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III‘III In' " IIIIIIII" . ..In.IIn.II‘II‘ IIIIIIIIIIII‘IIIJ III IIIIIIIII IIIIII' V II IIH'IHIIIII'II'I” I‘lIflI‘IIIIIIIIv‘I IIIWIHI' ‘.an 'I'II' I‘I.. ‘I- . ”In.” WIII'IIIIII .I.n ”I” ;I. .-.. ..lIn‘Ill nn'” “III I III'" .‘III'IIII :n' .Innn n.I"' III .nIIIIIYIII 'III fimflw - I. . II“ I'.1 IIIIII . ..nI'..n' IIII..III 'n (In; '1 .I'IInnIII'I'IIIIIILII '.I 'II"!I'1*.III.'II".' ' ... “III.“ IinInLI-IIHI. mN'III'InIIIMI"mIiLlnfiIIfl ‘ “I"! ... THFQ‘S ‘intuit;mmmmnmmnmmm 'i 3 1233 01005 2698 This is to certify that the thesis entitled PHYSICAL AND BIOLOGICAL FACTORS AFFECTING THE MOVEMENT OF WATER ACROSS ISOLATED PERFUSED FISH GILLS presented by William F. Jackson has been accepted towards fulfillment of the requirements for Ph. D. degree in W! gm! 0‘ PW Major professor Date—lunLZLJQZL. 0-7639 PHYSICAL AND BIOLOGICAL FACTORS AFFECTING THE MOVEMENT OF WATER ACROSS ISOLATED PERFUSED FISH GILLS By William F. Jackson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1979 ABSTRACT PHYSICAL AND BIOLOGICAL FACTORS AFFECTING THE MOVEMENT OF WATER ACROSS ISOLATED PERFUSED FISH GILLS By William F. Jackson The movement of tritiated water (3HOH) across isolated second gill arches of rainbow trout (Salmo gairdneri) perfused with or without 10 pmol/L epinephrine (Epi and No-Epi respectively) was studied under a variety of conditions. Variation of the osmotic gradient across gills, using either sodium chloride or sucrose as solute. had no effect on the permeability-surface area product (PdA), a measure of the transfer capacity of gills for 3HOH. In addition. the flux of tritiated water (Jw*) across trout gills was independent of the direction of the net tracer flux. These data indicate that 3 HOH moves across gills primarily by diffusion. Also, it was found that the transfer capacity (PdA) of gills was positively correlated with the degree of stirring 0f the bath solution in which gills were suspended suggesting that boundary layers at the surface of the gill epithelium offer a significant barrier to mass transport across fish gills. Increased perfusion flow resulted in an augmented PdA and decreased vascular resistance (R9) for both Epi and No-Epi gills. The effect of flow on PdA is probably mediated via recruitment of secondary lamellae (increased functional surface area, A). Recruitment and passive distension 0f the gill vasculature probably William F. Jackson accounts for the decreased Rg. When efferent pressure (Pe) was elevated PdA and R9 decreased. The lowered Rg probably resulted from passive distension of the gill vasculature and redistribution of perfusate flow between efferent arterial and venous pathways. The mechanism of action of Pe on PdA remains obscure. Epinephrine increased gill PdA and decreased R9. The action of epinephrine on PdA was by increased gill permeability (Pd) and by recruitment of secondary lamellae (increased functional surface area, A). Reduction of perfusate pH from 7.8 to 6.5 resulted in a sustained increase in PdA and a transient increase in R9 and perfusate leak from No-Epi gills. The data suggest that the changes observed resulted from altered distribution of perfusate flow (increased functional surface area, A) rather than from changes in gill permeabil- ity (Pd). Reperfusion of gills (Epi or No-Epi) at pH 7.8 resulted in a decrease in PdA. The mechanism of this hysteresis is unknown. Reduc- tion of bath pH from 7 to 3.5 had little or no effect on PdA and R9 for either Epi or No-Epi gill arches. The data from these studies indicate that: a) the transfer capacity of gills (PdA) is not a constant depend- ent solely on the magnitude of the tissue permeability (Pd) and anatomi- cal surface area, and b) calculation of Pd from tracer data is inappro- priate when applied to fish gills. ACKNOWLEDGEMENTS I would like to thank the members of my guidance committee: Dr. P. 0. Fromm for his support and guidance, Dr. J. R. Hoffert for his ever open door and ears, Dr. L. F. Wolterink for his thoughts and philosophy, Dr. J. B. Scott for his down-to-earth approach to science, and Dr. R. W. Hill for his interest and attentiveness. Special thanks are also due the members and friends of the fish lab especially Esther Brenke for her technical help and friendship. I would also like to extend a word of gratitude to other friends in the department of physiology: Paul Sorenson for his friendship over the past five years and for the many hours of discussion over countless pitchers of beer while listening to Barb Bailey-Hutchinson, Lynne Olson for her friendship and introduction to beer and nachoes at El Azteco, and Sam Rhodes for his parallel childhood. Finally, I would like to thank my best friend and wife, Debbie Budde-Jackson without whose constant support this dissertation would not have been possible. I am grateful for support from the National Science Foundation grant ENV 77-12300. ii TABLE OF CONTENTS Page INTRODUCTION ..................................................... 1 MATERIALS AND METHODS ............................................ 17 Experimental Animals ....................................... 17 Equipment .................................................. l7 Preparation of Gills for Perfusion ......................... 21 Perfusion Solutions ........................................ 22 Bath Solutions ............................................. 23 Radioactive Tracer Measurements ............................ 24 Measurement of Flow Rates .................................. 24 Viscosity of Perfusate ..................................... 25 Treatment of Data .......................................... 25 S. 1. Units ............................................. 25 Normalization of Data by Dry Weight of Filaments Perfused ............................................. 25 Calculation of Permeability Surface Area Product (PdA).. 26 Calculation of Gill Vascular Resistance ................. 3l Representation of Data .................................. 34 Experiment Protocols ....................................... 34 EXPERIMENTAL, RESULTS, AND DISCUSSION ............................ 36 General .................................................... 36 Osmotic Gradient Experiments .............. ‘ ................. 38 Efflux and Influx Experiments .............................. 41 Bath Tracer Activity Experiments ........................... 44 Stirring Experiments ....................................... 47 Flow Experiments ........................................... 53 Efferent Pressure Experiments .............................. 64 Perfusate pH Experiments ................................... 80 Bath pH Experiments ........................................ 85 Summary and Conclusions .................................... 89 LITERATURE CITED ................................................. 92 APPENDICES ....................................................... lOO LIST OF TABLES TABLE Page 1. Experimental Conditions .................................... 35 2. Osmotic Gradient Experiments ............................... 40 3. Parameters of Curve Fit to Resistance-flow Data ............ 