SODQUM UPTAKE BY ISOLATED, PERFUSED GiLLS 0F RAINBOW TROUT (SALMO GAIRDNERI), WITH SPECIAL REFERENCE TO PATTERNS OF GILL BLDDD FLOW Thesis for the Degree of Ph. D. MTCHIGAN STATE UNIVERSITY BRENT D. RICHARDS 1968 THESIS 0-169 EUN NTVERSIT TTTTTTTT TTTTT TT TTT 29310569 93 O MIC HT TTTTT “3 Ag: This is to certify that the thesis entitled SODIUM UPTAKE BY ISOLATED, PERFUSED GILLS OF RAINBOW TROUT (SALMO GAIRDNERI), WITH SPECIAL REFERENCE TO PATTERNS OF GILL BLOOD FLOW presented by Brent D. Richards has been accepted towards fulfillment of the requirements for _P_h_:_D_o___ degree in My WM 0 V/w‘ Major professor Date_AL1g2§_t_8.._l9_68.__ ' mam .v HOME & SITNS' 800K BINDEIIY INC. umnv mans-ac ABSTRACT SODIUM UPTAKE BY ISOLATED, PERFUSED GILLS OF RAINBOW TROUT (§ALMO GAIRDNERI), WITH SPECIAL REFERENCE TO PATTERNS OF GILL BLOOD FLOW by Brent D. Richards The pattern of fluid flow through the isolated, perfused rainbow trout gill can be altered from sinus flow to lamellar flow. Under control conditions and with acetylcholine, a majority of the fluid flows through the filamental sinus. 'When epinephrine is added to the perfusion fluid, the majority of the fluid flow is via the lamellae. These blood flow patterns were determined by perfusing the gills with india ink containing the various vasoactive substances, and then examining the gills histologically. The filamental sinus is apparently a portion of the lymphatic system and connects to the filamental lymph vessels at frequent intervals. There are also direct connections from the afferent filamental artery to the lymph vessels and sinus and from the sinus to the efferent filamental artery. The lamellar-afferent and lamellar- efferent vessels have muscular walls for at least part of their lengths and probably function in the regulation of Brent D. Richards 2 the pattern of fluid flow through the gill. Isolated, perfused rainbow trout gills are capable of actively taking up sodium against a concentration gradient of at least 60:1. This sodium uptake was a apparently stimulated directly by the low sodium content of the choline and sucrose Ringer solutions used. It could also be produced by the addition of epinephrine to perfusion fluid which was not low in sodium. The amount of sodium in the various solutions studied was determined by flame photometry. Studies using metabolic inhibitors suggest that the ATP necessary for the uptake of sodium is derived almost exclusively from oxidative metabolism. Although sodium and chloride can be taken up independently, evidence is presented that there is an interaction between sodium and chloride uptake. The rate of sodium uptake is not primarily dependent upon either the rate or the pattern of blood flow through the gill. SODIUM UPTAKE BY ISOLATED, PERFUSED GILLS OF RAINBOW TROUT (fiALMO GAIRDNERI), WITH SPECIAL REFERENCE TO PATTERNS OF GILL BLOOD FLOW By Brent D: Richards A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1968 ACKNOWLEDGMENTS The author wishes to thank Dr. P. 0. Fromm for his support and guidance during this study. Thanks are also extended to Dr. J. R. Hoffert for his many helpful suggestions. He also wishes to thank the other members of his committee, Dr.‘W. D. Collings, Dr. J. M. Dabney and Dr. 'W. L. Frantz for their interest and help in this project. In addition, the writer is indebted to the Department of the Interior, Federal Water Pollution Control Administration grant SIWP-00807 for support of this project. 11 TABLE OF CONTENTS Page INTRODUCTION 0 O O O C O O C O O O O O O O O O O 1 REVIEN OF THE LITERATURE . . . . . . . . . . . . 3 Circulatory Anatomy . . . . . . . . . . . . 3 Sodium Movement . . . . . . . . . . . . . . 5 MATERIALS AND METHODS . . . . . . . . . . . . . 11 Animals . . . . . . . . . Surgery . . . . . . . . . Perfusion Apparatus . . . Cannulation and Perfusion Circulatory Anatomy . . . Sodium Movement . . . . . Sodium Analysis . . Uptake of Sodium and Inhibitor Studi Statistics . . . . . . . . . . . . O O O O O O O O O N O 08 O N (7\ RESULTS 0 O O O O O O O O O O O O O O O O O Circulatory Anatomy . . . . . . . . . . . . 26 SOdium Movement 0 o o e o e o o o e o o o o 55 DISCUSSION 0 O O O O O O O O O O O O O O O O O O 62 Circulatory Anatomy . . . . . . . . . . . . 62 Sodium Movement . . . . . . . . . . . . . . 68 CONCIUSIONS o o o o e o e e e o o e o o e o o o e 72 APPENDIX 0 e e e o o o o e o o o o e o o e e o o 7Ll LITERATURE CITED 0 e e o o o e o o o e e e o e o 77 iii Table 1. LIST OF TABLES Page Sodium concentrations of the perfusion fluid and the fluid collected after passing through the gill, and the rate of sodium uptake for the various experimental perfusions . . . . . . . . . . 57 The rates of fluid flow through the gill during control and experimental perfusions and the percent change in rate from the control to the experimental perfusion . . . . . . . . . . . . . . . . . 60 iv LIST OF FIGURES Figure 1. IO. ll. 12. The locations of the incisions made in removing the opercula and anterior portion of the head . . . . . . . . . . . The locations of the incisions made in separating the first pair of gill arches from the remainder of the branchial apparatus . . . . . . . . . . . The perfusion apparatus . . . . . . . . . The major circulatory paths from the ventral to the dorsal aorta . . . . . Longitudinal section of a gill filament . A diagrammatic cross-section of a gill filament from a rainbow trout . . . . . . Photomicrograph of the afferent end of a cross-section of a filament from a rainbow trout gill which had been perfused with india ink . . . . . . . . . Photomicrograph of the efferent end of a cross-section of a filament from a rainbow trout gill which had been perfused with india ink . . . . . . . . . Photomicrograph of a longitudinal section of a gill lamella . . . . . . . . Photomicrograph of a longitudinal section of a gill filament, showing the lamellareafferent arterioles . . . . . . Photomicrograph of a longitudinal section of a gill filament, showing the lamellar-efferent arterioles . . . ._ Photomicrograph of a longitudinal section of a gill filament, showing the sinus-efferent vessels . . . . . . . V Page 1% l6 19 28 3O 36 38 38 Figure 13. 1%. 15. 16. 170 l8. 19. 20. 21. 22. 23. 2%. Photomicrograph of a cross—section of a gill filament in which the lymphatics had been perfused with india ink . . . . . Photomicrograph of a cross-section of a gill filament, showing the beginnings of the connection from the afferent filamental artery to the afferent collateral and the filamental sinus . . . . . . . . . . . Photomicrograph of the section serially succeeding that in Figure 1% . . . . . . . Photomicrograph of the section serially succeeding that in Figure 15 . . . . . . . Photomicrograph of the section serially succeeding that in Figure 16 . . . . . . . Photomicrograph of the section serially succeeding that in Figure 17 . . . . . . . Photomicrograph of a cross-section of a gill filament which had been perfused with india ink containing acetylcholine . Photomicrograph 03 a cross-section of a gill filament which had been perfused with india ink containing epinephrine . . Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in chiline Ringer solution. Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in sucrose Ringer solution. Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing cyanide . . . . . . . . . . . . Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing iodoacetate . . . . . . . . . . vi Page 1+0 #2 NZ Ah m. #6 M6 M8 #8 50 SO Figure 25. 26. Page Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing ouabain . . . . . . . . . . . . S2 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing atropine . . . . . . . . . . . 