was 0F w, 01- AND WATER ACROSS THE “SHErRT-CERCZHTED" mm 0229? MEMBRANE Thasis far the Degree of M. S. MICHIGAN STATE UNIVERSHY _ RICHARD c. ROSE 1967 _ ‘ "$45515 ABSTRACT .RLUXES.0F.NAT,CL‘~ AND WATER ACROSS THE "SHORT-CIRCUITED" PIGEON CROP MEMBRANE by Richard C. Rose A study of the history of physiology indicates a close association of bioelectrical phenomena with the ionic flux characteristics of biological membranes. For example, the electrogenic activity of amphibian skins results from a preferential movement of ions through the skin. In the present study the electrogenic activity of the pigeon crop membrane was investigated to determine if it results from a preferential ion movement through the membrane in a manner similar to that of the amphibian skin.' The muscle—free crop membrane was held in a Ussing- type chamber while the potential difference and short—circuit current across the membrane were alternately monitored. Under steady—state conditions, a solution of tritiated water 22 or 0136 was introduced into one side of the with either Na chamber and hourly samples were taken from the opposite chamber for 3-9 hours. Although there was a wide variability in the potential difference (17.4 i 16.5 mV), short—circuit current (20.3 t 13.2 uA) and total resistance (0.86 t 0.57K ohms) between membranes, the net Na+ flux through each membrane could always be equated to the short-circuit current developed by Fran-.4 Richard C. Rose the membrane. Many of the characteristics demonstrated by ion pumps of other biological membranes were also demon— strated to exist in the crOp membrane. It is postulated that the crop membrane possesses an ion pump mechanism capable of moving Na+ from mucosa to serosa against an elec— tro chemical gradient. The ratio of the potential difference to the short- circuit current for each membrane was found to be an index of the permeability of that membrane to water and ions. FLUXES OF Na+, Ci‘ AND WATER ACROSS THE "SHORT-CIRCUITED" PIGEON CROP MEMBRANE By Richard C. Rose A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1967 Dedicated to my Mother and my Father ii ACKNOWLEDGMENTS The author wishes to express gratitude to Dr. W. L. Frantz for his counsel and for providing funds in the form of a special graduate research assistantship. Thanks are extended to the members of the guidance committee, Drs. T. Adams, W. D. Collings and J. R. Hoffert, who helped in the preparation of this manuscript. Appreciation is expressed to Mr. J. H. Dunn for technical assistance and to Mr. K. Irish for help in construction of the perfusion chambers. The author is indebted to the National Science Founda- tion for providing funds in support of this work, (G.B. 3293). iii TABLE OF CONTENTS Page DEDICATION . . . . . . . . . . . . . . . ii ACKNOWLEDGMENTS . . . . . . . . . . . . . iii LIST OF TABLES. . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . vi Chapter I. INTRODUCTION . . . . . . . . . . . . 1 II. REVIEW OF LITERATURE 3 Ussing' 8 Short- Circuit Technique _5 Anatomy of the Frog Skin 7 Anatomy of the Pigeon Crop Membrane 7 Mechanism of Sodium TranSport 8 Metabolism. . . . . ll ATPase Activity . l3 Hormonal Action on Membranes. 15 III. MATERIALS AND METHODS . . . . . . . . . 18 EXperimental Animals . . . . . . . . 18 Apparatus . . . . . . . . . . . . 19 IV. RESULTS 1. Electrical Activity of the Crop Membrane . . . . 27 II. Evidence for an Active Ion Pump in the Crop Membrane. . . . . 28 III. Retention of Na+, C1“, and H20 in the CrOp Membrane. . 39 IV. Prediction of CrOp Membrane Permeability to Ions . . . - 39 V. Prediction of the Crop Membrane Per— meability to Water . . . . . . . . 41 V. DISCUSSION. . . . . . . . . . . . . AA VI. SUMMARY. . . . . . . . . . . . . . A9 REFERENCES . . . . . . . . . . . . . . . 51 iv LIST OF TABLES Table Page 1 Average Electrical Properties of Fifty CrOp Membranes . . . . . . . . . . . 27 2 Ion Flux and Short-Circuit Current . . . . . 29 3 Membrane Retention of Ions and Water . . . . 39 4 Independence of R1 and Short—Circuit Current. . 46 LIST OF FIGURES Figure Page 1. A Cross—Section of a Pigeon Crop Membrane. . . 9 2. Ion Pumping System Illustrated on the Cellular and Membrane Level. . . . . . . . . . lO 3. Model of Amphibian Epidermis . . . . . . . 16 4. Illustration of Perfusion Blocks and Electrical Circuits . . . . . . . . . . . . . 2O 5. Pictorial View of Perfusion Blocks . . . . . 21 6. Pictorial View of EXperimental Apparatus . . . 2A 7. Comparison of Na+ Flux and Short—Circuit Current in Pigeon Crops and Frog Skins. . . . . . 31 8. Short—Circuit Current and Sodium Flux Versus Time . . . . . . . . . . . . . . 33 9. Effect of a Potassium—Free Solution on the Potential Difference . . . . . . . . . 35 10. Effect of a Sodium-Free Mucosal Solution on the Short-Circuit Current . . . . . . . 35 ll. Dependence of Short-Circuit Current on Glucose . 37 12. Effect of DNP on the Short—Circuit Current . . 37 13. Effect of Ouabain on the Short—Circuit Current . 38 14. Ef‘fect of Temperature Changes on the Short— 3 Circuit Current. . . . . . . . . . 38 15. Chloride Flux Versus Ri' . . . . . . . . MO 16. Sodium Flux Serosal to Mucosal Versus Ri . . . 