63 4. Efferent Pressure Experiments .............................. 68 5. Perfusate pH Experiments ...... . ............................ 8l iv LIST OF FIGURES FIGURE Page l. Diagram of a transverse section through a gill filament... 9 2. Diagram of the apparatus used to perfuse gills ............ 19 3. Schematic diagram of a perfused gill ...................... 28 4. Schematic diagram of a resistance-capacitance analogy of an isolated gill .......................................... 33 5. Bath tracer activity experiments .......................... 46 6. Stirring experiments ...................................... 50 7. Flow experiments .......................................... 56 8. Perfusion pressure response to altered flow ............... 58 9. Efferent pressure experiments ............................. 67 lO. Diagram of possible relationship between veno-lamphatic vessels and lamellar arterioles ........................... 72 ll. Relationship between gill vascular resistance and leak.... 76 l2. Response of a gill to decreased perfusate pH .............. 83 13. Response of gills to decreased bath pH .................... 88 INTRODUCTION Fish represent an important natural resource by providing (among other things): (a) significant amounts of animal protein for human nutrition, (b) various medically and industrially important chemicals, and (c) abundant recreational activity for many people. This valuable resource is being endangered by the daily release of smoke and chemicals into the air and the discharge of effluent from sewers into our surface waters. Because of their intimate contact with the environment. the gills of fish are maximally exposed to any soluble and suspended pollutants present. Gill functions such as respiration and ionic, osmotic and acid- base balance (Evans, 1975), depend upon the relative permeability or impermeability of this organ. Thus, one action of a toxic substance on fish might be to alter gill permeability. Synthetic detergents, for example, have been shown to increase the permeability of trout gills to water (Jackson and Fromm, 1977). Before the effects of toxicants on gill permeability can be evalu- ated the role of physiological variables in determining gill transfer capacity must be studied. These factors include blood flow, blood pres- sure, and osmotic and ionic concentration gradients, which may determine the effective transfer capacity of the gill in addition to the perme- ability of the epithelium. The movement of water can be used to characterize the relative permeability of a biological membrane to small nonelectrolytes (Haywoodl et a1., 1977). Thus, by studying how various factors affect water transport across a membrane, information is gained with respect to the relative transfer capacity of the membrane. Theoretically there are several factors that directly affect water transfer across a biological membrane: the osmotic and hydrostatic pressure gradients, solute con- centration gradients across the membrane, and the permeability and surface area of the membrane. For a simple system composed of two well mixed compartments separated by a thin, homogeneous, semipermeable mem- brane, these factors can be combined into Equations 1 and 2 relating the flow of solvent (water) and solute across the membrane (Kedem and Katchalsky, 1958): C4 ll LpA (AP - An) (1) J s PsA ACS + (l - os) JVCS (2) where (see Appendix II for definition of units): av = Volume (bulk, hydraulic) flow (cm3/s) Lp = Hydraulic conductivity (cm/kPa.s) A = Effective (functional surface area) (cm3) Ap = Hydrostatic pressure gradient (kPa) An = RT2(oiyiAC1) = Effective osmotic pressure gradient (kPa) R = Gas constant (cm3.kPa/mol.K) T = Absolute temperature (K) Oi = Staverman's reflexion coefficient Yi = Osmotic activity coefficient AC1 = Concentration gradient for the ith (mol/cm3) solute 1n the system JS = Solute flow (mol/s) PS = Solute permeability (cmVs) ACS = Solute concentration gradient (mol/cm3) oS = Staverman's reflexion coefficient for the solute transported C; = Mean solute concentration within the membrane (mol/cm3) Solute flow (Equation 2) is composed of two terms: a diffusive term (PSA ACS) and a convective or solvent drag term ([1-os] Jv C;). If the volume flow (JV) is identically zero, any movement of solute across the membrane would be due to diffusion, and Equation 2 becomes Fick's equa- tion of diffusion, where the solute permeability (PS) is simply the diffusion coefficient (05’ cmZ/s) for the solute divided by the membrane thickness (Ax, cm), assuming that the concentration gradient across the membrane is linear. The reflexion coefficient (0) represents the amount of interaction between water and a solute and is a property of the membrane, not the solution (Staverman, 1951). For nonelectrolytes, o is thought to have a value between zero and one (House, 1974). When 0 = O, the solute is carried freely across the membrane by the solvent flow; if 0 = 1, Jv would have no effect on the movement of the solute across the membrane. In terms of osmotic pressure, a membrane with a reflexion coefficient of 0.5 for a solute would indicate that the effective osmotic pressure generated by that solute fer a given concentration gradient would be 0.5 the value calculated from the van't Hoff relationship, An = RTAC. When a net flux of electrolyte exists across a membrane an addi— tional component must be added to Equation 1 to account for any electro- osmosis that may occur (Kedem and Katchalsky, 1963). In most biological studies this component of water flow is neglected as it is much smaller than the other sources of water movement (House, 1974). If the solute in question is labeled water (w*), such as tritiated water (3HOH), Equation 2 can be rewritten (House, 1974), Jw* = PdA Acw* + LpA (AP - An) Ch, (3) assuming that the reflexion coefficient for 3 HOH (OW*) is zero, and that the surface area for diffusion and hydraulic flow are equal. Thus, data for the diffusive permeability (Pd), the surface area (A), and the hydraulic conductivity (Lp) are required to completely 3 characterize a membrane with respect to HOH and/0r H20 movement. In practice, L is replaced by the term osmotic permeability (Posm, cm/s) p which is given by Posm = LpRT/Vw (4) where V; is the partial molar volume of water (18 cm3/mol). Use of Posm allows direct comparison with Pd as the dimensions (cm/s) are the same. The osmotic permeability (Posm) can be determined experimentally by imposing either an hydrostatic pressure difference or an osmotic concentration difference of known magnitude across a membrane, measuring the resultant volume flow (JV) and applying Equation 1 to determine Lp and Equation 4 to calculate Posm. From Equation 3, Pd can be experimentally determined from the f1ux of labeled water across the system provided the convective component of the flux is either zero or very small with respect to the diffusive flux. This method of determining Pd depends on the assumption that 3HOH is an ideal tracer for H20, an assumption which appears to be valid in bio- logical systems (see House, 1974 and Curran et al., 1967 for references). The relationships presented