Cochlear function and sodium and potassium activated adenosine triphosphatase.

The maintenance of the cation gradients between endolymph and perilymph in the cochlea requires the operation of a cation pump. An adenosine triphosphatase system activated by sodium and potassium is present in high activity in the cochlear membranes (tegmentum vasculosum and stria vascularis). The cochlear microphonic potential is inhibited by perilymphatic perfusion of ouabain and erythrophleine. Since the microphonic potential depends on the high concentration of potassium ions in the endolymph, our findings strongly suggest the operation of an adenosine triphosphatase cation pump system activated by sodium and potassium, in the generation of cochlear cation gradients.

Life sciences: radiobiological advanced biostack experiment.

The radiobiological properties of the heavy ions of cosmic radiation were investigated on Spacelab 1 by use of biostacks, monolayers of biological test organisms sandwiched between thin foils of different types of nuclear track detectors. Biostacks were exposed to cosmic radiation at several locations with different shielding environments in the module and on the pallet. Evaluations of the physical and biological components of the experiment to date indicate that in general they survived the spaceflight in good condition. Dosimetric data are presented for the different shielding environments.

Gastric acid secretion in the lizard. Ionic requirements and effects of inhibitors.

1. The effects of ion substitution and various inhibitors on the transmucosal potential, short circuit current, mucosal resistance and acid secretion of the lizard gastric mucosa, incubated in an Ussing chamber, have been determined. 2. Ion substitution experiments indicate that the serosal potential step consists of a combined C1- and K+ diffusion potential, and that the mucosal potential step is Na+ dependent and behaves primarily as a Na+ diffusion potential. 3. Experiments with ouabain indicate that the major (Na+, K+)-ATPase activity responsible for maintenance of cation gradients is located on the serosal side of the mucosal cells, and that this pump activity is non-electrogenic. 4. Experiments with amiloride indicate that a passive sodium influx on the mucosal side is essential for the maintenance of the transmucosal potential and short circuit current. 5. Acid secretion requires the presence of sodium and chloride on the serosal side and the maintenance of a high intracellular potassium level through the (Na+, K+)-ATPase system. 6. The effects of acetazolamide and thiocyanate are compatible with an involvement of carbonic anhydrase and anion-dependent ATPase in acid secretion. 7. Upon initiation of acid secretion the serosal membrane permeability for chloride increases and that for potassium decreases.

Rat pancreas adenylate cyclase. VI. Role of the enzyme in secretin stimulated enzyme secretion.

1. The responsiveness of adenylate cyclase and enzyme secretin for secretin and the C-terminal octapeptide of pancreozymin has been investigated in particulate fractions of the pancreas of five different species. 2. The adenylate cyclase is sensitive to the C-terminal octapeptide of pancreozymin in all species investigated. 3. The enzyme is much more sensitive to secretin in rat and cat than in mouse and rabbit, whereas with guinea pig intermediate values are obtained. 4. The enzyme secretion is stimulated by secretin in pancreatic fragments of rat and cat, but not in those of mouse and rabbit. 5. These results suggest that in species where secretin stimulated enzyme secretion, it does so by stimulating the adenylate cyclase system.

Is there a plasma membrane-located anion-sensitive ATPase?

A study of the intracellular localization of HCO-3-stimulated, SCN--inhibited magnesium-dependent ATPase was performed in gill tissue of the rainbow trout (Salmo irideus), rabbit kidney and rabbit gastric mucosa. Tissue homogenates were subjected to centrifugal fractionation, and the microsomal (60 min 100 000 X g) and light mitochondrial (20 min 20 000 X g) fractions were further fractionated by density gradient centrifugation. Subfractions were characterized by marker enzyme assays and electron microscopic observation. In trout gill indications for an exclusively mitochondrial localization were found. In kidney no definite conclusions could be drawn. In rabbit gastric mucosa initially an apparently non-mitochondrial HCO-3-stimulated ATPase, in addition to a mitochondrial one, was found and its characteristics were studied. Further studies showed that this ATPase also appears to be of mitochondrial origin and probably represents mitochondrial inner membranes. Possible explanations for earlier conflicting reports concerning the localization of this enzyme in gastric mucosa and other tissues are discussed.

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.

Rat pancreas adenylate cyclase V. Its presence in isolated rat pancreatic acinar cells.