42 17. Water Flux Versus Ri' . . . . . . . . . A3 vi CHAPTER I INTRODUCTION Fundamental to all living processes is the interaction of molecules, atoms and ions. Gilbert (1965) suggested that the first form of life resulted from the random collisions of molecules under the proper environmental conditions. The future of life was dependent on the ability of organisms to control the internal concentrations of the various molecular Species Operational in the living processes. Every living cell, for instance, requires specific internal concentrations of the various ions to continue functioning. Membranes serve the cell, and the organism as a whole, in the selective entry of molecules by passive diffusion. A membrane may also function, with an investment of metabolic energy, in an active process to create the necessary chemical gradients between the intracellular and extracellular fluids. An investigation of the permeability characteristics of a membrane is facilitated when the environmental conditions of the membrane can be controlled. For this purpose Ussing and his co—workers developed an in_vitro perfusion system with which they were able to maintain sheets of tissue from cold blooded animals (frogs and toads) in a viable state long enough to measure accurately ion fluxes. 1 The present problem was to determine if the electro— genic activity of the pigeon crop membrane results from a preferential movement of ions. No technique described in the literature has been successfully used in ion perfusion studies on sheets of tissue from homeotherms, the author adapted the Ussing-type perfusion system to serve this pur- pose. The author's data were used to determine that the passive permeability of a crop membrane can be predicted on the basis of the electrogenic properties of that membrane, a technique thought to be general enough to have application to other membranes of similar structure and prOperties. CHAPTER II REVIEW OF THE LITERATURE The earliest investigation of electrical properties of biological membranes is attributed to Luige Galvani who discovered the electrical property of excised tissue by ob— serving muscle Spasms in frog legs suspended by copper hooks from an iron rod in 1791. DuBois-Reymond (1948) was the first to observe that the frog skin maintains a poten— tial difference between the inside and outside. In 1892 Reid postulated that the skin of some amphibians could help maintain the salt and water balance of the animals by secreting NaCl inward. This could not be attributed to osmosis because it occurred when a piece of skin separates two identical Ringer's solutions. In 1904 Galeotti made the important discovery, which was consistent with Reid's postulate, that Na+ was needed to maintain the skin potential. Galeotti incorrectly postulated, however, that the potential was due to a prefer— ential but passive permeability of Na+ from the outside to the inside of the frog skin. Francis (1933) found that electrical current was generated for many hours by the frog skin and attributed this to the movement of salts by active tranSport (energy dependent transfer of a solute against an electrochemical gradient.) Huf (1935) found that Cl. moved from the outside solu— tion to the inside solution of the in_vitrg frog skin bathed on both sides with identical Ringer's solution. Krogh (1937) further demonstrated the movement of Cl- through the frog skin. He depleted the body C1- of frogs by exposing them to a stream of distilled water and then measured a Cl_ uptake of 0.1 ueq/hr/cm2 from a solution of only 1 mM C1—. The rate of uptake increased as the external Cl- concentration increased. Nernst developed the following equation which describes the potential difference generated by an ion concentration difference between two solutions separated by a membrane: E = —— 1n —— (equation 1) where: E 2 potential difference across F 2 Faraday's constant membrane (mV) (96,500 coulombs/eq.) R = gas constant cl 2 ion concentration of (8.317 joules/mole-KO) inside solution T = temperature (degrees K) c2 2 ion concentration of z = valence outside solution Ussing (1949) modified the basic Nernst equation to describe the ratio of diffusion rates of an ion between two solutions of electrical difference E’: /_ __ in . E — zF 1n E—_ (equation 2) out where: M. = ion flux inward M = ion flux outward in out Using this formula Ussing demonstrated that I_ and C1- passively diffuse through the frog skin but the movement of Na+ cannot be explained in terms of diffusion. Huf and Parish (1951) replaced part of the internal Cl- concentration with phOSphate ion and found that the rate of movement of Na+ across the frog skin was unaltered. They postulated that the potential difference across the frog skin was due to an active (energy consuming) movement of Na+. The ground work was thus laid for the classical eXperiment of Ussing and Zerahn (1951) in which they showed that Na+ was the only species actively moved through the perfused in vitrg frog skin. Ussing's Short-circuit Technique The circuitry used by Ussing was essentially the same as that used in the present experiment, shown in Figure 4. The external (non-membrane) circuit was completed by a microammeter and a voltage supply (consisting of a battery and a variable resistor) connected in series with the plati— num electrodes. During the experiment the applied e.m.f. was adjusted by the variable resistor such that the potential difference across the skin, as read from the potentiometer, was maintained at zero. It is assumed that as each Na+ crossed the membrane it was neutralized by an electron from the external circuit. Consequently, the current in the ex— ternal circuit (short—circuit current, s.c.c.) was a measure of the rate of Na+ accumulation in the inside solution. The movement of Na+ (in ueq) was 105% of the s.c.c. (in ueq e‘) rather than the theoretical value of 100%. The slight dis- crepancy was