern of desensitization of
adenosine A, receptors.

Previous work established that confluent monolayers of LLC-PK, cells are
tight to the flux of ”C-sucrose, a sugar of comparable molecular weight to
adenosine. Additionally, adenosine deaminase was included in the buffer on the
conHalateral side of monolayers preHeated with adenosine. The adenosine
deaminase was present with a 40 fold higher activity than predicted adenosine flux
across the cellular monolayer (Appendix A). Therefore, the adenosine receptors
on the conHalateral side of the cell monolayer presumably were not
occupied/activated during the one hour exposure time course, yet this subpopulation

of receptors became desensitized. A heterologous pattern of desensitization could

107

explain such a phenomenon. However, the responsiveness of the stimulatory AVP
receptor was not affected by one hour exposure to 100 pM adenosine in the LLC-
PK, cell. Interestingly, AVP evoked cAMP production on either side of the cell
monolayer to an equal degree. Monolayers of LLC-PK, cells did not allow 3H-
AVP to cross at a time course of up to one hour (data not shown). Moreover, the
response to AVP exposed to both sides concurrently was the numerical sum of
either side stimulated independently. This suggests that there are equal numbers
of AVP receptors on either side of the LLC-PK, cell monolayer. The ability of 30
nM R-PIA to inhibit forskolin stimulated cAMP production via the adenosine A,
receptor also was not affected by a one hour exposure to 100 pM adenosine. These
results argue for an alteration of adenosine A, receptor function and not G-protein
or adenylyl cyclase function, as the responsiveness of other receptors coupled to .
the stimulation or inhibition of adenylyl cyclase are unaffected. Consequently,
these results suggest that subpopulations of unoccupied/non-activated adenosine A,
receptors can undergo homologous desensitization.

The homologous desensitization of the B—adrenergic receptor has been
associated with the phosphorylation of the carboxy-terminus tail of the receptor by
a receptor specific kinase termed BARK (Benovic et al. 1988, Hausdorff et a1.
1990). BARK has been shown to preferentially phosphorylate the occupied form
of the receptor. Additionally the B-adrcnergic receptor has also been shown to be

phosphorylated by PKA under conditions where heterologous desensitizatiOn occurs

108

(Bouvier et al. 1989). Although currently there is no information on the
phosphorylation state of adenosine A, receptors under either basal or desensitizing
conditions, it seems reasonable to postulate that the altered function of adenosine
A, receptors may involve their phosphorylation. Therefore, we chose to investigate
the possibilities that either PKA or PKC are involved in the homologous
desensitization of adenosine A, receptors.

It has been reported that protein kinase C can phosphorylate the adenylyl
cyclase-coupled B—adrenergic receptor in vitro (Bouvier et al. 1987). While the
phosphorylation of the B-adrenergic receptor by protein kinase A was stimulated
by receptor occupancy, protein kinase C mediated phosphorylation of the B-
adrenergic receptor was not. Previously, the stimulation of inositol triphosphate
turnover by 10 pM adenosine was shown to occur via a receptor mediated
mechanism in LLC-PK, cells (LeVier et al. in press). This adenosine receptor
mediated release of inositol triphosphate is thought to occur through the stimulation
of phospholipase C. The concenHation (100 pM) of adenosine used to desensitize
adenosine A, receptors would also elicit an adenosine receptor stimulated release
of diacylglycerol (DAG) via phospholipase C. Diacylglycerol is known to activate
protein kinase C. Additionally, cAMP has been shown to activate protein kinase
C via protein kinase A in the LLC-PK, cell line (Anderson and Breckon 1991).
However, inclusion of 1 pM staurosporine did not alter the pattern of homologous

desensitization of the adenosine A, receptor. This suggests that protein kinase C

109

is not involved in the homologous desensitization of adenosine A, receptors.
Forskolin was used to stimulate the inHacellular level of cAMP in LLC-PK,
cells for one hour. This elevation of cAMP level within the cell and presumed
increase in protein kinase A activity did not alter the pattern of homologous
desensitization of adenosine A, receptors. This suggests that protein kinase A is
not involved in the desensitization of adenosine A, receptors that are not
occupied/activated. In order to investigate the possibility that protein kinase A may
be involved in the desensitization of occupied/activated adenosine A, receptors, the
competitive protein kinase A inhibitor Rp-cAMPS was employed. Rp—cAMPS has
been shown in several cell systems to have a K, ranging from 1 pM to 10 pM
(Botelho et al. 1988). Preincubation with Rp-cAMPS at a concenHation of 10 pM
. or 100 pM followed by exposure of cells to 100 pM adenosine for 1 hour did not
alter the pattern of adenosine A, receptor desensitization. This implies that protein
kinase A is not involved in the desensitization of the occupied/activated adenosine
receptor either. Taken together, the results of these experiments suggest that the
homologous desensitization of adenosine A, receptors in LLC-PK, cells is not
primarily mediated by prOtein kinase A or protein kinase C.
Unfortunately, we were unable to investigate the possibility that a BARK-
like kinase may be involved in the homologous desensitization of adenosine A,
receptors. Other researchers have used heparin to inhibit BARK in permeabilized