were derived for a two compartment sys- tem with a homogeneous barrier. For such a system, the diffusive perme- ability (Pd) should be equal to the osmotic permeability (Posm) (i.e., Posm/Pd = l) as the fluxes of water originating from either a tracer con- centration difference or a pressure difference should both be diffusional across a uniform boundary (House, 1974). When Pd and Posm are measured in biological systems, it has often been observed that Posm is larger than Pd (Posm/Pd > 1) (Koefoed-Johnson and Ussing, 1953; Dainty and House, 1966b; Motais et al., 1969). Ussing (1953) attributed this dif- ference to the presence of water filled pores in the membrane and an actual bulk flow of water through the pores. Dainty (1963), on the other hand, suggested that Pd may be underestimated due to the presence of boundary ("unstirred") layers at the surface of membranes and deple- tion layers within the membranes. Concentrational boundary layers arise when mass transport occurs between a moving fluid and a body. The opera- tional thickness (6c) of such a boundary layer is inversely proportional to the square root of the fluid velocity and directly proportional to the square roots of the kinematic viscosity and the diffusion coefficient of the fluid for the substance being transported (Bennet and Meyers, 1962). Dainty and House (1966b) demonstrated that the estimated permeability coefficient (Pd) of frog skin varied directly with the rate of external stirring, indicating that boundary layers may be in- volved in limiting the rate of diffusional water movement across bio- logical systems. In certain cases even when boundary layers are taken into account the osmotic permeability (Posm) is found to be greater than the diffu- sive permeability (Pd) (House, 1974) suggesting that membrane pores or slits may also be present in epithelia. Thus, in more complex systems one must consider not only the permeability of cellular membranes, but also the presence of membrane pores and/or unstirred layers when study- ing the transfer capacity of biological membranes. For biological systems with relatively simple surface geometries, such as frog skin or toad bladders, the area across which water movement occurs (functional surface area, A) is equal to the anatomical surface area. However, in systems with more complicated geometries, such as gills, lungs, or systemic capillary beds, functional surface area cannot be equated with anatomical surface area. In such systems functional surface area is determined by the distribution of blood flow such that functional surface area is always less than or equal to anatomical sur- face area. Thus, the calculation of a permeability coefficient (Pd) of such systems is virtually impossible, rather a permeability-surface area product (PdA) is calculated. This product represents the effective transfer capacity of a system. The permeability of fish to water has been examined by many people, and it is well-established that the gills are the primary sites of water transfer (Krogh, 1939; Motais et al., 1969; also see Motais and Garcia-Romeau, 1972), the skin of teleosts being relatively impermeable (Fromm, 1968). Teleost fish have eight gill arches, f0ur on either side of the head. From each gill arch extend two rows of filaments. Each filament bears on its upper and lower surfaces rows of plate-like secondary lamellae across which exchange of gases, ions, and water can occur. Blood is delivered to the gill arches by afferent branchial arteries which originate bilaterally from the ventral aorta. Blood then flows from the afferent branchial artery into a filamental artery, and finally to a secondary lamella by way of lamellar arterioles (Figure 1). Lamellar arterioles may communicate with one to three secondary lamellae (Morgan and Tovell, 1973; Laurent and Dunel, 1976). The secondary lamellae of rainbow trout (Salmo gairdneri) have been examined and described in detail by Morgan and Tovell (1973), and, in general, are similar to those observed in other species (Hughes and Grimstone, 1965; Newstead, 1967). Trout lamellae consist of a pair of epithelial sheets, two cell layers thick, separated by pillar cells. Between the epithelium and the pillar cells lies a basement membrane. The body and flanges of adjacent pillar cells delimit the blood space. Along the outer edge of a lamella runs a marginal channel which is lined by typical endothelial cells, in contrast to the pillar cell channels which resemble the reticulo-endothelial network of the mammalian spleen (Hughes and Weibel, 1972). A basal channel has also been described (Smith, 1976). The cells of the epithelial layers are connected by tight junctions or desmosomes (Morgan and Tovell, 1973). The membrane of the outermost coppomLPG mumuwucp mzocc< . ee_c_Pas_m .oceEapwc _~Pm a g M m—memF agmucoumm u 4m ppmu gmppwa coco ppww cwm> Foucmseppm n >m II II <0 (50. ucmsm_wm _.pu u u mpomcmuce LeFFmEmp acmcmwmm u <4m acougm Foucmse_ww acmgmwwu u Pecucmu u m>u mampppgmo u u cmm> pewgomcm u >m m—owcmugm Loppmsmp ucmgmcm< u <4< apogee —mu:msm_wm newcomm< u mcmcu a mo Emcmmmo .P eezmca F «gamma (1‘42 \. mUIAu HWIHHUMMMMmHmumuIInMflIIIIIIAWIIunIIIAW I/II>u_ --...QJII E1] _... II lm\,0 I. ’, HI. H..\| we eoeeumwmmm u .Abxee meme pre age be bemused sew: egewe; mew mewmeeco he eewLe> we eweeo mwgw .p—wm e» Pepmwe meweeu we museumwmom u m—emme>___wm acecewwe we eoeeumwmem u e—eeeee we eeeeumwmmm u ee_wesep eee mwemme> F_wm veecewwe we eoeepmwmem u seww peeepwwe euemewcme u wwwm euew seww epemewcme u prm on wepmwe emeceomg egemmege see: u wwwm op weswxece emeseoec exemmece see: u Fwwm we Eeumam meeewe> we eoeeuweeeeu u mwmmme> pwwm uemcewwe we eueeuwoeeeu u eewpesew eee mwemme> ppwm uemcewwe we eoeeuwoeeeo u IUQJIUUGJ-H mucosa: CUQJ>¢U UQQQO'OO QU .meewuweeeo we now ee>wm e emcee “seameeo emceewmeeu use w—wm we meoeeuwoeeeo eee meoeepmwmmm .ppwm eeuewemw :e we xmeweee mueeuwoeeeoIeoeeumwmmg e we segmewe owuesmgum .e ecemwu 34 R9 = (pa/03) - [Rc + (09/03) (Rc + Re)] x g dry filaments (12) When efferent pressure (Pe) was varied resistance was calculated as R9 = [(9a - Pe)/Oa] - (1 + 08/0a) RC x g dry filaments (13) Resistances calculated using Equations 12 or 13 take into account any perfusate leak (Oa - Oe) from efferent venous pathways, although these resistances are not independent of such leak (see Figure 4). Representation of Data Results of experiments were expressed as means :_one standard error (number of observations) and statistical comparisons made at the a = 0.05 level unless otherwise stated. Experiment Protocols All experiments were performed using two second gill arches removed from the same fish: one perfused with solutions containing 10 umol/L epinephrine (Epi experiments) and one perfused without epinephrine (No-Epi experiments). The conditions under which each experiment was performed are outlined in Table 1. 