attributed to diffusion effects. Ussing found that K+ could not be substituted for Na+ in the outside solution; the s.c.c. was reduced but was still equal to the amount of Na+ transported when this change was made. Only Li+ was able to be substituted for Na+. When Li+ was added to the outside solution the s.c.c. was larger than the amount of Na+ transported, indicating that the new ion species was able to support some current (Ussing, 1954). The Na+ pump mechanism appears to be dependent on the presence of K+ in the inside solution. Fukuda (1942) found that if a K+—free solution was used in the inside solution, the potential difference drOpped very rapidly almost to zero. Harris and Maizels (1951) showed that under these circum- stances the active tranSport of Na+ was reduced. Rb+ was found to be able to replace K+ almost completely and Cs+ replaced it partially. Anatomy of the Frog Skin The frog skin consists of a mesodermal chorion and an ectodermal epithelium. The chorion contains blood vessels and connective tissue whereas the epithelium is made up of tightly packed cells of the stratum germinativum and one or more layers of cells undergoing keratinizatioh. Ottoson §t_al. (1953) have shown by electron micrOSCOpy that the germinativum is bordered by a basement membrane which is thought to represent a separation of electrical charge be— tween the corium and the germinativum. Anatomy of the Pigeon CropfiMembrane Dumont (1965) has used light and electron microsc0pes in a histological study of the pigeon crOp membrane. He reports the luminal surface of the crop membrane has a stratified squamous epithelium approximately twelve cells thick which is adjacent to a thin glycocalyx. The lamina prOpria is located beneath the glycocalyx and is composed of connective tissue, blood vessels and nerves. A transverse and a longitudinal layer of smooth muscle fibers is located external to the lamina prOpria. A thin serosa covers the entire organ. Beams and Meyer (1931) described the epithelium of the crOp in terms of two layers on a functional basis. They called the superficial layer lining the lumen of the crop the "nutritive layer." The stratum is 8—10 cells thick in the non-brooding bird and become 2-3 times thicker when the young hatch. The deeper or basal cells of the epithelium were referred to by Litwer (1926) as the "proliferating epithelium." A cross section of the crop membrane with the lamina propria and smooth muscle removed is shown in Figure 1. Mechanism of Sodium Transpprt Figure 2A illustrates the properties of the epithelial cell currently thought to account for its ion pumping ability. Koefoed-Johnsen and Ussing (1958) postulated a high permea- bility to Na+ and moderate permeability to C1’ of the outward-facing membrane of the germinal epithelial cell along with a very low permeability to K+. The inward—facing mem4 brane has a high permeability to KI and CI but a low permea- bility to Na+. If, as they postulated, the pump mechanism is located on the inward facing membrane it is easily ex— plained that NaCl is moved from outside to inside and the K+ concentration inside the cell remains high. Studies with different osmolarities of Na+ and K+ salts were done to confirm the permeabilities as described above. Figure 2B illustrates the carrier-based transport scheme that accounts for many of the facts known about the Na+--K+ pump. Hokin and Hokin (1960) studied the role phos- phatidic acid plays in the Na+ pump activity of avian salt glands. 0n the assumption that Na+ is the actively trans— ported ion in the salt gland as it is in the frog skin and nerve axon, they found a correlation between the amount of ..--9 Jeg ... ........ .. ...ibfimwfiwtxé - ... .1. . ... .u,>a.‘vi§uk.w1 w. ...‘Jn, $$4§7¥fl73 l ......xu...” won... r. .fifi vaH .\ fi\ wrr. J ....kah! 9%th ... . \.C¥‘3J\\l§t Oshx ....\. ..... ..\.. §n h a .“ 1. .L1 .A 1 310‘ . .\ . I...‘ _ .-.}... its... ‘ ,. ,. . ..wc son “-3.“ w. . u. .... a. a We..." ”Tr.“ nu.r..'|H rpfi.‘ “‘1‘ r «thfi! 3. c. I“\Hfl W. .— .p O “.36 . u ‘ +$Qu3?..mg.1, . . .. .n.‘ 91‘..Iy: \ .* .A’...* “yd-Hal“ ._‘. . . .. .. . o ...L. l . . H.... .. . .\ . . ...\. . . \.v.~b.v.‘ ‘ ...u. fi~ ..wa Vs. TWLVMLIVJ ...is. .. .\.. \..A.h....1.0|rv M . . w . a . ....mrcfifi.. .. .. . .. . .. nail... ...»? . .. a..A . Figure 1.--Cross-section of.a pigeon crep membrane. A ) ' ' Cell OutSide (r: (I ~ “\ Inside ' + ____. Na x”1 a ”I? ” K+ V / ‘ ‘\ + II \ \ Na\ I, ‘\¥ ‘\ \ \ L\ I \ \‘ \V l " \\ I \ | ’l \ | \ \ \ + K. K¥\‘Na+ ' ”‘~9 K 0.c.m. ximJ , I ,—" I ' ’ a ’ U I ’ ” ' ,,~' I I”’ I B) '. I I I I I Na+ \ T l \ Na+ Y Y Energy I l X X // I 1 K1 + K w\\-Kx 4 KX */ Figure 2.--Ion pumping system illustrated on the cellular and membrane level. ‘ Figure 2A illustrates cells as described by 0.c.m. is permeable (I.c.m.) is permeable Na+ from the cell and the ion pumping system of theépithelial The outward—facing membrane Ussing. to Na to K+. K+ into and the inward—facing membrane The pump (P) actively transports the cell. Figure 2B illustrates the carrier-based transport mechanism; X and Y represent the carrier molecules. 