cell systems (Hausdorff et al. 1990). When LLC—PK, cells were permeabilized the

110

adenylyl cyclase activity of subsequently isolated membranes could not be
stimulated using adenosine receptor agonists. Apparently the permeabilization
process interferes with the ability of adenosine A, receptors to stimulate adenylyl
cyclase in LLC-PK, cells. Therefore we were unable to investigate the possibility
that a BARK-like kinase may be involved in the homologous desensitization of
adenosine A, receptors. The development of cell permeant specific inhibitors of
BARK-like activity would be of great value in investigating this phenomenon.
Although our data cannot confirm the occupational state of conHalateral receptors
there is, nonetheless, the exciting possibility that homologous desensitization of
adenosine A, receptors in LLC-PK, cells is accompanied by the altered function of

unoccupied/non-activated adenosine A, receptors on the conHalateral side.

VI. Summary and Conclusions

The research presented in this thesis suggests that:

1. Desensitization of adenosine receptors in primary cultures of RCCI‘
cells involves the altered function of adenosine A, and A, receptors.
Additionally, altered function of the regulatory G-protein, G,. confers a
heterologous component to this desensitization.

2. The adenosine receptor mediated pathways were functionally localized to
either the apical or basolateral side of the LLC-PK, renal cell line.
The adenosine receptor mediated pathway that stimulates inositol phosphate
turnover was localized to the basolateral aspect of LLC-PK, cells. The
inhibitory adenosine A, receptor pathway was localized to the basolateral
aspect of the cell monolayer, although a minor apical component could not
be ruled out. The stimulatory adenosine A, receptor pathway was found to
reside on both sides of the monolayer. However, the major component of
the adenosine A, receptor pathway was apical with a smaller, albeit
significant, component residing on the basolateral side of LLC-PK, cells.

3. Desensitization of stimulatory adenosine A, receptors on both sides of LLC-
PK, cells, following selective exposure of adenosine to either the apical or

basolateral side of the monolayer, was found to be homologous. This

111

112

pattern of homologous desensitization was the same irrespective of the side
on which the receptors were exposed to adenosine. This suggested that
adenosine A, receptors on the conHalateral side of adenosine exposure,
which were presumably unoccupied/unactivated, could be desensitized

independently of other unoccupied/unactivated G-protein linked receptors.

The desensitization of adenosine receptors in primary cultures of RCCT cells
did not occur until concenHations of agonist reached levels known to
occupy/activate the stimulatory adenosine A, receptor. Lower concenHations of
agonist which occupy/activate the inhibitory adenosine A, receptor did not induce
desensitization out to a time course of 4 hours. Neither adenylyl cyclase nor the
regulatory G,-protein appeared to be involved in adenosine receptor desensitization,
suggesting that altered function of both adenosine A, and A, receptors had
occurred. Cholera toxin stimulated cAMP production was attenuated following
exposure to adenosine agonists. This decreased stimulation by cholera toxin
suggested an altered function of the regulatory G,-protein, and forms the basis for
the observed heterologous desensitization of AVP receptors.

The functional localization of adenosine receptors in cultured renal epithelial
cells is an important first step in the physical localization of these receptors both
in culture and in viva. The functional localization of adenosine receptor mediated

pathways in the LLC-PK, cell line revealed that the majority of inhibitory

113

adenosine A, receptors were basolateral, while the majority of stimulatory
adenosine A, receptors were apical. The adenosine receptor mediated pathway
responsible for the stimulation of inositol phosphate turnover was basolateral.
Additionally, concurrent stimulation of adenosine A, receptors on both sides of the
monolayer produced cAMP levels that were the numeric sum of either side when
stimulated independently. This, along with the presence of adenosine dearrrinase
on the conHalateral side in sufficient amounts to give an enzymatic activity 40 fold
higher than the predicted flux of adenosine across the monolayer (Appendix A),
suggests that adenosine receptors on the conHalateral side were not
occupied/activated during the course of an experiment.