35 wwweuee Lew axe» cw weeswceexe peeew>weew mew .eewce> Levee; u a max .wpwm ee egemmmge uemgmwwm u w m.m\_s .mppwm euew ope; zeww muemewcee u o m\w .geee :w Lee mewggwum we :ewpe—e>ec we open u e cexemx .gaee we zew>wuoe owwwoeem :o:m u o ~s\emx .wpwm mewgeuee maemewcee we zuw>wuoe ewwwueem :o:m u e ex\mee .sewpepeEme even I e > m.w m.w m~.o m.mp m.m o m ze seem e.w > m.. m~.e e.e_ m.e. o m 2e ebemeweea e.w e.w > m~.e e.ep m.e e m eesmmece beecewwe o.w m.w m._ > w.ww m.m o m sewn e.w e.w m.F m~.e > m.e e m eeeecwem o.w m.w m.w m~.o w.m~ > o m auw>wuoe Looms» seem o.w m.w m.w mm.o w.w— o w.e— m xewwwm o.n m.w m._ m~.o m.wp m.w o m xe—weH o.w m.w m.p m~.o m.mw m.w o m> ueeweecm ewuesmo 1e seem In wcea wee bee eewem one nee fleece beeeweeexm 225:8 55528: g 2E EXPERIMENTAL, RESULTS, AND DISCUSSION General The preparation used in the present study appeared to be more stable than that originally described by Bergman (1973) and those used in other laboratories (Wood, 1974; Wood et al., 1978; Haswell and Randall, 1978) in that tritiated water flux and vascular resistance remained constant for periods up to 4 h after cannulation. Thus, the reported rapid deterioration of isolated gill preparations reported by other investigators (see Haswell et al., 1978 for references) was not a problem in this study. This may be the result of several factors. First, the use of albumin in the perfusate appeared to increase the stability of the gills as compared to the use of no colloid (Bergman, 1973) or dextran (Jackson, 1976) in previous studies in this laboratory. The use of albumin in artificial perfusion solutions has also been reported to stabilize perfused mammalian organ systems (Landis and Pappenheimer, 1963; Rippe, 1978). The strict control of perfusate pH to a physiological level at 10 C may also have been important. In addition, during the 30 min pre-experimental period the volume flow rate into gills was increased to a maximal value and then reduced to the level to be used during the first experimental period. This pretreatment resulted in more consistent data and may be related to wash out of metabolites or to an opening up of gill vessels after 15 min ischemia. Finally, the 36 37 reduction of the concentration of calcium in the perfusate may have influenced the stability of the preparation (Rankin and Maetz, 1971). Reduction of perfusate oxygen tension as suggested by Haswell et al. (1978) appeared, at least for resistance and tritiated water flux studies, to be unnecessary. The permeability-surface area product (PdA) determined for gill arches removed from 52 trout was found to be 15.78 :_2.77 ul/s.g for gills perfused with epinephrine and 5.65 :_l.l7 p1/s.g for arches per- fused without epinephrine. Samples were collected after 30 min perfu- sion at 0.25 m1/s.g with stirring revolution rate of 18.8/s and post- branchial pressure of 1.9 kPa. Using the values of PdA above and data from wood (1974) for lamellar surface area (2561.5 cmzlkg body weight) and unpublished data collected by Bergman in our laboratory (2.125 g dry filaments/kg body weight) an estimate of the lower bound on the true Pd for gills can be calculated as (13.09 :_2.298) x 10"6 cm/s and (4.69 :_ 0.97) x 10'6 cm/s for Epi and No-Epi groups respectively. These values are lower bounds on Pd because the anatomical surface area is the abso- lute maximum functional surface area available for mass transport. The values above confirm data for Pd obtained for trout gills by Haywood et a1. (1977) in vivo and in vitro (15.9 x 10'6 cm/s for Epi gills and 6.6 x 10"6 cm/x for No-Epi gills) and by Isaia et a1. (1978) in isolated gills ([16.3 and 9] x 10'6 cm/s for Epi and No-Epi conditions respec- tively). In every experiment epinephrine significantly increased permeabil- ity-surface area product (PdA) above, and decreased gill vascular 38 resistance (R9) below that of gill arches perfused with solutions not containing Epi (see Flow Experiments for representative values of R9). Osmotic Gradient Experiments The osmolality of the bath solutions was varied in two steps with sodium chloride or sucrose, and the effects on PdA observed. After the 30 min pre-experimental period in a 1% Ringer (control) bath (3 mOs/kg), flow into gills was measured, and 0.5 ml samples of the bath and perfu- sate effluent taken. The osmolality of these solutions was determined and aliquots taken for tritium determinations. Sodium chloride (1 g) or sucrose (30 g) was then added to the bath increasing its osmolality to 319 :_3 (6) or 305 :_3 (7) mOs/kg respectively. After 15 min the sam- pling procedure was repeated and the same amount of the appropriate osmolyte added to the bath. This increased the bath osmolality to 632 :_4 (6) and 620 :_2 (7) mOs/kg for sodium chloride and sucrose respectively. The osmolality of the perfusate entering gills was 278 :_2 (6) mOs/kg for the sodium chloride experiments and 271 :_3 (7) mOs/kg in the sucrose study. Osmolalities were measured using a microsample vapor pressure osmometer (Model 51008, Nescor, Inc., Logan, Utah). Treatment effects were analyzed using a blocked analysis of vari- ance with Student-Newman-Keuls multiple comparison method to compare treatment means (Sokal and Rholf, 1969). 39 The results of experiments in which the osmotic concentration difference across rainbow trout gills (ACosm) was varied using either sodium chloride or sucrose as solute are presented in Table 2. Neither osmolyte produced any consistent significant change in the permeability- surface area product (PdA) of gills perfused with (Epi) or without (No-Epi) 10 pmol/L epinephrine in the perfusate. For No-Epi gills exposed to varied sucrose concentrations the calculated F statistic was close to the tabular F statistic (3.00 versus 3.89 respectively, see Table 3). A critical number of runs test (Steel and Torrie, 1960) was performed on this set of data and indicated that if a true difference existed it should have been detected after only four experiments. An analysis of variance was performed on the data from the first four No-Epi sucrose experiments and the treatment F value was computed as 3.85. Thus, the F value actually decreased as the number of experiments increased from four to seven. Also, there was no consistent trend in the data f0r these experiments. For example, comparing the PdA values obtained for osmotic gradients of -266 and 351 mOs/kg, of the seven experiments, three did not change, two increased and two decreased. Thus, there was no indication of any relationship between Anosm and PdA. Comparison of sodium chloride data with sucrose data (Table 2) indicated a significant difference in the magnitude of PdA even at Acosm = -275 or =266 mOs/kg where both groups were suspended in 1% Ringer solution. These differences may be due to seasonal variations in gill permeability as sodium chloride experiments were performed during August. 1977 while sucrose experiments were performed during January, 1979. 40 mewsmeoewem peesuwz eemewcee mpwwm Eecw epem u ; mewcgemewee m\wes: ow mpw: comewgwe wwwwm Eecw euem u m Ammm_ .wwmem eee wememv mo.o u e .Eeeeesw we eesmme eee ewumwueum m Leweeew u w ewpmwpeum m peesaeesa empeweeweu u e meewue>semee we geese: u e meees we Lemme eseeeeum u u m.m\w: .ueeeese emse eoewsemrmuwwweeeELem n e mx\mos .zuwwewesme epemewcee I seem u e m.m Pmm ANF.NV mm.m ee.m N.e ~.m em wam-oz m.e mmm- emecoem m.ew wmm ANP.NV ae.m ee.