52 vii INTRODUCTION Since Smith (1930) postulated an osmoregulatory role for the teleost gill, there have been numerous studies which support the idea that ions, particularly sodium and chloride, are actively transported across the surface of the gill. Marine teleosts transport sodium from the blood, which has a sodium concentration of about 200 mEq/l, into sea water, with a sodium concentration of about #50 mEq/l. Sodium transport by freshwater fish is from the water, with a sodium concentration of 0.1 to 20 mEq/l, into the blood which has a sodium concentration of about 150 mEq/l. Thus, the concentration ratios against which sodium transport takes place range from about 2:1 (water to blood) in marine teleosts to about 300:1 (blood to water) for freshwater fish. There are four major factors which must be taken into account in analysing the net uptake or excretion of ions by the gill. They are: (l) the mechanism by which ions are moved (active transport, exchange diffusion, etc.), (2) the effective surface area available for exchange, (3) the duration of exposure between the blood and the water, and (h) the concentration gradient against which the ions must move. The purpose of this study was to examine the net 1 2 sodium flux across isolated, perfused rainbow trout gills, with respect to each of the above parameters. Some information on the mechanisms involved was obtained by examining the effects of metabolic inhibitors on the net flux of sodium across the gill membranes. The effective surface area available for exchange was studied by histological examination of gills which had been perfused with india ink containing various vasoactive agents. The rate of flow of fluid through the branchial vasculature was used as a measure of the duration of exposure between the blood and the water. For all perfusions, flow rates were used as an index of the vasoactive nature of the various experimental solutions. The concentration gradients were kept relatively constant in all experiments. Since two separate and distince approaches were used in this study, the major sections of the thesis have been divided into at least two parts. The first part of each section deals with the circulatory anatomy and the second part with sodium movement. REVIEH OF THE LITERATURE Circulatory Anatomy Although there have been numerous studies dealing with the osmoregulatory and respiratory functions of the teleost gill, only a few detailed studies of the functional circulatory anatomy have been made. The general path of blood flow through the gill has been well established by Allis (1912, from Goodrich, 1958) and by Mbtt (1950 a, 1951) and is as follows: ventral aorta, afferent branchial artery afferent filamental artery, lamellar lacunae, efferent filamental artery, efferent branchial artery, and then to the dorsal aorta. The counter current nature of the lamellar blood flow and the flow of water across the gill surface was demonstrated by van Dam (1938, from Steen and Kruysse, 196%). Steen and Kruysse (196%) examined the pattern of blood flow through isolated teleost gills and found that there were three routes by which blood could flow from the afferent to the efferent filamental arteries. In addition to the previously established lamellar pathway, they found that blood could go directly from afferent to efferent filamental artery at the apical end of the filament and that it could also flow through the large sinus which occupied the central portion of the filament. Hughes and Grimstone 3 T, (1965) suggested further that lamellar blood flow might occur along two separate paths. The first, and perhaps "preferred" path is along the margin of the lamella by a direct channel formed by the pillar cells and the lamellar epithelium. The second path is through the body of the lamella among the pillar cells. These authors also suggested that the pattern of blood flow through the gill is regulated by the state of contraction or elongation of the pillar cells, a view which is shared by Newstead (1967). The effects of various pharmacological agents on the branchial vascular resistance have been studied using both isolated gills (Keys and Bateman, 1932; Dstlund and Fange, 1962) and in intact fish (Mott, 1951b; Randall and Stevens, 1967; Stevens and Randall, 1967). It is difficult to interpret the results of the lg 1129 experiments since other portions of the circulatory system, in addition to the branchial vasculature, were undoubtedly affected by the injected substances. No quantitative comparisons could be made among the results presented in the above studies since only one paper (Keys and Bateman, 1932) gave the actual concentrations of the vasoactive substances tested. In the other papers, the doses administered were reported but since the volumes of blood or perfusion fluid in which the agents were dissolved were not specified, the actual concentrations could not be determined. Qualitatively, however, these workers found that epinephrine decreased the resistance and acetylcholine increased the resistance to 5 branchial blood flow in all of the species studied. Histamine decreased the resistance to branchial blood flow in one species of fish and apparently increased it in another. It was also found that the response to acetyl- choline could be blocked by atropine (Dstlund and thge, 1962) and the response to epinephrine could be blocked by phenoxybenzamine (Randall and Stevens, 1967). The only fish in which the branchial vascular resistance has been shown to be under some direct nervous control is the eel, which was shown by Mott (1951b) to have a vagal depressor reflex. Steen and Kruysse (196%) presented some evidence that epinephrrne and acetylcholine not only change the resistance of the branchial vasculature, but also change the pattern of blood flow through the gill. Sodigm Movement Both marine and freshwater teleosts have the problem of maintaining the osmotic concentration of the blood against large concentration gradients. To prevent excessive dehydration, marine teleosts must continually drink sea water and excrete salts both renally and extra- renally (Smith, 1932). The freshwater teleost, on the other hand, must constantly take up salt and excrete a copius volume of dilute urine (Pitts, 193%; Keys, 1937) in order to maintain a relatively constant osmotic concen- tration of its body fluids. 6 With very few exceptions, (notably the work of Keys and Bateman, 1932 and of Bateman and Keys, 1932), the information available relative to sodium and chloride uptake and excretion by teleost gills is based on studies of osmoregulation by intact fish maintained under a variety of conditions. By comparing the amount of water ingested and absorbed to the volume of urine excreted per unit time, Smith (1930) demonstrated that there was a significant extra-renal water loss from the sculpin and eels adapted to sea water. 'With another group of fish, he also determined that the amount of chloride which was absorbed along with the water exceeded renal chloride excretion and thus obtained indirect evidence for extra-renal chloride excretion by marine fish. In order to actually demonstrate the extra-renal excretion of salts, eels which had been adapted to fresh water were used. They were injected with salt solutions, fecal salt loss was prevented by ligating the anus and the urine was collected from free swimming fish in fresh water with a retention catheter and a balloon. ‘With this preparation, any increase in the salt concentration of the bathing solution was an indication of extra-renal salt loss and Smith (1930) refers to this loss as extra-renal excretion. The possibility still remained, however, that this extra-renal loss represented merely passive diffusion or leakage for which the fish could not compensate by active uptake of salt from the surrounding medium. 