11 Na+ pumped and the turnover rate of phosphatidic acid. They postulated a phosphatidic acid-diglyceride cycle capable of carrying Na+ from one surface of the membrane to the other. The enzymes necessary for catalysis of the reactions of this cycle have been found in the membrane fraction. Metabolism Francis and Gatty (1938) suggested that NaCl movement through the frog skin was dependent on metabloism since 1 mM cyanide, a known metabolic inhibiter, abolished the net flux. Bromo-acetate reduced the net inward Cl- movement which was restored with the addition of pyruvate or lactate. Huf and Parish (1951) reasoned that an increase in metabolic substrates to the frog skin should increase the net flux of NaCl. However, when they added the sodium salts of ATP or glycerOphosphate to the inside solution of the perfused frog skin there was no increase in the rate of move- ment of Na+. They assumed the increased Na+ gradient coun- teracted any increase in Na+ transport. Zerahn (1956) and Leaf and Renshaw (1957) compared oxygen consumption rates with the active tranSport of Na+ in the frog skin. Zerahn used the Winkler method for 02 determination. He compared 0 consumption of the normal 2 perfused membrane with that of a skin in a Na+—free solution (inhibited Na+ pump activity). Leaf and Renshaw used the polarographic method with vibrating electrodes to measure oxygen consumption. They used posterior pituitary hormone 12 to stimulate Na+ transport, and then compared the increases + in Na tranSport and 02 consumption. Both experiments in- dicated that 1 equivalent of 02 was consumed for each 16—20 equivalents of Na+ transported. Leaf and Renshaw placed the skin in an oxygen—free Ringer's solution to determine whether any tranSport would occur from anaerobic metabolism. The Na+ transport decreased to 10% of its original value within 30 minutes. The following factors were reported by Zerahn (1956) to affect Na+ tranSport and oxygen consumption: 1. Na+ concentration in the outside solution 2. Potential difference across the skin 3. Oxygen tension 4. Time of year 5. Species of the animal 6. Temperature 7. Additions of posterior pituitary hormone 8. Method of blank determination Metabolic inhibitors have been reported to reduce the potential difference and the short-circuit current of the frog skin and toad bladder. Schoffeniels (1955) found that dinitrophenyl (DNP) caused the frog skin potential to fall and finally to reverse. In the inactivated membrane, Na+ movement is passive and with Ringer's solution on each side of the membrane there is no net flux. The presence of ouabain reduces the frog skin potential difference (Koefoed—Johnsen,l957). This was reported to be 13 due to its inhibitory effect on ATPase activity (Schatzman, 1953). Schatzman suggests that in the Ussing model of the Na+-K+ pump ouabain has an inhibitory action at the site for + K activation near the outside surface of the cell membrane but does not compete with Na+ at its activating site on the inside surface of the cell membrane. ATPasejActivity Consistent with the theory that the Na+ pump activity is dependent on energy derived from oxidative phosphoryla- tion, Skou (1957) found in crab nerve a Na+ and K+ dependent ATPase necessary for Na+ and K+ transport. Post_etlgl. (1960) found an insoluble ATP—Splitting enzyme system in human erythrocytes. The activities of the enzyme system and the Na+ pump had the following group of prOperties in common: 1. Both were located in the membrane. 2. Both utilized adenosine triphosphate in contrast to ionsine triphOSphate. 3. Both required Na+ and K+ together. Either Na+ or K+ alone was ineffective. 4. K+ activation was competitively inhibited by high concentration of Na+ in both systems. 5. Ammonium ion substituted for K+ but not for Na+ in both systems. 6. Oubain inhibited both systems. l4 Farquhar and Palade (1966) have studied the locali- zation of ATPase activity in the epidermis of Rana pipiens, Rana catesbiana and Bufo marinus. They incubated skin sections in a Wachstein-Meisel medium (0.83 mM ATP, 10 mM MgSOu, 2.4 mM Pb(N0 ) and then treated each section with 3)2 (NHM)2S to convert the ATPase reaction product (lead phos- phate) into black lead sulfide which could be seen with an electron microsc0pe. They found the enzyme present in all cell membranes that face the labyrinth of epidermal extra- cellular spaces. No activity is indicated in the outer and inner fronts of the epidermis. From their results Farquhar and Palade suggest modifi- cations be made on the Koefoed—Johnsen and Ussing model (1958) of the frog skin membrane. The recently proposed model allows the membrane a larger surface area for pumping activity. The geometry of the extracellular spaces may help restrict the diffusion of ”captured" Na+ towards the interstitial fluid. Koefoed-Johnsen and Ussing suggested that the outward facing membrane, permeable to Na+ but not to K+, was localized on the outer aSpect of the s. germinativum. Farquhar and Palade suggest that this membrane should be located nearer the outer front of the epidermis since there appears to be no structurally continuous barrier on the inner side of the s. germinativum as Koefoed-Johnsen and Ussing suggested. Farquhar and Palade propose that the pump mechanism is located in all cell membranes facing the 15 extracellular spaces. The most likely area for free diffu- sion of K+ is the inward facing membrane of the epidermis. Studies done on the development of the p.d. across the frog membrane may help to determine whether the theory of Farquhar and Palade is more accurate than that of Koefoed- Johnsen and Ussing. Engback and Hoshiko (1957) inserted a micro-electrode into the frog skin from the outside and found that the potential across the skin was established in two steps. Immediately upon entering the skin the electrode recorded a negativity in relation to the outside medium. This was assumed to be the resting potential of a superficial epithelial cell. A positive potential of about 60 mv ap- peared at a depth of 50p . The full potential of the mem— brane was recorded when the micro—electrode reached a depth of about 100m The results of Engback and Hoshiko appear to be more consistent with the theory of Farquhar and Palade than that of Koefoed-Johnsen and Ussing. The two step development of the p.d. may be due to the diffuse distribution of the Na+ pumps as described in Figure 3. Hormonal Action on Membranes The neurohypophyseal hormones (ADH) have been found to alter the rate of movement of salts and water across many biological tissues. ADH increases the Na+ pump activity of the frog skin (Fuhrman and Ussing, 1951) and the permea— bility of the skin to Na+ (Ussing, 1955). The s.c.c. of Z O I ——-—HM OFM-———- ._ la ‘ 1’ '1 I 4- ‘- I R‘ s "’ / PM ““ )Na+ - 4- IM I ~ - -..