~ m.e m.ep em wee m.mw mew- w.o_ emm we.~m ee.e ww._ e._ w.e_ _e gene-oz w Pp mum eewLewmu N.- emm seweem mew.me e..e no.F a.o e.m~ we ewae F.P~ mum- . see wfiwe wee owsou om omm emea e ea geese oowpoeme muemewceexm useweecm owuesmo .N epeew 41 Addition of either solute to bath solutions had no effect on the osmolality of the perfusate effluent nor did it affect the vascular resistance of gill arches in either group as perfusion pressure remained unchanged in all experiments when either sodium chloride or sucrose was added to the bath solution. Efflux and Influx Experiments Efflux and influx experiments were performed as follows: gills. were perfused during the last 15 min of the pre-experimental period with 3HOH (16.7 kBq/ml). At the end of the pre- perfusate containing experimental period perfusate inflow was determined and samples of the perfusate effluent and bath taken. The perfusate was then switched to one not containing tracer, and 3HOH was added to the bath (8.3 kBq/ml). Gill arches were then allowed to attain a new steady state (15 min), flow determined, and samples collected. The clearance of 3HOH (V, m1/s) was calculated as: v = Qa(Ca - ce)/(ca - Cb) (14) where Qa’ C , and Cb are as above and Ca is the specific activity of the e perfusate entering the gill. The ratio of clearances for tracer influx and efflux was then calculated. Clearances were used to calculate this ratio rather than the actual tracer fluxes (Bq/s) because the clearances are normalized with respect to the absolute specific activity of the tracer used. Clearances calculated in this fashion are directly propor- tional to their respective fluxes, such that the ratio of influx to 42 efflux clearances was equal to the actual ratio of influx/efflux for similar tracer gradient. These ratios were compared to 1 using a 2-tailed Students-t test. There was no significant difference between influx and efflux of 3HOH as judged by the ratio of influx/efflux in spite of a measured 272 mOs/kg osmotic concentration difference across gills. The values of influx/efflux were 0.94 :_0.09 (5) and 1.03 :_0.29 (5) for Epi and No-Epi experimental groups respectively. The flux of tritiated water (Jw*) across isolated trout gills was insensitive to the magnitude of osmotic gradients across gills and was independent of the direction of the net tracer flux in the face of an existing osmotic gradient. These results indicate that tritiated water moves across gills by diffusion, a fact that has simply been assumed in 3HOH flux across fish gills whether in vivo or every other study of in vitro. A similar conclusion is reached based on calculations using Equation 3. Consider a simple two compartment system separated by a semipermeable membrane with the following characteristics (representative values of Pd and Lp for epithelia after House, 1974, other values are hypothetical, symbols as in Introduction): Pd = 10'5cm/s Lp = 10'9cm/kPa.s A = 100 an2 AP = -4 kPa ACW* = 50 kBq An = RTo(-275 mOS/kg) = -647.3 kPa CW* = 25 kBq o = 1 and aw. = PdA ACW* + LpA(AP - Air) Cw. (3) 43 Thus Jw* = 50 Bq/s + 1.61 Bq/s Thus, the tracer flux due to hydraulic water flow across the system is at most 3.12% of the total tracer flux. If the effective osmotic pres- sure is less than its theoretical maximum (0 < l) the convective transfer of 3HOH across the membrane would be even less. Further, in this example, the osmotic permeability (Posm) calculated from Lp (Equation 4) is 1.308 x 10"4 cm/s which is 13.08 times Pd (Posm/Pd = 13). For the hydraulic component of the net tracer flux to be as high as 15% of the net flux, the ratio of PosmVPd would have to be approximately 65. Fish gills are much more complex than the simple two compartment system for which Equation 3 was derived, however, such calculations should serve as an approximation of the conditions. Estimates of the ratio of Posm/Pd for freshwater fish have ranged from 1 to 10 (Motais et al., 1969) indicating that the contribution of bulk flow to any net tracer flux would be negligible. Thus, variations in the osmotic concentration difference across gills or changes in perfusion pressure (i.e., the hydrostatic pressure difference across secondary lamellae) would have no detectable effect on 3HOH flux (Jw*) and the calculated permeability-surface area product (PdA). Also, from theoretical and experimental evidence it can be con- 3 cluded that the flux of HOH (Jw*) across gills is diffusional and that it is valid to calculate a transfer coefficient (PdA) from the 3HOH flux (Jw*) and gradient (ACW*). 44 Bath Tracer Activity Experiments The response of gills to successive stepwise increases in the 3HOH activity of the bath solution were performed as follows: tracer was added to the bath and at the end of the 30 min pre-experimental period, the flow rate into the gill was determined and samples of the bath and perfusate effluent taken. Tritiated water was again added to the bath solution and after 15 min, the sampling procedure outlined above repeated, and 3HOH added to the bath again. This procedure was per- formed for 4 different specific activities of the tracer (1.8 - 26 kBq/m1). Tracer flux (Jw*’ Bq/s.g) was calculated from the specific activ- ity of the perfusate effluent (Ce, Bq/ml) and the flow rate into the gill (0a, m1/s.g) as: Jw* = Qa Ce (9) The logarithmic mean concentration difference of tritiated water across the gill (ACW*, Bq/ml) was calculated from the bath activity (Cb, Bq/ml) and Ce (as above) as: ACw* = -Ce/1n(l - ce/cb) (10) A plot of Jw* against ACW* should be linear, the slope being PdA (ml/5.9) as in Equation 11. Thus linear regression analysis was applied to the data. Experiments consisting of stepwise increases in the tritiated water specific activity of the bath solution were performed to test not only 45 Figure 5. Bath tracer activity experiments. Jw* = Tritiated water flux ACW* = Logarithmic mean difference of tritiated water across g1ll Epi = Gills perfused with 10 pmol/L epinephrine No-Epi = Gills perfused without epinephrine in the perfusate 0.60: O.|5 « 46 o Epi (ifll‘S) A No Epi (§,n=5) — regression --- 95% confidence interval Is 00,,“ (k Bq lrnl) Figure 5 2'5 47 the model presented above (Equation 8), but also to demonstrate the stability of the isolated gill preparation as the step increases were separated by 15 min intervals. These experiments (Figure 5) indicate that there was a linear relationship between the tracer flux (0",) and the tracer gradient (ACW*) f0r both Epi and No-Epi experimental groups. The slope of the lines in Figure 5 equals the permeability-surface area product (PdA) and, as can be seen, the slope of the Epi line (19.04 1 1.40 ul/s.g) was significantly greater than that of the No-Epi group (4.00 :_0.57 pl/s.g). Stirring Experiments The role of boundary layers in limiting the rate of 3HOH movement across isolated perfused trout gills was examined by randomly varying the rate of mechanical stirring of the bath in which the gills were suspended. During the last 15 min of the pre-experimental period the stirring motor was adjusted to the stirring rate to be used during the first experimental period. At the end of the pre-experimental period the flow rate into gills was determined, and samples of the perfusate effluent and bath taken. The stirring rate was then changed to the next rate, and, after 15 min, the sampling protocol repeated. This procedure was followed for 5 stirring rates, PdA being calculated at each rate. Stirring rates were determined stroboscopically. Treatment effects were assessed using a split-plot analysis of variance (Cochran and Cox, 1957) with Student-Newman-Keuls test to compare means. 