7 Keys (1931), Keys and Bateman (1932) and Bateman and Keys (1932) used a perfused heart-gill preparation (in which the heart pumped the fluid through the gills) and a perfused gill preparation to study the secretory activity and some of the branchial vascular responses in the salt water adapted eel. They found that adrenaline increased the rate of flow of fluid from the ventral to the dorsal aorta and that the rate of chloride excretion decreased with an increase in the rate of fluid flow through the branchial vasculature. Changes in weight as well as changes in the freezing point depression and chloride and protein concentrations of the blood of Anguilla vulgarig during adaptation from fresh water to salt water and back to fresh water were examined by Keys (1933). He found that there was an initial stage during which the fish responded passively and gained or lost salts and/or water in response to the concentration gradient between the blood and the surrounding medium. The duration of this stage depended on whether the fish was transferred from salt to fresh or from fresh to salt water. This passive stage ended when the fish began to actively control the composition and osmotic concentration of the blood, and the weight was returning toward its pre- treatment value. Krogh (1937) measured the uptake of chloride by seven species of freshwater fish which had been previously "washed out" in distilled water. He found large variations among the species studied but, in general, the fish were able to 8 absorb chloride from millimolar salt solutions. Using a divided chamber which kept the salts lost in the urine and feces from mixing with the water which surrounded the head of the fish, Krogh (1938) found that fish can absorb sodium and chloride separately. He also hypothesized that the sodium uptake was at least partially in exchange for ammonium ions and that the chloride must exchange for bicarbonate. Romeu and Maetz (196%), using radioactive tracers, provided additional evidence for the independence of sodium and chloride transport by the in yiyg gills of goldfish (Cara§sig§ aurgtug). Maetz and Romeu (196%) re-examined the theory that sodium exchanges for ammonium and that chloride exchanges for bicarbonate. They found that increasing the internal ammonium ion concentration increased the uptake of Na2l+ by goldfish and that increasing the external ammonium ion concentration inhibited the uptake of N32%. These treatments, however, did not affect the chloride balance. If the internal bicarbonate concentration was increased, however, Cl36 uptake increased and if the bicarbonate concentration in the external medium was increased, 0136 uptake was inhibited. These changes in C136 uptake occurred with no significant change in the sodium balance. They also found that injection of Diamox (a carbonic anhydrase inhibitor) caused an inhibition of both Na2“ and 0136 uptake. A significant increase in blood chloride occurred after inhibition of carbonic anhydrase in the marine teleost 9 §erranus gp. (Maetz, 1953) and a decrease in blood chloride was observed in the freshwater teleost‘ggggg‘gp, (Maetz, 1956). Gordon (1963) compared the losses of Cl36 from rainbow trout adapted to fresh water, 1/7, 1/3, 2/3, and 3/3 sea water. In one group of fish the renal papillae were ligated, the other group was unoperated. By comparing the rates of appearance of the injected 0136 in the bathing medium for both ligated and unoperated fish, he determined that the total 0136 exchange in fresh water and in 1/3 sea water took place via the gills. Kamiya (1967) found that cyanide and dinitro-phenol inhibited the excretion of sodium by isolated gills of salt water adapted eels, however, fluoride and iodoacetate had little or no effect. At least a portion of the excretion of sodium by the gills of salt water adapted eels is apparently dependent upon oxidative metabolism. In addition, it has been found that the level of Na-K activated ATPase is about six times higher in the gills of sea water adapted fish than in gills from fresh water adapted fish of the same species (Epstein, Katz, and Pickford, 1967). After making histological examinations of the gills from 10 species of fish, Keys and Willmer (1932) put forth the theory that there was a specific type of cell (the "chloride cell," acidophil cell, Kestdillmer cell, or Granel cell) which was responsible for chloride transport across the gill epithelium. Bevelander (19%5) examined 10 the gills from 36 species of fish and could find no histological evidence for specialized chloride secretory cells. Virabhadrachari (1961) found that the number of "chloride cells" increased during adaptation from 0-50% sea water, did not change during adaptation from 50-75% sea water and decreased during adaptation from 75-100% sea water. Philpott (1965) used electron microscopy and electron diffraction of silver acetate treated gills and found that there was a greater density of silver salts in the area of the "chloride cells" than in other areas. From these studies it can be seen that, although fish gills are certainly involved in osmoregulation and active transport of sodium and chloride, it is not at all certain whether this ability to transport ions is a general property of gill epithelial cells or is reserved for certain "transport cells" which may or may not have been identified. MATERIALS AND METHODS Animal; The fish used in this study were rainbow trout (figlmg gairdneri) weighing between 130g and 250g. They were obtained from the Michigan Conservation Department Hatchery in Grayling, Michigan, and were transported from the hatchery to Michigan state University in a large, well insulated metal tank, the inside of which had been painted with non-toxic paint. The water in the tank was mechanically agitated during the entire trip in order to provide adequate aeration. The holding facilities at Michigan State University consisted of two rooms maintained at a temperature of 12 to 13°C with 1% hours of light and 10 hours of darkness per day. The main cold room contained six 300 liter wooden tanks which were lined with fiberglass. The smaller room contained a variable number of smaller (100 liter) tanks into which groups of fish were transferred shortly before use. The fish were kept in aerated, flowing water from which the chlorine and excess iron had been removed. Fish in the main holding tanks were fed twice a week, those in the smaller tanks were fasted. ll 12 Man Isolated gills were surgically prepared in the following manner. An unanesthetized fish was removed from a tank, wrapped in a cloth towel and weighed. Heparin was then injected into the caudal portion of the dorsal aorta and the fish was returned to the water for at least one minute to allow the heparin to mix with the blood. The fish was then removed from the water and the spine was cut at the level of the pectoral fins. A mid-ventral incision was made from the anus to a point just anterior to the pectoral fins. The head was removed and the caudal portion of the fish was discarded. The opercula and anterior portion of the head were removed by cutting as indicated by the dotted lines in Figure 1 (A and B). The branchial apparatus was then dissected free from the remaining section of the spinal column and the dorsal musculature. Care was taken to prevent damage to the efferent branchial arteries and the dorsal aorta. The right and left halves of the branchial apparatus were separated by a mid-dorsal incision as shown in Figure 2 (incision A). The first pair of arches was then removed by cutting as indicated by lines B, C, and D (Fig. 2). The remaining gills were placed in a beaker of dechlorinated tap water. The right and left first arches were separated ventrally by a midline incision and, if one of the first arches failed to perfuse, it was replaced by 13 Figure l The locations of the incisions made in removing the opercula and anterior portion of the head. 