The above results allowed for the selective exposure of a subset of adenosine
A, receptors on a monolayer of LLC-PK, cells. Following a one hour exposure of
adenOsine receptors on either the apical or basolateral side to a stimulatory
concenHation of adenosine, the adenosine A, receptors were desensitized. The
pattern of desensitization was the same regardless of the side of exposure, with the
basolateral adenosine A, receptors being completely desensitized and the apical
adenosine A, receptors desensitized by ~50%. Neither adenosine A, receptors nor
AVP receptors were desensitized, marking this as a homologous desensitization of
adenosine A, receptors. Various kinases have been implicated in the altered
function of receptors (B-adrenergic receptors for example) following prolonged

exposure to agonists. Further investigation revealed that protein kinase A and

114

protein kinase C were not primarily involved in the homologous desensitization of
adenosine A, receptors. As outlined in the literature review, a BARK-like kinase
may be involved in the homologous desensitization of occupied G-protein linked
receptors, however we have no evidence of this in LLC-PK, cells. Nevertheless,
the exciting possibility exists that homologous desensitization of a subset of
adenosine A, receptors that are unoccupied/unactivated can occur in the LLC-PK,
renal cell line. Because neither PKA or PKC are involved, and BARK
preferentially phosphorylates occupied receptors, a novel second messenger system
may be implicated in the homologous desensitization of unoccupied/unactivated
adenosine A, receptors.

In conclusion, the findings summarized above indicate that the adenosine
receptor systems present in renal epithelial cells have a polar distribution and are
regulated by prior exposure history to agonist. The polar distribution of adenosine
receptor mediated pathways on a renal epithelium has functional consequences
dependent upon the side on which such an epithelium might be presented with
adenosine. That is, adenosine on the interstitial side could bring about functional
changes that are different than those brought about by adenosine present on the
lumenal side of renal tubular epithelium. It should be Cautioned, however, that the
localization and regulation of these adenosine receptor systems in cell culture might
not be directly reflective of those that exist in viva. Nonetheless, the results of

these three projects will serve as a solid data base upon which future in viva work

115

can draw.

Appendix

1 16
Appendix 'A

The velocity of an enzymatic reaction is defined as:

v = (Va... x (Cl) / (K,n + [0])

Va“, is the velocity of the enzymatic reaction at infinite subsHate concenHation.
K, is the concenHation of subsHate where v = VM/Z.

[c] is the concenHation'of subSHate at time t.

The K"I of adenosine deaminase (EC 3.5.4.4) is 35 pM.

The VM of adenosine deaminase is 1 unit = 1 pmole of adenosine deaminated per
minute at pH 7.4 at 25°C.

Starting with an adenosine concenHation of 100 pM, the predicted concenHation
of adenosine that may have crossed from the apical (upper) side of the monolayer
to the basolateral (lower) side at 30 minutes was 5% (1.88 pM) of the steady state
concenHation of 37.5 pM.

The concenHation of adenosine deaminase used was 1.5 U/ml.
The basolateral volume was 0.5 ml, thus 0.75 units of ADA was present.
v = 0.75 pmole/min x 1.88 pM / 35 pM + 1.88 pM = 0.038 pmole/min.

Amount of adenosine predicted to have crossed at 30 minutes;
1.88 pM x 5x10“’L = 0.00094 pmole.

Ratio of ADA activity to adenosine flux:
0.038 pmole/min / 0.00094 pmole = 40 min".

A similar set of calculations for the ADA activity added to the apical (upper) side
of the monolayer versus the predicted flux of adenosine from the basolateral

(lower) side gives a ratio of;
0.037 pmole/min / .00093 pmole = 40 min".

Therefore there was ~ 40 fold more adenosine deaminase activity present on the
conHalateral side than adenosine flux to that side.

Literature Cited

VII. Literature Cited

Altschuld, R.A., L.M. Gamelin, R.E. Kelley, M.R. Lambert, L.E. Apel, and GP.
Brierley (1987) Degradation and resynthesis of adenine nucleotides in adult
rat heart myocytes. J. Biol. Chem. 262:13527-13533.

Anand-Srivastava, M.B., M. Cantin, M. Ballak, and S. Picard (1989)
Desensitization of the stimulatory A, adenosine receptor-adenylate cyclase
system in vascular smooth muscle cells from rat aorta. Mol. Cell.
Endocrinol. 62:273-279.

Anderson, R.J., and R. Breckon (1991) cAMP stimulates protein kinase C activity
in cultured renal LLC-PK, cells. Am. J. Physiol. 261:F945-F950.

Ando, Y., K. Tabei, and Y. Asano (1991) Luminal vasopressin modulates Hansport
in rabbit cortical collecting duct. J. Clin. Invest. 88:952-959.

Arend, L.J., W.K. Sonnenburg, W.L. Smith, And w.s. Spielman (1987) A, and A,
adenosine receptors in rabbit cortical collecting tubule cells: modulation of
hormone-stimulated cAMP. J. Clin. Invest. 79:710-714.

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