48 Increasing the rate of revolution of the stirring bar from 0 to 18.8/s resulted in a 2.5 and 2.9 fold increase in PdA for Epi and No-Epi experimental groups respectively. Over the stirring range tested, PdA increased asymptotically (Figure 6a) as the rate of mechanical stirring increased f0r both experimental groups. A split-plot analysis of vari- ance revealed that there was a significant interaction term between drug (Epi or No-Epi) and the rate of stirring. This could be the result of one of two phenomena: (a) either epinephrine altered the shape of the response curve to stirring rate, or (b) the variability of the response curve to stirring rate differed between the two treatments. To test these hypotheses, the data were normalized to the largest value of PdA observed during each-experiment for each gill such that all data were expressed as a number between zero and one, for each experiment. The split-plot analysis was recomputed and this time the interaction term was not significant (Figure 6b), although the main treatment (stir- ring rate) effect was still significant. This implies that the shape of the response curves was similar for both Epi and No-Epi gills and that the significant interaction term noted when the raw data were analyzed involved the variability of the response being different between the two groups. The highest stirring rate (18.8/s) represented quite P violent mixing: gill filaments were observed to oscillate in the vortex of the stirring bar with a maximum amplitude of approximately 5 mm. This agi- tation had no effect on vascular resistance: perfusion pressure remained constant when stirring rate was changed in all cases. 49 Figure 6. Stirring experiments. See text f0r details. PdA = Permeability-surface area product Epi = Gills perfused with 10 umol/L epinephrine No-Epi = Gills perfused without epinephrine in the perfusate a. Raw data f0r Epi and No-Epi gills (n = 6) Revolution Group 0 3.9 9.1 13.9 18.8 rate (1/s) Epi 7 12 15 17 18 PdA (pl/5.9) No-Epi 3 5 6 6 8 b. Normalized data (see text) (n = 6) Revolution Group 0 3.9 9.1 13.9 18.8 rate (l/s) Epi 0.30 0.62 0.77 0.91 0.93 PdA (relative) No-Epi 0.37 0.61 0.75 0.85 1.00 Means underscored by the same line are not significantly different (a = 0.05) (ul/s-g) PdA PdA relative 50 0 Epi 20- A No Epi '6‘ )1 issue) 12‘ . 4 4 0 5 10 I5 éo 1.0- l 0.8- T T 0.6« O.4i 0.2. i, , , , E O 5 IO 15 20 stirring rote (l/s) Figure 6 51 The contribution of boundary layers as a rate limiting factor f0r diffusion of water across gills has been assumed to be negligible (Motais et al., 1969; Isaia et al., 1978). However, in the present study homogenation of the external medium did significantly increase PdA. The data presented are in agreement with those obtained by Dainty and House (19660) for frog skin, and indicate that some boundary layer does exist at the surface of the epithelium which potentially can limit the rate of diffusion. In biological systems unstirred layers have been analyzed as a diffusion barrier in series with the membrane in question (Dainty and House, l966a,b; House, 1974). According to Dainty and House (1966b) the relationship between the true permeability of the membrane (Pt), the measured apparent permeability (Pa), and unstirred layer thickness (6C) is: 1/Pa = 1/Pt + (Sc/Dw (15) where Dw(cm2/s) is the free diffusion coefficient of the tracer. From Equation 15 it is seen that if the effective permeability of the boundary layer (Ow/6c) is much larger than the true permeability (Pt) then, the apparent permeability (Pa) would not be affected to a large extent by the boundary layer. However, if the true permeability (Pt) is of the same order of magnitude as the effective boundary layer perme- ability (Dw/oc) the apparent permeability (Pa) would be much smaller than the true permeability (Pt) and transfer across the membrane would be rate limited by the unstirred layer. 11111:. 52 With the following assumptions: (a) the permeability-surface area product (PdA) was 99% of its maximum value at the highest stirring rate (i.e., Pa/Pt = 0.99), (b) in Epi experiments 95% of the maximum surface area was perfused and (c) in No-Epi experiments 58% of the maximum sur- face area was perfused (Booth and Holeton, observation reported in Hoar and Randall, 1979) the calculated values of Pa are 15.6 x 10'6 and 6.5 x 10'6 cm/s for Epi and No-Epi experiments respectively. Using 5 3H011 free diffusion these data and the value of 1.61 x 10' cm2/s fer a coefficient (Dw*) at 10 C (interpolated from Table 1.2, House, 1974) Equation 15 predicts that the unstirred layer thickness (6c) for Epi experiments was 106 um and 250 pm for No-Epi experiments. Based on calculations by Hills and Hughes (1970) the resistance of a boundary layer to oxygen transfer at the surface of gills was 5 to 10 times that offered by the gill tissue. In the present context this would indicate that the membrane permeability (Pt) is 5 to 10 times greater than the boundary layer permeability (Box/6c). Using the value of the thickness of the gill tissue for trout (7.5 um) given by Hills and Hughes (1970) and assuming that diffusivity of gill tissue for oxygen (Dox) was the same as that of the boundary layer, Equation 15 predicts that the bound- ary layer thickness (6c) would range from 75 to 37.5 pm. Considering that the mode of ventilation in the present study was vastly different than the in vivo case considered by Hills and Hughes (1970) the two ranges of be calculated above are not incompatible. Thus, boundary layer effects can not be neglected when considering diffusional mass transport across fish gills. 