1% A" venTreT View B“ Te’reraT VTET.) Figure l 15 Figure 2 The locations of the incisions made in separating the first pair of gill arches from the remainder of the branchial apparatus. muster//:\\ FFFFFFF 17 the corresponding second arch. During surgery, the gills were kept moist by periodic immersion in dechlorinated tap water. We“ 122m The gills were perfused using a constant pressure perfusion apparatus shown diagrammatically in Figure 3. It consisted of a pair of plastic 10cc syringe barrels connected by a piece of plastic tubing and a 3-way valve. The valve was attached to a blunt-tipped 25 gage needle. Leading from the needle was a piece of PE 20 tubing 50cm long, into which had been inserted a blunt-tipped, cut-off barrel of a 25 gage needle. This needle served as the afferent cannula and was inserted into the afferent branchial artery at the ventral end of the gill. The efferent portion of the perfusion apparatus consisted of a blunt-tipped, cut-off 25 gage needle barrel inserted into a 25cm piece of PE 20 tubing. This cannula was inserted into the afferent branchial artery at the dorsal and of the gill. The inflow and collection pressures could be altered by changing the height of the syringe barrel and the free end of the efferent cannula with respect to the gill. The pressures used were 5% and 20cm of Ringer solution (%0 and 15mm Hg) for the inflow and collection pressures respectively. These approximate the pressures found by Stevens and Randall (1967) for the ventral and dorsal aortic blood pressures in intact, swimming rainbow trout. 18 Figure 3 The perfusion apparatus which consists of two 10cc syringe barrels connected by a piece of plastic tubing and a 3-way valve. Leading from the valve is the afferent cannula which consists of 50cm of PE 20 tubing, into which is inserted a cut-off, blunt—tipped 25 gage needle. FFFFFFF 20 Caggulation agd Perfgaion The dorsal and ventral ends of the gill were trimmed of excess bone and tissue, exposing the afferent and efferent branchial arteries respectively. Prior to cannulation of a gill, the valve on the perfusion apparatus was Opened and a steady, rapid flow of fluid was established. The afferent cannula was inserted into the afferent branchial artery and tied in place with No. 30 cotton thread. The ligature was tied around the arch rather than directly around the artery, thus preventing loss of fluid due to leakage at the cut end of the arch. The efferent artery was cannulated in the same manner and the gill was immersed in 50ml of 1% Ringer solution. Cirgulatggy Agatomy The gill was first perfused with Ringer solution (Appendix) containing the test substance (control, epinephrine, acetylcholine) in the required concentration, or with one of the experimental solutions used in the sodium uptake studies. This perfusion was carried out with the free end of the efferent cannula at approximately the same height as the gill, and was continued until no more blood could be seen entering the collecting tube. The volume collected, however, was never less than lOQpl. At the end of this initial perfusion, the free end of the efferent cannula was raised to a height of 20cm above the gill and 21 was inserted into another 10Qpl pipette. The valve on the perfusion apparatus was turned and the india ink (Appendix), which contained the test substance or was diluted with the experimental solution, was allowed to flow through the gill until at least lOQpl of ink solution had been collected from the efferent cannula. Additional ligatures were placed around the ends of the gill and were tightened as soon as the cannulas were removed, reducing the loss of ink from the branchial ‘vasculature to a minimum. Fixation was carried out over night in Dietrich's fixative (Appendix), and the gill was dehydrated, infilatated with and embedded in Paraplast (Arthur H. Thomas 00., Philadelphia, Pa.) using standard procedures. The gills were sectioned at 8p, the sections were mounted on glass microscope slides and stained using standard procedures for either hematoxylin and eosin or Masson's trichrome stains. Only gills from which there was no visible leakage of india ink were prepared for histological examination. Sodium Movement After cannulation, the gills were placed in 50ml of 1% Ringer solution and perfused with 100% Ringer solution until no more blood was observed entering the collecting tube. As in the anatomical studies, the free end of the outflow tube was at approximately the same level as the gill and the perfusion was continued until at least 10Qpl of 22 fluid had been collected from the outflow tube. At the end of this initial perfusion, the free end of the outflow tube was raised to about 20cm above the gill, which was placed in 50ml of fresh 1% Ringer solution. A clean lOQpl pipette was placed over the end of the outflow tube and the stopwatch started. The time required to collect lOQpl of fluid was recorded. A 5Qpl aliquot of the fluid collected was diluted 1:200 for sodium analysis, and a 5ml sample of the bathing medium was also placed in a vial for sodium analysis. The valve on the perfusion apparatus was turned, and a lOQpl sample of the experimental fluid was collected from the efferent cannula and discarded. The gill was then placed in 50ml of fresh 1% Ringer solution, a clean lOQpl pipette was placed over the free end of the outflow tube, and the time required for the collection of 100ul of fluid was determined and recorded. Samples of the bathing solution and fluid collected from the outflow tube were again taken for sodium analysis. In addition to the four samples already mentioned, a 5ml sample of the bathing solution stock solution and 5Qpl aliquots of the control and experimental perfusion fluids were also taken for sodium analysis. Both the perfusion fluid (fluid which had not yet passed through the gill, P) and the fluid collected after passing through the gill (F) were diluted 1:200 for sodium analysis. Since the bath fluid had a very low sodium concentration, samples were only diluted 1:5 for sodium analysis. 23 Sodium Analyaia Sodium analysis was performed using a Coleman Model 21 Flame Photometer and a Coleman Model 22 Galv-o-meter galvanometer (Coleman Inst. 00., Maywood, Ill.). A11 dilutions were made using 0.02% Sterox SE (Scientific Products, Evanston, Ill.) in distilled water. Sodium concentration was read as percent transmission and converted to mEq/l using a standard curve. As a constant check on the accuracy of the readings, the solutions were read in the following order: blank, l50mEq/1 standard, blank, sample, blank, etc. The blank also served as a cleaning solution, washing any remaining sodium out of the aspirator and burner assembly of the flame photometer. The sodium concentrations of the perfusion fluid (P) and the fluid collected after passing through the gill (F) were determined directly from the standard curve. Since bath solutions were diluted 1:5 rather than 1:200, the sodium concentrations determined form the standard curve had to be divided by %0 to determine the actual sodium concentration in the bath. The accuracy with which the sodium concentration of a given sample could be determined was,:l.0mEq/l. Uptake 9f Sodium agd Ingipitor.