(1) In order to determine the cellular localization of the secretin- and pancreozymin-sensitive adenylate cyclase in rat pancreas, the occurence of this enzyme system has been investigated in isolated pancreatic cells. (2) Digestion of rat pancreatic lobules with collagenase yields a preparation of isolated cells which upon differential morphological analysis appears to consist for 97% of acinar cells and to contain for fewer centro-acinar and ductal cells than undissociated lobules. (3) Expressed per mg protein, the isolated cells contain the same amount of DNA, chymotrypsin and lactic dehydrogenase as the undissociated tissue. The stimulated adenylate cyclase activity is nearly entirely recovered in the isolated acinar cells, as is also the case for the low Km adenosine 3',5-cyclic monophosphate phosphodiesterase activity and the adenosine 3',5'-cyclic monophosphate (cyclic AMP) content. Marked losses are noted for the basal adenylate cyclase and the high Km cyclic AMP phosphodiesterase activities. (4) Washing the isolated acinar cells in Krebs-Ringer bicarbonate medium containing 10 mM 1-methyl-3-isobutylxanthine causes a cyclic AMP level 2.6 times that in cells washed in Krebs-Ringer bicarbonate alone. The cyclic AMP level is further increased by subsequently incubating the cells for 10 min in the presence of 3-10(-7) M pancreozymin-C-octapeptide or secretin to values 1.7 or 4.7 times the control level in cells incubated for 10 min with 1-methyl-3-isobutylxanthine alone. (5) It is suggested that the adenylate cyclase of the acinar cells may be involved, with another factor, in the stimulation of enzyme secretion, whereas a ductular cyclase would function in the regulation of the bicarbonate-dependent fluid secretion.

Role of negatively charged phospholipids in highly purified (Na+ + K+)-ATPase from rabbit kidney outer medulla studies on (Na+ + K+)-activated ATPase, XXXIX.

1. The requirement for specific polar head groups of phospholipids for activity of purified (Na+ + K+)ATPase from rabbit kidney outer medulla has been investigated. 2. Comparison of content and composition of phospholipids in microsomes and the purified enzyme indicates that purification leads to an increase in the phospholipid/protein ratio and in phosphatidylserine content. 3. The purified preparation contains 267 molecules phospholipid per molecule (Na+ + K+)-ATPase, viz. 95 phosphatidylcholine, 74 phosphatidylethanolamine, 48 spingomyelin, 35 phosphatidylserine and 15 phosphatidylinositol. 4. Complete conversion of phosphatidylserine into phosphatidylethanolamine by the enzyme phosphatidylserine decarboxylase has no effect on the (Na+ + K+)-ATPase activity of the purified preparation. 5. Complete hydrolysis of phosphatidylinositol by a phospholipase C from Staphylococcus aureus, which is specific for this phospholipid, has no effect on the (Na+ + K+)-ATPase activity. 6. Hydrolysis of 95% of the phosphatidylcholine and 60--70% of the spingomyelin and phosphatidylethanolamine by another phospholipase C (Clostridium welchii) lowers the (Na+ + K+)-ATPase activity by about 20%. 7. Combination of the phospholipid-converting enzymes has the same effect as can be calculated from the effects of the enzymes separately. Only complete conversion of both phosphatidylserine and phosphatidylinositol results in a loss of 44% of the (NA+ + K+)-ATPase activity and 36% of the potassium 4-nitrophenylphosphatase activity. 8. These experiments indicate that there is no absolute requirement for one of the polar head groups, although in the absence of negative charges the activity is lower than in their presence.

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.

Movement of calcium through artificial lipid membranes and the effects of ionophores.

The calcium efflux from multi-layered vesicles (liposomes) of different lipid composition has been studied. Liposomes composed of lipids extracted from cattle retinas are compared with liposomes which consist of phosphatidylcholine or a 1:1 phosphatidylcholine/phosphatidylserine mixture. The percentages of 45-Ca capture by these three types of liposomes are 10, 1 and 4% respectively; The efflux rates are 2.5-10- minus 6, 2-10- minus 6 and 4-10- minus 5 S- minus 1 respectively. The semilogarithmic efflux curves for phosphatidylcholine and phosphatidylcholine/phosphatidylserine liposomes are linear with time, but those for the retinal lipid liposomes are discontinuous. The activation energy for the calcium efflux from the latter liposomes is about 10.5 kcal/mol, both before and after the discontinuity. The ionophores X537A and A23187 enhance the calcium leakage from retinal lipid liposomes, the latter ionophore being much more effective than the former. At high concentrations both ionophores seem to transport calcium as a 1: 2 Ca-ionophore complex. At low ionophore concentrations, however, X537A appears to transport calcium as a 1:1 complex, but A23187 as a 2:1 complex.