53 The effects of mucus on water diffusion across gills has not been studied, although the diffusion coefficients of fish mucus for water and oxygen (Ultrsch and Gros, 1979) and sodium and chloride (Marshall, 1978) have been measured and are not significantly different than the respective coefficients in water or saline. The viscosity of mucus is probably greater than that of water, hence, mucus may have an important effect in terms of a boundary layer at the surface of the secondary lamellae as pointed out by Ultrsch and Gros (1979). One might question whether or not the boundary layer data in the present study have any quantitative or qualitative relevance to in vivo conditions because the mode of ventilation of the isolated gill in this study was vastly different from that of the intact animal. In this regard, Sorenson and Fromm (1976) and Wood et a1. (1978), reported that increased ventilation of perfused heads and whole trout preparations increased heat and oxygen transfer respectively. Thus, boundary layers nay play a significant role in determining the transfer capacity of fish gills. In addition to recruitment of ventilated units (Hughes, 1972; Hughes and F105, 1978) altering boundary layer resistance may be an effective means of varying the transfer capacity of the gill to water, ions and oxygen. Flow Experiments The flow rate (0a) into gills was varied in a random, stepwise fashion at 5 or 7 different flow rates (8.3 x 10" - 1.67 x 10'2 m1/s). This range was used regardless of the size of the fish from which the 54 gills came. Thus, the above range corresponded to a range of flows from 0.01 to 0.68 ml/s.g dry filaments. The total range of cardiac output in vivo is 0.039 to 0.78 ml/s.g dry filaments with the normal range - 0.11 - 0.39 ml/s.g (calculated from data in Hoar and Randall, 1970). Pulse rate ranged from 0.167 to 1.5/s. At the end of the 30 min pre-experimental period, the flow rate into each gill arch was determined, and samples of the perfusate effluent and bath collected. The pump was then adjusted to the next flow rate, perfusion pressure allowed to stabilize (5-10 min) and the sampling protocol repeated. This procedure was repeated for each flow rate used. Permeability-surface area product (PdA) and gill vascular resistance (Rg) were calculated at each flow rate. An increase in the volume flow rate into gills resulted in an asymptotic increase in the transfer capacity (PdA) of gill arches per- fused with 10 pmol/L Epi or without, the response being more pronounced in the Epi than in the No-Epi experimental groups (Figure 7a). In addition, vascular resistance decreased as volume flow increased, the resistance of Epi treated gills always being less than their No-Epi counterparts (Figure 7c). Thus, PdA responded inversely to R9 as flow was increased. To examine the shape of the Epi and No-Epi PdA response curves to varied flow rates, data from Figure 7a was normalized to the mean value of PdA calculated from values obtained at the three highest flow rates. Normalized data are presented in Figure 7b. As can be seen, the Epi and the No-Epi normalized data fall on the same smooth curve (fitted by eye) indicating that the relative response of both groups to variations 55 Figure 7. Flow experiments. See text f0r details. Epi = Gills perfused with 10 umol/L epinephrine No-Epi = Gills perfused without epinephrine in the perfusate PdA = Permeability-surface area product 0a = Perfusate flow into gills R9 = Gill vascular resistance Number of observations (n) of means are as indicated f0r Epi means in part a. a. Qa versus PdA b. Data from a. normalized to asymptotic values of PdA. See text. C. Qa versus Rg (ul/s-g) PdA 56 ° Epi 4 No Epi n - +=xise o 20‘ 5 1.0* 8 6 7 9 06« /{ E ‘ 0.6: 10‘ m in E 0.4%, U 5’ ,H’l’H—fiI' 3' + 0.24 0.1 012 0.3 o'.4 6.5 0'1 0'2 013 0.4 0.5 00 (ml/s-g) o. (mI/s-g) c 250 '5. 200‘ J: \ O O. :‘5 ISO‘ 100« °= i 504 + \ N“~ .-.—.fifi*_‘ 0.I 0.2 0.3 (f4 0.5 do (ml/s-g) Figure 7 57 in perfusate flow was the same. Because there was no maximum limiting value of resistance by which gill vascular resistance could be normalized, the technique used above to compare PdA response curves could not be applied to gill vascular resistance data. When the pumping rate was increased perfusion pressure for both groups increased rapidly and then declined over a 10 min period to some new steady-state value (Figure 8). If flow was decreased the inverse of the above occurred: pressure initially declined, then over a 10 min period it gradually increased to some new steady-state. These new steady-state values were higher or lower respectively than those recorded for flow rates prior to the change in pumping rate. Increased perfusion flow rate increased the conductance of isolated gill arches with respect to perfusate flow and to diffusion of 3HOH across the gills. These results confirm the reports of Sorenson and Fromm (1976) for heat transfer and Wood et a1. (1978) fer oxygen trans- fer and similar data have been reported for mammalian capillary beds (Renkin, 1959). Increases in gill transfer capacity for 3HOH (PdA) resulting from increased perfusate flow could be due to variations in l) boundary layers in the lamellar blood space, 2) changes in the perme- ability (Pd) of the gill due to the varied pressure gradients as flow varied, 3) increased surface area (A) due to distension of lamellar channels, and/or 4) recruitment of exchange units and hence increased surface area (A). The contribution of any boundary layers in the blood space in altering PdA would be insignificant. This can be seen by consideration of the relationship of a momentum or velocity boundary 58 Am.m\pev wwwwm epew maemewcee we 3e—m eoeeemwmec Leweeeee mew eeueecceo me: Aegemmece sees no: .awee emee mwzm :wv «Lemmece eewmeweme maemewgee mm» cw mewgzeeewee peemuwz emmewcme wwwwm oewceeoewao e\_aes e. new: eonswcoa m__we wam-oz wee .m_weume Lew axe» mom .emueoweew me mzerw .ueeswgeaxm wee sesw museums e>wueueemmsemm .3eww emceuwe ea emceemmc egemmmse :ewmewgem .m msemwm 59 m ecemwm tea on _ neeueo 11— easeeeeeeeeeeeeessemeégggeemesmwsgmemm . Em o2 .m e é_§§e§e§§§e§§e§e§e§m§§§§§§ 4 Eu .4 . 60 layer (6) and a concentration boundary layer (BC) with respect to a fluid's kinematic viscosity (0) and the diffusion coefficient for labeled water (Dw*)' This relationship (Bennet and Meyers, 1962) is given by - 1/3 6/6c - (v/Dw*) (15) The momentum boundary layer in the gill blood space can be no larger than the radius of a lamellar channel which can be estimated as being no larger than 3 pm (Wood, 1974). The kinematic viscosity (v) of the perfusate in the present study was 0.01566 cm2/s (see Methods) and the 3HOH estimated as 1.61 x 10"5 diffusion coefficient for cm2/s (inter- polated from Table 1.2 in House, 1974). Application of Equation 16 predicts that the permeability of a boundary layer (Dw*/6c) would be 0.399 cm/s which is at least 2000 times greater than the maximum value of gill permeability (Pd) estimated in the present study (Figure 7a). From Equation 15 (see Stirring Experiments) it can be seen that such a large boundary layer permeability would have no effect on an estima- tion of gill permeability (unlike the presence of relatively thick boundary layers at the external surface of the secondary lamellae). Thus, internal boundary layer effects can be neglected. Increased gill water permeability, pgr_§g, due to increased flow and hence increased pressure is unlikely. Such changes would result in an increased hydraulic water flow (JV) across gills which, theoretically, would actually decrease the measured influx of 3HOH (see Equation 3) and decrease the estimated PdA. As indicated in the discussion of osmotic gradient experiments above, changes in hydraulic water flow across gills 61 would have no noticeable effect on PdA. Distension of pillar cell channels and the resultant increase surface area overlying the channels can also be discounted as a possible source of the increased PdA. By analogy with Burton's (1962) argument for capillaries, the small size of the lamellar channels (3-8 pm) pre- cludes significant distension of these channels within the physiological pressure range (l.25—4.5 kPa). Thus, the most likely explanation for the increased transfer capacity (PdA) as flow increased is recruitment of additional exchange units. Wood (1974) used a simple n parallel channel model of gills (Muir and Brown, 1971) to analyze gill vascular resistance changes resulting from varied pressure gradients. Using experimental resistance, pressure, and morphometeric data he compared measured vascular dimensions and numbers to those predicted by the model, and found that variations in gill resistance could be best explained by changes in the number of units perfused rather than changes in the caliber of existing channels. The discussion presented above for gill transfer capacity support this conclusion. Recruitment may occur due to passive changes in pressure gradients across the gill and/or passive distension of prelamellar vessels (filamental arteries or lamellar arterioles) from the increased perfusion pressure as flow increased. Booth (1978), using vitally stained red blood cells in vivo, demonstrated that only 58% of the secondary lamellae were perfused in resting rainbow trout. He reported that in some cases whole filaments did not show the presence of labeled cells. Thus, recruitment of individual secondary lamellae and even whole filaments of secondary lamellae is physiologically possible. 62 The data presented in the present study suggest that fish can vary the transfer capacity of their gills passively, by varying cardiac output, or actively, through the action of neural and hormonal mechanisms. The parallelism of the PdA versus flow response curves (Figure 7b) for gills perfused with or without 10 pmol/L epinephrine suggest that the mechanism by which PdA is altered (i.e., recruitment) is the same in both instances. This implies that Epi data differs from No-Epi data only by some constant multiplying factor such that the relative response of the two groups, with respect to PdA, is similar, with Epi data simply increased by some constant factor. This could result from the action of epinephrine on the gill epithelium increasing permeability (Pd) and/or on the gill vasculature increasing the number of units perfused (i.e., increased A). Resistance data (Figure 7c) were analyzed in an attempt to determine the relative contribution of these two phenomena. Data presented in Figure 7c were fitted to a decreasing hyperbolic function of the form Rg=MNaib on The results of the curve fit are given in Table 3. The asymptotes (K2) of the flow-resistance curves for the two groups were not significantly different while the slopes of the curves (K1) were significantly differ- ent. This indicates that if flow were increased enough, vascular resistance of Epi and No-Epi gills would be identical. Presumably at this point the number of units perfused would be identical for Epi and No-Epi gills. Thus, if the effect of epinephrine on PdA was only to increase the number of units perfused (i.e., A) one would expect that 63 Table 3. Parameters of Curve Fit to Resistance-flow Data d a b c 2 Group n K1 S(K1) K2 S(K2) r Epie 10 1 18 0.04 5 18 1 29 0 99 No-Epif 10 2 61 9 0 15 11.20 4 36 0 98 a = number of points used in the curve fit 0 = parameter of fitted equation, see text c = standard error of the parameter d = coefficient of determination e = data from gills perfused with 10 pmol/L epinephrine f = data from gills perfused without epinephrine g = significant difference by Student's t-test, a = 0.05 64 the curves for raw flow versus transfer capacity for Epi and No-Epi gills (Figure 7a) would have the same asymptotes, the magnitude of which would depend solely on the absolute number of exchange units that could be perfused. This was not observed: the asymptote of the epinephrine gill PdA data was about 2.5 times greater than the PdA asymptote for gills perfused with solutions not containing epinephrine (Figure 7a). Thus, the effect of epinephrine on PdA cannot be explained simply on the basis of its effect on A (i.e., recruitment of lamellar units). However, Booth and Holeton (observations reported in Hoar and Randall, 1979) have found that, in vivo, the number of secondary lamellae perfused increases from 58%, at rest, to 95% after administration of epinephrine. Thus, it appears that epinephrine produces a mixed effect on gills resulting in both recruitment (increased A) and altered permeability (increased Pd). Efferent Pressure Experiments In this series of experiments changes in in vivo systemic vascular resistance and alteration of dorsal aortic pressure were mimicked by varying the height of the outflow cannula from the gills and effects of this manipulation on transfer capacity (PdA) was determined. Efferent pressure (Pe) was varied in a latin square design (Cochran and Cox, 1957). During the last 15 min of the pre-experimental period the height of the outflow was adjusted to the appropriate level for the first experi- 'mental period. At the end of the pre-experimental interval the flow rate into gills was measured, and samples of the perfusate effluent and bath collected. Efferent pressure (Pe) was then changed to the 65 appropriate value, the pressure allowed to reach a new steady state, and the sampling regime repeated. The permeability-surface area product (PdA) and gill vascular resistance (Rg) were calculated for each pres- sure used (1.9, 3.7, 5.5, 7.2 and 9.0 kPa). Dorsal aortic pressures (i.e., Pe) range from 2 to 4 kPa in vivo (Satchell, 1971). A latin square analysis of variance was used with the Student-Newman-Keuls test for comparison of treatment means. Increased postbranchial pressure (Pe) resulted in a significant linear decrease in the permeability-surface area product (PdA) for gills perfused with or without 10 umol/L epinephrine, the slopes of the lines being similar f0r both treatment groups (Figure 9a). Since a latin square experimental design was used, any time or position dependent effects were taken into account and were not significant. Thus, the effects seen were related to the increase in efferent pressure (Pe) (Table 4). Vascular resistance (R9) also decreased as efferent pressure (Pe) increased (Figure 9b), the effect being slightly more pronounced in gills perfused without epinephrine compared to gills perfused with 10 umol/L Epi (see Table 4). In addition, the outflow/inflow ratio (OE/06) decreased as Pe increased (Figure 9c and Table 4). This indi- cates that elevated Pe increased the loss of perfusate from gills (i.e., leak). No differences were noted between Epi and No-Epi gills with respect to leak (De/03). The ratio of pulse pressures recorded distal (APe) and proximal (APa) to gills for each efferent pressure were calculated. By analogy with the resistance network (Figure 4) used to calculate vascular resistance, the ratio of pulse pressures (APE/APa) represents the amount 66 eae\oae menses as .e eo\eo memce> me .e um memce> me .e me .e eueeumwmes Leweome> wwwm u mm mwpwm euew 3e_w ea :eww meme—wwe muemewgee we ewpem u eo\eo Awwwm ea weumwe emecouem ecemmecm eeesv esemmese weegewwm u em . . mppwe e o op Feswxece eee weumwe eeeceoeg mmcemmece emwee we ewuem u me me woeeese eece woewgem-AawwweemEgme u