§tggia§ To stimulate sodium uptake, gills were perfused with Ringer solution in.which 50% of the sodium had been replaced 2% by choline (choline Ringer solution, Appendix) and with Ringer solution in which 50% of the sodium (as NaCl) had been replaced by sucrose (sucrose Ringer solution, Appendix). The inhibitors studied (ouabain, cyanide, and LIM in iodoacetate) were all used at concentrations of 10' choline Ringer solution. Atropine (10'3M and lO'hM)'was also used in some experiments in an attempt to block any acetylcholine-like effects of the choline in the Ringer solution. Perfusions were also performed using sucrose Ringer solution containing 10'3M atropine and served as controls on any effects of atropine in addition to its anti-cholinergic effects. Statistica All statistical analyses in this study were performed using nonparametric statistical tests. These tests have a number of advantages of parametric tests, such as Student's t test. The assumptions associated with nonparametric tests are fewer and less critical than those associated with parametric tests. Nonparametrics can be used with very small sample sizes (N56) and the tests are generally much more rapidly and easily performed. If all of the underlying assumptions for parametric statistical analysis are met £11219 independent observations, normal distribution, equal variances, additivity of effects), than the parametric test is more powerful. That 25 is, it takes fewer observations to achieve a given significance level with the parametric test than with its nonparametric counterpart. If, as in the present study, the differences observed are large enough to show significance using nonparametric tests, the greater power of the parametric test is of no particular advantage. The three tests used in this study were: the Sign test, which was used to compare related samples when N was large, theTflalsh test, which was used to compare related samples when N was small, and the MannAdhitney U test, which was used to compare independent samples. The first two tests correspond to the parametric paired t test and the third corresponds to the unpaired 3 test. RESULTS Circulatory tom The results of the studies of the circulatory anatomy of the rainbow trout gill are presented in figures % through 26. The orientation of the gills in all figures is approximately the same, with the afferent artery at the top or right-hand side of the figure. Figure % is a diagrammatic representation of the major circulatory paths from the ventral to the dorsal aorta. Figure 5 is a diagrammatic representation of a longitudinal section of a gill filament (a cross-section of a gill arch) showing the various paths by which blood can flow from the afferent to the efferent filamental artery. Figure 6 shows a diagrammatic cross-section of a filament from a rainbow trout gill. The major vascular connections are also shown in figures 7 through 9 which are photo- micrographs of control gills perfused with india ink. Figure 7 shows the ink in the afferent filamental artery, the lamellar-afferent arteriole, part of the "preferred path" along the lamellar margin, the filamental sinus, and the right afferent collateral. In Figure 8 the ink is again present in the filamental sinus and the "preferred path" but it can also be seen in the lamellar lacunae, the 26 27 Figure % The major circulatory paths from the ventral to the dorsal aorta. 28 : ousmfih mallow ’mbesm) #506 T \ m .zrvfm. ’0 7)U¢0.vfi 15.3% \‘m.-. \‘N \... 1 ATI rsw’Lm ’07.? mep V, +cwyouum m4som ,mnsow T % 29 Figure 5 Longitudinal section of a gill filament. 9“"9‘” branchTaT l ar'AErW q’ 2(‘evem+ “‘\ EulamenTQT \ tI ‘, Grier\\ , ".l' O,/a§§cren1’ 0 branchiaj It," In' I! a")! V\' 23$?cren‘t xdameniaT arterT lamella Figure 5 Figure 6 A diagrammatic cross-section of a gill filament from a rainbow trout. 32 Figure 6 33 smaller-efferent arteriole, the efferent filamental artery and in the left efferent collateral. Although there is little ink in the section shown in Figure 9, two important features of lamellar circulation are apparent. The first is the "preferred path" which can be seen along the upper edge of the lamella and the second is the close contact between the pillar cells and the red blood cells in the lamellar lacunae. Figures 10, 11, and 12 are photomicrographs showing the lamellar-afferent, lamellar-efferent, and sinus—efferent blood vessels respectively. As can be seen from Figures 10 and 11, the lamellar-afferent and lamellar -efferent arterioles definitely contain muscular elements within their walls. It has not been possible to clearly demonstrate the presence of contractile elements in the walls of the sinus-efferent vessels (Fig. 12), but neither the dimensions of the vessels themselves nor the thickness' of the vessel walls precludes such a possibility. The photomicrograph in Figure 13 is the result of accidentally inserting the afferent cannula into what was apparently the branchial lymph duct rather than the afferent branchial artery. It is a representative section from this tissue showing the ink in the filamental sinus and afferent and efferent collaterals, but with no ink in the afferent filamental artery or the lamella and little or no ink in the efferent filamental artery. Ink was also absent from the afferent branchial artery, there was, however, some ink in 3% the efferent branchial artery. Figures 1% through 18 are photomicrographs of serial sections and show the direct connection from the afferent filamental artery to the afferent collateral and then to the filamental sinus. Such connections are only evident near the base of the filament. The sections shown in Figures 19 and 20 are from gills perfused with india ink containing acetylcholine and epinephrine respectively. They represent the extremes of fluid flow patterns through the gill. The ink in the acetylcholine treated gill is concentrated in the filamental sinus and the collateral vessels with very little ink appearing in the "preferred"channel or the lamellar lacunae. In the epinephrine treated gill, on the other hand, the ink is concentrated in the lamellae with little or no ink in the filamental sinus or collateral vessels. Figures 21 through 25 are photomicrographs of cross— sections of gills perfused with india ink in choline Ringer solution (Fig. 21), sucrose Ringer solution (Fig. 22), choline Ringer solution containing cyanide (Fig. 23), choline Ringer solution containig iodoacetate (Fig. 2%), and choline Ringer solution containing ouabain (Fig. 25). By comparing these figures with Figures 19 and 20, it can be seen that the pattern of ink distribution in these experimental gills resembles that seen in acetylcholine treated gills much more closely than it does that seen in epinephrine treated gills. 35 Figure 7 Photomicrograph of the afferent end of a cross-section of a filament from a rainbow trout gill which had been perfused with india ink. (Masson's trichrome x 53%) Figure 8 Photomicrograph of the efferent end of a cross section of a filament from a rainbow trout gill which had been perfused with india ink. (Masson's trichrome x 53%) 36 Figure 8 37 Figure 9 Photomicrograph of a longitudinal section of a gill lamella. (Masson's trichrome x 1066) Figure 10 Photomicrograph of a longitudinal section of a Bill filament, showing the lamellar—afferent arterioles. (Masson's trichrome x 1066) 38 Figure 10 39 Figure 11 Photomicrograph of a longitudinal section of a gill filament, showing the lamellar-efferent arterioles. (Masson's trichrome x 1066) Figure 12 Photomicrograph of a longitudinal section of a gill filament, showing the sinus-efferent vessels. (Masson's trichrome x 1066) %0 Figure 12 %1 Figure 13 Photomicrograph of a cross-section of a gill filament in which the lymphatics had been perfused with india ink. (Masson's trichrome x 53%) Figure 1% Photomicrograph of a cross-section of a gill filament, showing the beginnings of the connection from the afferent filamental artery to the afferent collateral and the filamental sinus. (Masson's trichrome x 266) %2 Figure 13 Figure 1% H3 Figure 15 Photomicrograph of the section serially succeeding that in Figure 1%. (Masson's trichrome x 266) Figure 16 Photomicrograph of the section serially succeeding that in Figure 15. (Masson's trichrome x 266) %% '. "\‘leg.’ I -‘{jr=;‘ . .. T g. . . N. W'i'ti‘l‘fialg Figure 15 “A ‘:-‘ v . ‘ ‘ g '7 v ‘ .’