-42'4-DK4' ‘ ~~~ - -------------. I ”Noun.HOHUHNHIHIHOIHUHWHHH“.H'H|y....H'H'MHIH|,I|HIIHIH| I l nun... Na+ Permeable membrane K H H -_Membrane provided with Na+ and K+ pumps Figure 3.—-Mode1 of amphibian epidermis. This figure depicts the modified model of amphibian epidermis by Farquhar and Palade (1966). The three cell layers repre— sent schematically, from left to right, the s. corneum, s. spinosum, and s. germinativum. The intercellular space is shown in white and the intracellular space in gray. The nuclei appear in strippled gray. EM, external medium, OFM, o ward fac'rg membrane; IFM, inward facing membrane; BM, basement membrane; IM, internal medium; d, desmosome. 1+v Us 17 the toad bladder is increased (Leaf, Page, and Anderson, 1959), as is the membrane permeability to water (Leaf, 1960), urea, acetamide, propionamide, butyramide, dimethyl formamide, cyanamide and nicotinamide (Maffly, Hays, Lamdin and Leaf, 1960) although the permeability to many other molecules is unaffected. At least two different theories are currently defended concerning the action of ADH on the permeability of mem— branes. Schwartz et a1. (1960) hold that ADH increases the pore size of the outward—facing membrane. They point out that the increased active transport of Na+ in the frog and toad bladder may be due to increased amounts of Na+ available to the pump as the permeability of the outward-facing mem- brane increases. Orloff and Handler (1961) believe that adenosine 3‘,5'-mon0phosphate (cyclic AMP) is the agent that directly regulates the membrane permeability and that the role of ADH is to regulate the concentration of cyclic AMP. Crabbe (1960) found a stimulatory effect of aldo- sterone on the toad bladder similar to that of ADH on the fr“0g skin but his system required an incubation time of about one hour before the effect became evident (Kleinzeller and Kotyk, 1960). Edelman, Bogoroch and Porter (1963) demonstrated that in the toad bladder aldosterone is located in the nucleus of the epithelial cells and acts by promoting DNZAMdependent RNA systhesis that produces proteins involved in the coupling of metabolism with Na+ transport. CHAPTER III MATERIALS AND METHODS Experimental Animals White King pigeons of both sexes, one to seven years old, were purchased within two weeks of their use from the Cascade Squab Farm, Grand Rapids, Michigan. There was no attempt to regulate the amount of light hours the birds received each day; the present experiments were done during the summer months. The birds were kept in continuous supply of water and feed. The birds were killed by cervical dislocation. The feathers over the crOp area were plucked and then the skin was teased from the crop membrane. Two samples of membrane were obtained from each bird; each was held in an inter— locking ring system two inches in diameter. The serosal surface and musculature of the membrane were separated from fine mucosa under a dissecting microsc0pe. The mucosal mem- kfl‘ane (2.54 cm2) was then placed between the halves of a lucite perfusion chamber. The Operation took approximately ten minutes during which the membrane was constantly bathed in warm bird Ringer—glucose solution (see page 26). 18 19 Apparatus Two types of lucite perfusion chambers were used to hold the membranes. The first type used was an electrically driven stirring device. Oxygen was released into an air Space above the medium. The second type of block (Figures 4 and 5) used an air-lift siphon for stirring. This block system was first used by Knoll (1962) to hold frog skins and was later modified by Edelhauser for perfusion studies on trout corneas (1966). The block system was again modi- fied by the author to make s.c.c.—p.d. readings across frog skin and pigeon crop membranes. Pressurized air was passed through an air purifier and flow equalizer (Koby Corporation, Melrose, Mass.) and then combined with 95% oxygen and 5% carbon dioxide. The resulting mixture (72% nitrogen, 27.5% oxygen, 0.5% carbon dioxide) was passed through a series of buffered wash bottles to saturate it with water.‘ This mixture was used to Operate the air—lift siphon and also supply the perfusion medium with oxygen and regulate its pH. Lucite caps were used to prevent evaporation and Splashing of the perfusion medium. Both types of lucite per— fusion chambers were found to furnish adequate stirring and oxygen to maintain the electrical activity of the membrane for up to twelve hours. The temperature of the medium was maintained at 38-400 C. Miniature calomel electrodes (E.H. Sargent & CO., Detroit, Michigan) were used to measure potentials in both perfusion systems. The saturated electrolyte in these 20 AL E R DT If U Ch '" RT RS - ‘3- \\ M /\/v VR III O —. - E Figure 4.