Absence of (Na+,K+)-ATPase involvement in lactose production by lactating guinea pig mammary gland.

The role of the (Na+, K+)-ATPase system in lactose production by the lactating guinea pig mammary gland has been studied in vitro with slices of the gland. In this system there is an initial fast lactose release, mainly representing secretion of preformed lactose, followed by a continuous slow lactose release, representing mainly lactose synthesis. The latter process occurs at a rate of 1.6 to 2.4 g lactose/kg wet wr/h, which value is about half of the lactose production in vivo (3.9 g/kg set wt/h). Incubation of slices in the presence of 10-4 M ouabain does not influence the rate of overall lactose production. When determined separately, it does not change either the rate of secretion or that of synthesis. This pleads against a role of the (Na+, K+)-ATPase system in lactose secretion or synthesis, in particular it seems to rule out control of the rates of these processes by the intracellular potassium concentration. An explanation for the generally observed correlation between the lactose and potassium concentrations in milk, may be that both the maintenance of the intracellular potassium concentration and the lactose synthesis rate require the presence of ATP.

Inhibition by cyclic AMP of lactose production in lactating guinea pig mammary gland slices.

Addition of the cyclic AMP phosphodiesterase inhibitors theophylline (10- minus 2 M) or papaverine (10- minus 4 M) leads to a complete inhibition of lactose synthesis in incubated guinea pig mammary gland slices. Addition of 10- minus 5 M cyclic AMP or dibutyryl cyclic AMP results in 1 30-40% inhibition of the synthesis, which effect is not increased by applying higher concentrations of these compounds. A 30-40% inhibition can also be obtained with epinephrine (5 - 10- minus 5 M), or isoproterenol (10- minus 4 M), but the polypeptide hormones glucagon (10- minus 7 M), insulin (1 munit/ml) and relaxin (10 mug/ml) do not significantly affect lactose synthesis. Cytochalasin B (5 mug/ml) inhibits lactose production by 58and colchicine (10- minus 5 M) by 25%. These experiments suggest that an increase in the intracellular level of cyclic AMP either through its addition, through hormonal stimulation of its synthesis, or through inhibition of its intracellular breakdown, leads to an inhibition of lactose production in lactating mammary gland. This effect of cyclic AMP is similar to that of progesterone, which is known to inhibit lactation in vivo and the withdrawal of which at parturition has been postulated to initiate lactogenesis.

Biochemical aspects of the visual process. XXVII. Stereospecificity of ocular retinol dehydrogenases and the visual cycle.

A comparative study is made of the stereospecificity of two particulate retinol dehydrogenases from bovine eyes and of horse liver alcohol dehydrogenase. The particulate retinol dehydrogenase of outer segments reacts with the all-trans isomers of retinaldehyde and retinol but not with the 11-cis compounds. In contrast, a particulate retinol dehydrogenase present in pigment epithelium reacts preferentially with the 11-cis compounds. Horse liver alcohol dehydrogenase (EC 1.1.1.1.) can convert both isomers, but the all-trans isomers are clearly preferred. Differences with regard to cofactor preference and stability are also noted. The outer segment enzyme clearly functions in the rhodopsin cycle. It is unlikely that the 11-cis retinol dehydrogenase from pigment epithelium is directly involved in providing 11-cis retinaldehyde from rhodopsin regeneration, but it may serve to make available 11-cis retinaldehyde from rhodopdsin, digested in phagocytized rod sacs, for the synthesis of visual pigment by the visual cells.

Rat pancreas adenylate cyclase. III. Its role in pancreatic secretion assessed by means of choleara toxin.