.;r,‘ ‘ "It.” . (7.53.: a, Figure 16 %5 Figure 17 Photomicrograph of the section serially succeeding that in Figure 16. (Masson's trichrome x 266) Figure 18 Photomicrograph of the section serially succeeding that in Figure 17. (Masson's trichrome x 266) %6 Figure 18 “7 Figure 19 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink containing acetylcholine. (Masson's trichrome x 53%) Figure 20 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink containing epinephrine. (Masson's trichrome x 266) %8 Figure 19 Figure 20 %9 Figure 21 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution. (Masson's trichrome x 266) Figure 22 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in sucrose Ringer solution. (Masson's trichrome x 266) 50 Figure 21 Figure 22 51 Figure 23 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing cyanide. (Masson's trichrome x 266) Figure 2% Photomicrograph of a cross—section of a gill filament which had been perfused with india ink in choline Ringer solution containing iodoacetate. (Masson's trichrome x 266) 52 Figure 23 Figure 2% 53 Figure 25 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing ouabain. (Masson's trichrome x 266) Figure 26 Photomicrograph of a cross-section of a gill filament which had been perfused with india ink in choline Ringer solution containing atropine. (Masson's trichrome x 266) Figure 26 55 A Figure 26 shows the distribution of india ink in gills perfused with choline Ringer solution containing 10'3M atropine. The high concentration of ink in the lamellae and its virtual absence from the filamental sinus and collaterals resembles the pattern seen with epinephrine perfusion rather than that seen.when gills were perfused with india ink containing acetylcholine. Sodigm Movemegt The results of the experiments on sodium uptake are presented in Table l which gives the concentrations (mEq/l) of the perfusion fluid (P) and of the fluid collected after passing through the gills (F), as well as the rates of sodium uptake (qu/min). The most rapid uptake of sodium occurred when gills were perfused with sucrose Ringer solution. Perfusion with choline Ringer solution resulted in a significantly (p=.025) slower uptake of sodium compared to the rate of uptake during perfusion with sucrose Ringer solution. No significant net uptake of sodium was observed when the gills were perfused with Ringer solution containing a normal concentration of sodium (158mEq NaI/l). With all of the other perfusion solutions tested, both the amount and the rate of sodium uptake were significantly (p(}10) greater than zero. The rates of sodium uptake by gills perfused with choline Ringer solution containing ouabain or cyanide were 86% and 65% lower respectively than the rates of uptake by 56 Table 1 Sodium concentrations of the perfusion fluid (P) and the fluid collected after passing through the gill (F), and the rate of sodium uptake for the various experimental perfusions. 57 uma.o«wmm.o Hwo.OHwHN.o ouo.oummm.o :ao.onaom.o mmo.onomo.o uao.on:mo.o o:a.onm:m.o amo.onwmm.o wmo.ouum:.o -T---.ooo.o seemmfl.oxmuab no uhom n.mfl:.uma o.:um.uo s.muc.mm e.auw.mo H.mflm.ms m.dflm.mm m.muw.uoa n.ano.os u.muw.moa m.ou:.mma *smmflw w.afl:.oma m.ou«.sa o.ou:.aw m.anm.mw a.aflm.:m m.aflm.aw o.onw.mm m.oum.am o.ono.mm m.ouw.mma *emmflm H taste 2 CHE\+NZ g‘ *** H\ mz uma a* henchmen mHHau mm genes: * mnfinzaocaao 2mm.o + ammsfim gooa mafiaonpw EJTOH + enamonpm Emuoa + mumpoomocofi + ocacmmo + cfimnmso + ocHQoupm EMTOH + nmmnfim newcam nomcfim nowawm nmmcam ammnam newcam nommam osaaoso ocaaono ocaaono osaaoho ouaaoeo ouaaoso omouosm omonosm nomcfim Rooa coausaom 58 gills perfused with choline Ringer solution which contained no inhibitor. These differences were significant at the =.025 and p .10 levels respectively. The rate of sodium uptake by ouabain perfused gills was significantly less (p<.l5) than the rate of uptake by cyanide perfused gills. The rates of sodium uptake by gills perfused with choline Ringer solution containing iodoacetate or 10'3M or lO’hM atropine were not statistically different from the rates of uptake by gills perfused with choline Ringer solution alone. Addition of atropine (lo-BM) to sucrose Ringer solution did not result in a statistically significant change in the rate of sodium uptake. The rates of fluid flow through the gills are given in Table 2 which compares the flow rates during perfusion with 100% Ringer solution (control perfusion rate, R1) to the flow rates during perfusion with the various experimental solutions (experimental perfusion rate, R2). ‘When the experimental solution was the same as the control solution (100% Ringer solution), there was a significant (p(.001) increase in the flow rate. There was also a significant (p=.03l) increase in the flow rate when the experimental solution was 100% Ringer solution containing epinephrine (0.27M). This increse was significantly greater (p(.01) than when the experimental solution was 100% Ringer solution without epinephrine. There was no significant change in flow rate with the sucrose Ringer solution alone, with sucrose Ringer solution containing 10'3M atropine, or 59 Table 2 The rates of fluid flow through the gill during control (R1) and experimental (R2) perfusions and the percent change in rate from the control to the experimental perfusion. 60 .mosam> omwno>m one so no: .mpwc HwGHMHuo co comma has GHE\H3 ** comsmnom madam mo nonasc * m.osno.uea+ o.mnxamm u.m«x.mm m ocansdosado 25m.o + somcam mooa u.m HH.AH n o.mnd.mm H.mMN.wm m maggoaum 2:Toa + nomcfim mafiaonu 0.:Hum.a - H.muo.mm o.mum.mm m odanouam ZmTOH + somsam osaaoho a.maflo.am . a.:u4.wa w.mfla.am a onshoomoooa + tomcam ohaaono m.: “4.:5 T m.HHN.m m.NHw.mH m ocHCmmo + ummcfim ocfiHoso :.Nafli.ww I u.aflm.: :.:flv.ma m Cfiwnmso + nmmcfim mafiaoso m.u flw.mm T m.aflm.u N.mflm.mm wa nomcfim mafiaoso w.m HM.H u N.mflf.:m u.uflN.mm m mcfiaoupm EMTOH + nomcfim omonosm m.wafl4.m u w.mflw.mm :.:HH.Nm w nomcwm mmonozm :.: HM.AH + m.mum.wm w.mflu.:m mm humane mooa .ssmmnumcsnos *smmumm simmn.m *z coaosaom N magma , A. r- O ~nv 61 with choline Ringer solution containing 10'3M or lO'hM atropine. The flow rates of all of the other experimental perfusions were significantly less (p(.10) than the control rates. The rate of sodium uptake by gills perfused with choline Ringer solution was plotted as a function of gill weight, and flow rate. There appeared to be a slight decrease in the rate of sodium uptake with increasing gill weight and no change as a function of flow rate. There was also no relationship between the rate of sodium uptake and the time, during the study, when the experiment was performed. DISCUSSION Circ tor Anatomy According to Steen and Kruysse (196%), the two main paths by which blood can pass through the filament of a teleost gill (the lamellar path and the filamental sinus path) were first described by Riess (1881). It was also Riess (1881) who suggested that the anatomical and cyto- logical nature of the vessels within the filamental sinus and of the sinus itself, would classify them as lymph vessels rather than as blood vessels. The investigations by Steen and Kruysse (196%), which involved primalily the common eel (Anguilla yglgagia), supported and expanded these earlier studies. They found the direct connections between the filamental sinus and the afferent and efferent filamental arteries. They also pointed out that there is a direct connection from the sinus to the major lymph vessel at the base of the filament, which provided further evidence for the lymphatic nature of the filamental sinus. Evidence is presented herein that, in the rainbow trout, as opposed to the eel, the lymphatics (or afferent collaterals) are directly connected to the filamental sinus at frequent intervals along the filament. These lymph vessels are also connected directly to the afferent 62 63 filamental artery near