—-Illustration of perfusion blocks and electrical circuits. AL - Air line R - Reservoir B — Battery (1.5V) RS -- Ring system C - Cap RT - Recovery tube for fluid Ch — Chamber SH - Sample hole DT - Delivery tube for fluid SSB - Short-circuit bridge E - Electrode VR - Variable r Sistor M - Membrane (o—ll x 10 Ohms) mV - Millivoltmeter (0-100v) uA - microammeter PW - Platinum wire (0 - ll pA) 2]. l O. 0.0!“ ..‘I 1M. t: V P ... ..J ..7. ‘ 0:“ A 0 on n. .OOQndoOchw? 6 ~ on no noonn ct non ... 0 04:3?!“ no no. nu on . n n a o. a . 9. nonunion. 0*: ¢ rcpt. o. t: .o« ..non.n .nnvn n a . o... nfluounnsan no . \ 22 electrodes was changed from KCl to NaCl because the K+ released by the electrodes was found to alter the membrane potentials after 2—3 hours. Each set of converted electrodes was checked before and after use and confirmed to have an asymmetry of less than 0.3 millivolts and 1.0 microampere. The electrodes were checked periodically in a NaI crystal well counter to insure that no Na22 or Cl36 had been absorbed into the NaCl solution. Two systems for short—circuiting the membrane were used in this experiment. The first was the manual type Similar to that first described by Ussing (Figure 4). A counter-e.m.f. of three 1.5 volt batteries in series was con— nected to a variable resistor. Contact with the perfusion solution was made through platinum wire seated in a Ringer— agar mixture. The external resistance was adjusted so that the membrane potential as read from a recording electrometer (YSI model 81, Yellow Springs Instrument Co., Antioch, Ohio) was reduced to zero. The exogenous current required to main— tain the potential difference equal to zero was read from a microammeter. A constant adjustment of the external resist— ance was required as the membrane's potential or resistance (Zhanged. A second method of recording the short-circuit current Was devised by Frantz and Holland (1967, in preparation) and was used in these experiments. The p.d. is constantly mulled to zero by a Sargent model SR recorder converted to a cathode follower and calibrated to record 4.0 mV full 23 scale. When a 40K Ohm resistor is intermittently introduced into the circuit by a timing device, the s.c.c. is read directly off the chart as l bA/scale unit. This method of recording the s.c.c. eliminates the need for manual adjust— ment of the external resistance. The perfusion apparatus was surrounded by grounded COpper wire screening to prevent Spurious magnetic or elec— trical impulses from affecting the p.d. and s.c.c. measure- ments. The volumes, and therefore the hydrostatic pressures, of the bathing solutions on the two sides of the membrane were maintained constant to prevent changes in the p.d. After the perfused membrane reached a condition of steady p.d. and s.c.c. 100 pl of a radioisotOpic solution containing tritiated water (HTO) with either NagZCl or NaCl36 was added to the mucosal or serosal solution. Two samples, one at 3 min and one at 10 min, were taken from the inoculated solution using 20 ul micropipettes (Drummond microcaps, Drummond Scientific Co., Broomall, Pa.). Hourly (20 pl) samples were taken from the opposite solution. At the end of each experiment the perfused section of the mem— brane was put in a counting vial in toto. Each counting vial (Packard Instruments Co. Inc., Downers Grove, Ill.) contained 15 ml of a scintillation solution, composed of a primary scintillator (5 gm PPO, 2.5—diphenyloxazole), a secondary scintillator (50 mg a- naphthylphenloxanole), 80 gm napthalene and enough dioxane to make one liter. The HTO and Na22 or Cl56 content of Figure 6.-—Pictorial View of experimental apparatus. 25 M O‘\. each sample was assayed for 100 minutes in a Nuclear Chicago Mark I Liquid Scintillation Spectrometer. The counting efficiency was 30% for HTO and 90% for Na22 and C136. The counting error was less than 2% on each sample. Each sample was corrected for background activity and quenching. The bird Ringer's solution used in these experiments contained the following concentrations Of ions: Na+ 150.0 meq/i 01‘ 128.0 meq/l K+ 3.25 meq/l SO“ = 1.0 meq/l Ca++ 2.20 meq/I H003“ 29.1 meq/l Mg++ 2.50 meq/l Glucose 5.55 meq/l The K+-free solution contained all the ions listed above with Na+ substituted for K+. The choline chloride bird Ringer's solution contained 150 meq/l choline and less than 5 meq/l Na+. + + + . . Na , K and Ca concentrations were determined on a Coleman Model 21 flame photometer. Cl_ concentrations were determined on a Coleman microtitrator using the method Of Shales and Shales (1947). CHAPTER IV RESULTS 1. Electrical Activity of the Crop Membrane A potential difference exists between two identical Ringer's solutions separated by the crop membrane (Table 1). Because this membrane generates a cationic current in yitgg, the serosal solution acQuires a positive charge with reSpect to the mucosal solution. The electrogenic properties can be expressed in the Ohm's law relation, V 2 IR, adapted to the perfused membrane: V is the potential difference; I is the short-circuit current (equivalent to the cationic cur— rent), and R (total resistance of the circuit) can be cal- culated as p.d./s.c.c. TABLE l.