1. The role of adenylate cyclase in rat pancreas is further investigated by means of cholera toxin, which is known to activate the enzyme in several tissues. 2. Cholera toxin activates rat pancreatic adenylate cyclase in vitro upon preincubation of tissue slices with the toxin for more than 30 min, but not when it is merely present during the enzyme assay. The maximal effect is reached after 90 min pre-incubation. The half-maximally activating concentration is 3.5 mu-g/ml upon pre-incubation for 90 min. 3. After pre-treatment of pancreatic tissue slices with 2 mu-g/ml cholera toxin, further stimulation of adenylate cyclase activity can be obtained by adding pancreozymin-C-octapeptide, secretin, or fluoride to the assay medium, but the final activity with maximally effective concentrations of the hormones is not higher, and with fluoride even less, than that without the toxin pre-treatment. 4. The in vivo effects of the two hormones and of cholera toxin have been studied after cannulation of the pancreas. Pancreozymin-C-octapeptide (intravenously) markedly stimulates both flow rate and rate of protein secretion. Synthetic secretin (intravenously), in addition to its expected effect on flow rate, slightly stimulates protein secretion, which is not due to a wash-out effect. Cholera toxin, topically applied to the cannulated rat pancreas, causes a steady increase of the flow rate after a delay of 20--30 min. The rate of protein secretion is not affected or slightly decreased by the toxin. Pancreozymin-C-octapeptide, given intravenously 1 h after cholera toxin application, causes the same increase in flow rate and rate of protein secretion as would be expected without cholera toxin treatment. 5. The sodium and potassium levels in the pancreatic fluid after administration of secretin or cholera toxin do not change, while the chloride level decreases in both cases. 6. These observations indicate that the rat pancreas adenylate cyclase activity is a rate-limiting factor in the regulation of water and electrolyte secretion. A possible auxiliary role in the regulation of enzyme secretion cannot yet be excluded.

Studies on (Na+ plus K+)-activated ATPase. XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg1+ and inorganic phosphate.

Dissociation of the (Na+ + K+)-ATPase ouabain complex, formed in the presence of Mg2+ and inorganic phosphate (Complex II), is inhibited by Mg2+ (21-45%) and the alkali cations Na+ (25-59%) and K+ (27-75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0-11% by Na+ and 11-19% by K+. The degree of inhibition is not directly related to the size of the dissociation rate constant (k-) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues. Inhibition curves for Na+ and K+ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30-40 mM Na+ and 6-10 mM K+, and the maximally inhibitory concentrations are 50-150 and 15-20 mM, respectively. Maximal inhibition by Na+ or K+ for these tissues is the same. For guinea pig kidney cortex Na+ and K+ are almost equally effective, but 150 mM K+ or 200 mM Na+ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations. The inhibition curve for Mg2+ is not sigmoidal. In the kidney preparations Mg2+ inhibits half-maximally at 0.4-0.5 mM, maximally at 1-3 mM. Maximal inhibition by Mg2+ is higher than by Na+ or K+ for rabbit kidney cortex and lower for guinea pig kidney cortex. There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg2+ and the alkali cations. Complex II differs in stability in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na+ and K+, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.

Biochemical aspects of the visual process. XXVIII. Classification of sulfhydryl groups in phodopsin and other photoreceptor membrane proteins.

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards rho-chloromercuribenzoate and rho-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the rho-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and isprobably located in the vicinity of the chromophoric center. The differences between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the rho-chloromercuri-derivatives.

Studies on the catalytic hydrogenation of biomembranes. Hydrogenation of phosphatidylcholine liposomes by two water-soluble rhodium-phosphine catalysts.

The reactions of two water-soluble amphiphiolic rhodium-phosphine hydrogenation catalysts, chlorotris(sodium diphenylphosphinobenzene-m-sulfonate)rhodium(I) and chlorotris(bissodium diphenylphosphinoundecylphosphate)rhodium(I), with aqueous dispersions of unsaturated phosphatidylcholines have been studied. Under mild reaction conditions (pH2 = 1.2 atm, 37 degrees C, pH 6.9) hydrogenation of aqueous dioleoylphosphatidylcholine dispersions, prepared by sonication, was achieved. This reaction was not affected by the presence of high salt concentrations. The rate of hydrogenation was independent of catalyst concentration. The reaction with dioleoylphosphatidylcholine dispersions, prepared by the ethanolic injection method, was preceded by a lag period of about 5 h. The reaction with dioleoylphosphatidylcholine dispersions, obtained by vigorous shaking, was rather slow, suggesting the presence of a penetration barrier. The reaction with dioleoylphosphatidylcholine proceeds via the isomerisation of the oleoyl to the elaidoyl moiety, followed by hydrogenation of the elaidoyl moiety. The possible interactions of the catalysts with the bilayer are considered and the implications of these findings are discussed.

Transbilayer distribution of phospholipid fatty acyl chains in photoreceptor membrane.