the base of the filament. The presence of numerous red blood cells in the filamental sinus and the afferent collaterals (or lymphatics) and their virtual absence from the major lymph vessels at the base of the filament, indicates that there must be some sort of filtering mechanism whereby the red cells are removed from the lymph and returned to the blood. The precise nature and location of this filtering system are unknown. The presence of such a well developed lymphatic- blood vascular system would seem to be advantageous in the maintenance of the ionic and osmotic composition of the blood in both marine and freshwater fish. In both environments, the large filamental sinus would serve to decrease the amount of exposure between the blood and the surrounding environment. This would decrease the amount of salt lost and water gained by freshwater fish and the amount of salt gained and water lost by marine fish. It is also possible that the branchial lymphatic system acts as a blood volume buffer during acute osmotic stress in both freshwater and marine teleosts. In the freshwater fish, the branchial lymphatics undoubtedly serve to drain off some of the excess fluid formed by the osmotic uptake of water across the gill epithelium. In the marine teleost, the flow of fluid might concievably be from the lymphatics into the blood, tending to restore any fluid which had been lost across the gill epithelium. 6% In their studies of the pattern of blood flow through the gill, Steen and Kruysse (196%) placed freshly excised gill filaments into physiological salt solutions on a microscope slide. They then placed a cover slip over the filament and observed the patterns of blood flow when pressure was applied to the cover slip. Using this method, they found that in untreated gills blood flowed, often simultaneously, between the afferent and the afferent filamental arteries by way of the lamellae, by way of the filamental sinus and directly by way of the connection between the afferent and efferent filamental arteries at the tip of the filament. ‘When acetylcholine was added to the salt solution bathing the filament, they found that the blood flowed through the filamental sinus and around the tip of the filament. Addition of adrenalin, on the other hand, caused all of the blood to flow through the lamellae. The absence of a normal, unidirectional, pressure gradient from afferent to efferent, however, makes it difficult to determine the true physiological significance of the flow patterns obtained in these experiments. In the present study, using approximately normal pressure gradients from afferent to efferent, and with the acetylcholine or epinephrine in the perfusing fluid rather than applied to the outside of the gill, the results obtained were similar to those of Steen and Kruysse (196%). The effects of epinephrine and acetylcholine, however, were not as absolute as those reported by these investigators. 65 Perfusion with india ink containing acetylcholine, for example, did not completely eliminate the flow of fluid through the lamellae, and perfusion with epinephrine did not result in 100% lamellar flow. Since there was very little difference between the india ink distribution in the control gills and that in acetylcholine treated gills, it would appear that the pattern of blood flow through the gills is primarily under adrenergic control. The effect of atropine on the flow pattern through gills perfused with choline Ringer solution, however, indicates that there is probably also some tonic cholinergic control of gill blood flow. Thus, the pattern of blood flow through the rainbow trout gill seems to vary from "purely cholinergic" flow (exclusively through the filamental sinus) to "purely adrenergic" flow (exclusively through the lamellae. The similarity between the india ink distribution in the gills perfused with sucrose Ringer solution and that in the control gills, indicates that a low internal sodium concentration does not tend to favor lamellar perfusion. No conclusions can be drawn with respect to the effects of cyanide, iodoacetate or ouabain on the pattern of fluid flow through the gills. This is true since any effects which they might have were probably blocked or overshadowed by the cholinergic effect of the choline Ringer solution. In his studies on the juvenile coho salmon (Ogaorgynchus kisutch) and seven other species, Newstead 66 (1967) was unable to find any structural specialization in the walls of the afferent or efferent arteries which could account for the variations seen in the pattern of blood flow through the gills. Based on this apparent absence of arterial control sites, and on electron microscopic studies of the pillar cells, he suggested that the pattern of blood flow through the teleost gill is regulated by active alteration of the cross-sectional area and thus the resistance of the lamillar lacunae (an hypothesis previously advanced by Hughes and Grimstone, 1965). There are two major objections to accepting this as a general theory. They are: (l) the fact that the arterioles which branch off of the afferent filamental artery and those which enter the effernet filamental artery do possess muscular elements within their walls (at least in‘fialgg‘gaigggazi), (2) the very large changes in lamellar resistance which must exist in order to produce the observed changes in the pattern of fluid flow through the gill. The following diagram is a simplified schematic representation of the circulatory paths through the gill, showing the apical path (A), the lamellar path (L), and the sinus path (S). As can be seen from this diagram, the paths 67 are arranged in parallel and therefore, the amount of flow through a given path will depend not only on the resistance of that path, but also on the resistance of the other paths. If, as is suggested by Newstead (1967), the lamellar resistance (L) is the only variable resistance in the circuit, then it must by able to vary from near zero to infinity, with respect to the sinus (S) and apical (A) resistances in order to change the circulatory pattern from completely lamellar to completely sinus flow. If, on the other hand, there are two variable resistances in the w.— 0” u-"~ “~. 5 on m.--w-vdv ‘-'~ "" -" - '1‘ Sc-“ %~ cm circuit, than much smaller changes in the sinus and the lamellar resistances could result in the observed changes in flow pattern. Thus, the anatomical sites for the control of the blood flow pattern are probably some combination of the lamellar-afferent and lamellar-efferent arterioles and the sinus-afferent and sinus-efferent vessels. Acetylcholine, since it not only causes blood to flow through the filamental sinus, but also decreases the flow rate (Estlund and FSnge, 1962), probably acts solely to cause vaso- constriction of the lamellar-afferent and lamellar-efferent arterioles. Epinephrine, which stimulates blood flow through the high resistance lamellar circulation, also increases the flow rate. Thus, epinephrine must cause both 68 vasoconstriction of the sinus-afferent and sinus-efferent vessels and vasodilation of the lamellar-afferent and the lamellar-efferent arterioles. __.12_30d I! mm The results of these experiments clearly indicate that the isolated, perfused gills of rainbow trout are capable of taking up sodium against a concentration gradient of at least 60:1. The differences between the rates of sodium uptake by gills perfused with sucrose Ringer solution and by gills perfused with choline Ringer solution may have been brought about by: a stimulatory effect of sucrose of sodium uptake, an inhibitory effect of choline on sodium uptake or, an interaction between the uptake of chloride and that of sodium. The first of these possibilities seems unlikely since, to the best of my knowledge, there is no evidence for any direct effect