--Average electrical properties of fifty crop membranes. n 2 50 Potential difference 17.4 i 16.5 mV Short—circuit current 20.3 I 13.2 uA Total resistance 0.86 f 0.57K Ohms IX) “d R- CD II. Evidence for an Active Ion Pump in the Crop Membrane The net Na+ flux across the membrane as determined with Na22 accounts for all of the s.c.c. develOped by the membrane. This is evidence for a Na+ pump according to Ussing's theory (see Chapter II, p.5). Table 2 Shows that after the membrane pumping mechanism becomes saturated with Na22 (4 hours) the amount of Na+ (in ueq.) moved from the mucosal solution to the serosal solution is essentially equivalent to the s.c.c. (in Meq. e‘) developed by the mem— brane. The amount of Na+ reaching the serosal solution is actually slightly greater than can be accounted for on the basis of the s.c.c. because there is a small diffusion of Na+ across the membrane in both directions (see Results IV, p. 39). To account for this diffusion, Na22 was put in the serosal solution of three membranes and the diffusion rate from the serosal to the mucosal surface was measured (Table 2). The methods used for calculating the amount of Na+ crossing the membrane and the amount of s.c.c. during the same time period are as follows: The assumptions made in these calculations are: l. the tissue reacts the same to Na22 as to Na23; 2. the bathing medium is completely and instan— taniously mixed by the stirring apparatus; the injection of the isotopic Na+ does not Do produce a significant Na+ gradient. 29 HH.m mw.m we.m mo.m mo.a sfl.n mm.o Ameo\-m omav Hemoosa on mm.o Hm.o om.o Sn.o on.o so.o o.o Am2o\-no amsv ammonmm -Ho mo.m mm.n mm.H mm.H 00.9 no.0 :m.o Ameo\um omav Homonmm ow am.o mm.o mn.o mn.o on.o on.o o.o Amao\-no amsv Hemoosa -Ho sw.m mu.m am.m mw.n sa.a no.0 Hm.o Amao\-o omsv Hmnooss on mm.o mm.o sm.o sm.o o.o mo.o o.o Ameo\+mz omsv Hmnoson +mz ®O.: ww.m mm.m :m.m OQ.m m@.H m:.H ©O.H no.0 AmEo\Io Gonv HmmOnom Op mm.a om.m o@.m mm.m mo.m os.a mm.a sw.o mm.o Ameo\+mz omiv ammoosa +mz .mms .mmn .mMs .nwc .mmn .nwe .nme .nwn .mn cospoomcn maoooma stone mane .pconnso pflsonflOIpnozm USO xsam COHII.m mqm< 0.50“ :5 H CH + CU Z 3.25 ‘- Figure 16.--Sodium flux serosal to mucosal versus Ri' wal- 43 16 fir? 1a «) H20 flux (meq/cmg/hr.) H1 m 001 4:4 mxnl ow+ ”1 Figure 17.——Water flux versus Ri' CHAPTER V DISCUSSION The first demonstration of active transport of ions through a biological membrane was made by Ussing (1951) . working with frog skins. A. C. Brown (1965) summarized the conditions necessary for active transport as the following four points, (evidence is recalled under each point to sup— port the author's hypothesis of active transport in the cr0p membrane): (1) "the force is located within the membrane"; The force which moves Na+ across the cr0p membrane must be located within the membrane since there is no con- centration gradient between the perfusing solutions of the in_vitro_preparation. (ii) ”the force directly influences particle motion"; The force which accounts for the Na+ accumulating in the serosal solution must be direct since under s.c.c. condi- tions there are no electrochemical or osmotic gradients in- fluencing particle motion; (iii) “the force tends to increase the free energy of the particle as it passes through the membrane"; The free energy of the Na+ is greater in the serosal solution because they have a higher electrochemical energy AM “5 (positive p.d. under non-s.c.c. conditions) than the Na+ in the mucosal solution. (iv) "the force is established by and maintained through the consumption of free energy made available by metabolism"; The s.c.c. is dependent on the glucose available to the membrane, and it is inhibited by metabolic poisions. Brown (1962) characterized the behavior of frog skins to ion diffusion by measuring what he referred to as "mean internal conductance” ( 0 = s.c.c./p.d.). He observed that 0 usually falls for a short time immediately after the skin has been perfused in_vi£rg. He hypothesized that 0 become smaller because the p.d. is raised due, perhaps, to increased passive resistance. Data from fixapresent experiment support the idea that a change in passive ionic resistance can change the relation between the s.c.c. and the p.d. In this thesis, however, the above relation has been referred to as "internal ionic resistance," Ri = p.d./s.c.c. This term seems more appli- cable since the passive resistance to ions is being described; it has the additional qualities of being distinct and inde- pendent of the Na+ pump activity. To verify that the Ri describes the passive ionic resistance of the membrane independent of the s.c.c. the following experiment was done. A membrane was perfused in the usual manner for one hour during which the s.c.c. and p.d. (and therefore the Ri) were constant. Ouabain was then put in the serosal solution to decrease the s.c.c. According to Schatzmann (p. 13) ouabain inhibits the Na+ pump without altering the passive permeability of a membrane. The s.c.c. and p.d. were alternately measured as they decreased to zero. As seen in Table 4 the Ri did remain constant as the s.c.c. decreased and therefore is independent of the s.c.c. TABLE 4.