The transverse distribution of the fatty acyl chains of the major phospholipids over the two faces of the photoreceptor membranes has been determined in bovine rod outer segment (stacked disk) preparations. For this purpose, the fatty acid composition of the phospholipids has been analyzed before and after treatment with trinitrobenzenesulfonic acid and phospholipase D. The latter agents are used under conditions in which they have been demonstrated to attack only the outer (cytoplasmic) face of the membrane. After treatment with trinitrobenzenesulfonic acid or phospholipase D, the fatty acid composition of the unreacted phospholipids is the same as that before treatment, regardless of the extent of modification or hydrolysis attained. The fatty acid composition of phosphatidic acid, resulting from phospholipase D action, also remains unchanged during progressive hydrolysis. These results indicate that the fatty acyl chains of the major phospholipids have the same composition on either side of the disk membrane. Together with our previously published evidence for the distribution of the major phospholipids in rod outer segment disk membranes, this means that both the phospholipids and their fatty acyl chains have a remarkably symmetrical distribution over the two membrane faces. On the basis of literature data it is concluded that this approximate symmetry reflects the high mobility of the entire phospholipid pool of disk membranes, thus including appreciable transbilayer movements of the phospholipids.

Transbilayer distribution of phospholipids in photoreceptor membrane studied with various phospholipases.

The distribution of the three major phospholipids of bovine rod outer segment disk membranes over the two faces of the membrane has been studied by means of treatment with phospholipase C, phospholipase A2 and phospholipase D. Two different preparations of rod outer segment disk membranes have been used, which are called 'stacked disks' and 'disk vesicles' on account of their morphological appearance. The hydrolysis patterns obtained by phospholipase treatment of these preparations have been compared to those of a retinal lipid suspension or detergent-solubilized disk membranes, which serve as control preparations with a similar phospholipid composition but a random availability of the phospholipids. Special attention is given to the early phase of enzyme treatment in order to eliminate secondary effects on the molecular organization of the membrane due to appreciable phospholipid hydrolysis. Analysis of the hydrolysis patterns for all three phospholipases in stacked disks, as compared to those in randomized control preparations, suggests a slightly asymmetrical distribution of phosphatidylcholine (40--45% at the outer face) and phosphatidylethanolamine (55--60% at the outer face) and a symmetrical distribution of phosphatidylserine in rod outer segment disk membranes. Extensive treatment with phospholipases C and A2 leads ultimately to nearly complete hydrolysis of all phospholipids, but with phospholipase D a final level of 40% phospholipid hydrolysis is observed in stacked disk preparations. This suggests that in the latter case the inner face of the membrane is inaccessible to the enzyme. Further work will be necessary in order to substantiate these conclusions.

Transepithelial permeability in the rabbit pancreas.

1. The transepithelial permeability in the isolated rabbit pancreas has been studied with the aid of radioactive markers added to the bathing medium. 2. After addition of these compounds in 2 mM concentration to the medium, they equilibrate within 30 min to a steady-state concentration in the secreted fluid. The latter concentrations, expressed as percent of those in the bathing medium, are: urea 100%, glycerol 90%, erythritol 95%, mannitol 60%, lactose 5%, sucrose 4% and inulin 3%. 3. Addition of 10(-5) M carbachol to the bathing medium after 60 or 90 min of incubation results in an increase of the concentrations of mannitol, lactose sucrose and inulin in the secreted fluid. Maximal concentrations, reached about 35 min after addition of the stimulant, are: mannitol 65%, lactose 31%, sucrose 23%, inulin 8%. 4. No change in the concentration of urea is observed, while the concentrations of glycerol and erythritol increase always to 100% after addition of 10(-5) M carbachol. 5. For sucrose and lactose the increase in permeability appears to be dependent on the concentration of carbachol. 6. There is no increase in the extracellular space for lactose, sucrose and inulin after incubating fragments of the rabbit pancreas with 10(-5) M carbachol. 7. Addition of atropine 5 min or more after carbachol stimulation has no effect on enzyme secretion, but markedly inhibits the increase in sucrose permeability. 8. These results indicate that: (a) the permeability of the transcellular transport route in the isolated rabbit pancreas is determined by the size of the permeating molecules, (b) this route is probably extracellular, (c) its permeability is increased by a cholinergic agent in dose-dependent fashion, (d) the increase in permeability is not caused by the enzyme secretion as such.