of sucrose on the transport of sodium by membranes in general or by fish gills in particular. The studies in which gills were perfused with choline Ringer solution containing atropine, suggest that any inhibitory effects that choline might have on sodium uptake by the gill are either very slight or are particularly insensitive to inhibition by atropine. This is particualrly apparent when the effect of atropine on flow rate is compared to its effect on sodium uptake. Atropine, at a concentration of lO‘hM blocked the effects of choline Ringer solution on the rate of fluid 69 flow through the gill, but the rate of sodium uptake was not increased even when the concentration of atrOpine was raised to 10'3M. Thus, any inhibitory effects of choline on the rate of sodium uptake are probably insignificant. ‘With respect to the third possibility, an interaction between the uptake of chloride and the uptake of sodium, Krogh (1938) found that either sodium or cholride could be taken up independently by goldfish (Caraaaiga aggatga). ‘When this occurred, however, the chloride had to exchange for bicarbonate and the sodium had to either exchange for ammonium or be accompanied by bicarbonate. This theory has since been supported by Maetz and Romeu (196%) and by Romeu and Maetz (196%). Since no source of ammonium ions was supplied in the perfusion fluid and no bicarbonate was added to the bathing solution, most of the sodium taken up by gills being perfused with choline Ringer solution was probably taken up as sodium chloride. Lesser amounts may have exchanged for endogenously available ammonium ions or may have been tsken up in combination with bicarbonate ions derived from the carbon dioxide present in the bathing solution. However, any uptake of sodium chloride would tend to increase the chloride level of the perfusing fluid above the original value. An increased chloride concentration.would tend to increase the passive loss of chloride ions, but since there is little bicarbonate available in the bath for exchange, maintenance of electrical neutrality requires that the chloride loss be 70 accompanied by an equivalent loss of cations, probably sodium ions. This sodium-chloride interaction would tend to decrease the rate of sodium uptake by gills being perfused with choline Ringer solution. In sucrose Ringer solution, however, both the sodium and the cholride concentrations were below normal blood levels. This would not only decrease the passive losses of sodium as described above, but might actually result in an increase in the rate of sodium influx, due to sodium ions "passively" following the actively transported chloride ions. In either case, the overall effect would be an increase in the net uptake of sodium ions by the gill. The inhibition of sodium uptake by ouabain and cyanide, indicates that sodium uptake by isolated, perfused rainbow trout gills is an ATP-dependent process and that much of the ATP used is derived from oxidative metabolism. The complete lack of inhibition of sodium uptake by iodoacetate suggests that glycolysis is not required for sodium uptake by the gill. These results, along with those of Kamiya (1967) using salt water adapted eels, suggest that glycolysis may generally be unimportant as a metabolic path in the gills of fish. As yet, no attempt has been made to discover what other paths of energy production may be of significance in this tissue. Comparisons of the rates of sodium uptake by gills perfused with choline Ringer solution, sucrose Ringer solution, and 100% Ringer solution containing epinephrine 71 indicate that the rate of sodium uptake is not primarily dependent upon either flow rate or flow pattern throuth the gill. CONCLUSIONS 1. The filamental lymphatic system connects directly to the blood circulatory system at frequent intervals along the filament. 2. The patterns of filamental blood flow found in gills perfused using a normal pressure gradient vary from mostly sinus flow ("purely cholinergic") to mostly lamellar flow ("purely adrenergic"). 3. The pattern of blood flow through the gill is probably regulated by vasoconstriction and vasodilation of the lamellar-afferent and lamellar-efferent arterioles and the sinus-afferent and sinus-efferent blood vessels. %. Isolated, perfused rainbow trout gills are capable of taking up sodium against a concentration gradient of at least 60:1. 5. Sodium uptake by gills is at least partially dependent upon the availability of other cations in the perfusion fluid for which the sodium can exchange or the avail- ability of anions in the bathing medium which can accompany the transported sodium. 6. Sodium uptake by isolated, perfused rainbow trout gills is ATP dependent and the ATP used is derived mainly from oxidative metabolism. 72 73 The rate of sodium uptake is not primarily dependent upon the pattern or rate of fluid flow through the gill and is probably a function of the internal sodium concentration. APPENDIX 75 SOLUTIONS USED Camposition.gf 109% Rigger Sglutiog NaCl 7.37 gm/liter KCl 0.31 gm/liter CaCl2 0.10 gm/liter Mgsog 0.1% gm/liter KHQPOg 0.%6 gm/liter Na2HPOh 2.02 gm/liter Glucose 0.90 gm/liter Composition 9; CQoline Riggar Solution Choline Cl 6.91 gm/liter NaCl 3.85 gm/liter KCl 0.31 gm/liter CaC12 0.10 gm/liter MgSOu 0.1% gm/liter KH’ZPO)+ 0.%6 gm/liter NaZHPOh 2.02 gm/liter Glucose 0.90 gm/liter Compositian12f Spagoaa.§inga;.§glptign Sucrose 23.96 gm/liter NaCl 3.85 gm/liter KCl 0.31 gm/liter CaC12 0.10 gm/liter MgSOh 0.1% gm/liter KH2P0h 0.%6 gm/liter NaZHPOg 2.02 gm/liter Glucose 0.90 gm/liter 76 All of the Ringer solutions used were adjusted to pH 7.% and 300m03M L: SmOSM). Dietrich's Fixative Tap‘Water _ 150 ml 95% Ethanol 75 ml h0% Formalin 25 m1 Glacial Acetic Acid 5 ml India Ink The india ink used was Pelikan Biological India Ink and was obtained from the John Henschel Co., New York. According to Peterson, Ringer, Terzaloff and Lucas (1965), this ink is 10% carbon with a particle size of 0.02 to 0.03». It contains #.3% fish glue, 1.0% phenol and none of the shellac or ammonia normally found in other india ink preparations. The sodium content of the ink was adjusted to equal that of 100% Ringer solution and the resulting osmotic pressure was 30% mOSM. LITERATURE CITED Bateman, J. B. and A. Keys. 1932. Chloride and vapour- pressure relations in the secretory activity of the gills of the eel. J. Physiol., 75: 226-2k0. Bevelander, G. J. 19H5. A comparative study of the branchial epithelium in fishes, with special reference to extrarenal excretion. J. Morphol. 57: 335-397. Epstein, F. H., A. I. Katz, and G. E. Pickford. 1967. Sodium- and potassium-activated adenosine triphosphatase of gills: Role in adaptation of teleosts to sea water. Science, 156: 3779. Goodrich, E. S. 1958. Studies on the structure and development of vertebrates. Vol. II. Dover Publications Inc., New York. Gordon, M. S. 1963. Chloride exchange in rainbow trout (Salmo aird1 21) adapted to different salinities. Biol. Bull. :MS-W Hughes, G. M., and A. V. Grimstone. 1965. The fine structure of the secondary lamellae of the gills of G d Bollgchiug. Quart. J. Microscop. Sci. 10 = 3 3-3 3. Kamiya, M. 1967. Changes in ion and water transport in isolated gills of the cultured eel during the course of salt adaptation. Annot. Zool. Jap. #0: 123- 129. Keys, A. 1931. The heart- gill preparation of the eel and its perfusion for the study of a natural membrane in sit . Z. VergL Physiol., 15: 352- 363. Keys, A. 1933. The mechanism of adaptation to varying salinities in the common eel and the general problem of osmotic regulation if fishes. Proc Roy. Soc. London, Ser. B., 112: 18h-199. Keys, A. 1937. Properties of the gill membranes of fishes. Trans. Faraday Soc., 33: 972-98h. \l “J 78 Keys, A. and J. B. Bateman. 1932. 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