-—Independence of R1 and short—circuit current. Time (min.) 0 2 u 6 8 10 12 1M 16 18 p.d. (mV) 22 21 17 15 14 12 9 8 6 5 s.c.c. (MA) 13 13 11 10 9 7 6 5 4 3.3 R1 (K ohms) 1.69 1.62 1.55 1.50 1.55 1.71 1.50 1.60 1.50 1.52 The concept of measuring the permeability of a bio- logical membrane to ions on the basis of its electrical proper— ties may be useful in evaluating the effects of other agents on membrane permeability. For instance, the activity of the Na+ pump in the frog skin is known to be regulated by ADH (Koefoed—Johnsen and Ussing, 1953). Schwartz et_al, (1960) have proposed that ADH stimulates the Na+ pump activity of the frog skin by increasing mucosal permeability to ions which will cause increased amounts of Na+ to be available to the Na+ pump. It may be possible to evaluate Schwartz' theory of ADH action by observing the R1 as the hormone acts M7 on the skin. If ADH does increase the mucosal permeability of the frog skin a concurrent decrease in Ri would be ex- pected. In the_inivitro crop membrane preparations ADH in low concentration (1 IU/ml serosal solution) has not increased the Na+ pump activity. If the application of increased con— centrations of ADH stimulates the Na+ pump activity due to increased ion permeability of the epithelial cells, then this stimulation should be accompanied by a decrease in Ri' It was reported (p. 39) that the crop membrane retains 89% less Na22, 88% less Cl36 and 45% less HTO when the iso- topes are introduced into the mucosal solution rather than the serosal solution. This different saturation ratio of Na+, Cl- and H20 may be due to a functional permeation barrier in the membrane. The barrier in the crop membrane is assumed to be the germinativum as it is in the toad bladder (Edelman SI: 31., 1963, and Leaf and Hays, 1962), the mucosa of the stomach (Rehm, 1963) and the intestine (Wilson, 1962) or the combined germinativum and Spinosum as in the frog skin(Farquhar and Palade, 1966). A cross section of the crop membrane (Figure 1) has a low layer of cornified epithelium (8n ) overlying the spinosum and germinativum (M u) and a thick submucosa (50 p). Because the mucosa is one sixth the thickness of the sub- mucosa it would be eXpected to have about 86% less Na+ and 01— to exchange with the bathing medium than the submucosa has. The reported values for Na22 and C136 retention are 48 in agreement with the eXpected value assuming that the germinativum is the diffusion barrier. The mucosa should also have 86% less H,0 to exchange 2 with the bathing medium than the submucosa has. The results indicate, however, that only 45% leSSHTO is retained when this isotOpe is introduced into the mucosal solution than when it is introduced into the serosal solution. Evidently the permeability of the membrane to water is high enough that it becomes nearly saturated with HTO during the three to nine hour experiments. A series of short (10-60 minute) eXperiments should help determine whether HTO is more rapidly exchanged with the submucosa than the mucosa. The literature reveals no successful methods of pre— dicting H20 diffusion rate through a biological membrane on the basis of electrical properties. Garby and Linderholm (1954) unsuccessfully attempted to correlate D20 movement across the frog skin with electrical resistance used as an index of the permeability to ions. The present experiments have indicated that H20 dif- fusion through the crop membrane can be predicted on the basis of the R1 (Figure 17). There is no electrochemical or structural model which explains why the H20 permeability is related to the Ri’ However, from the similarity between Figures 15, 16, and 17 it is reasonable to assume some common factor determines the permeability of the membrane to both ions and Hi0. CHAPTER VI SUMMARY An Ussing type perfusion system has been used to demonstrate that a sheet of tissue from a homeothermic animal (pigeon cr0p membrane) can be maintained in a viable state for up to twelve hours. The crop membrane is electrogenic and may be com— pared with the frog skin and toad bladder as units which have ability to move Na+ against an electrochemical gradient, i.e., this membrane has a "Na+ pump." The pump activity has been shown to be decreased by DNP or by the lack of glucose. It is dependent on the presence of Na+ in the mucosal solution and K+ in the serosal solution. The perfused crop membrane takes up Na+, C1- and H20 more rapidly from the serosal solution than from the mucosal solution. This has been explained in terms of a diffusion barrier located near the mucosal surface. A method of predicting the permeability of a crOp membrane to cations (Na+) and anions (C1-) on the basis of the p.d./ s.c.c. has been presented. A membrane for which the p.d./ s.c.c. is greater than 1K ohm has a low perme— ability. It is expected that this method would be applicable to other membranes with electrogenic ion pump activity. 49 50 The permeability of a crop membrane to H20 is also predictable on the basis of the p.d./s.c.c. Membranes for which this ratio is large have relatively low permeabilities. The research described in this thesis has led to the following questions pertaining to the pigeon crOp membrane: 1. What is the size of the Na+ Space and H20 space and where is the permeability barrier located? In what part of the membrane is the Na+ pump ATPase located? Is Li+ or any other cation able to substitute for Na+ in the pump mechanism? How much oxygen is consumed in the transport of each Na+ ion? What is the effect of aldosterone and ADH on the Na+ pump activity? + What effect does Ca+ have on the permeability of the crop membrane to ions and water? REFERENCES Beams, H. W. and R, K. Meyer, 1931. The formation of pigeon "milk." Physiol. Zool., 4: 486. Brown, A. C., 1962. In vivo frog skin short—circuit current and Open circuit voltage. Fed. Proc., 21: 144. Brown, A. C., 1965. Ch. 43, Physiology and Biophysics. Saunders Company. Crabbe, J. Quoted by Kleinzeller and Kotyk, (1960). Dumont, J. N., 1965. Prolactin—induced cytologic changes in the mucosa of the pigeon crop during 'Milk" forma— tion. Zeitschrigt fur Zellforschung, 68: 755. Dubois-Rewnond,E., 1848. 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