Active sodium uptake by the skin of foetal sheep and pigs.

1. Simultaneous measurements of unidirectional sodium fluxes across foetal skin incubated in vitro with identical solutions ([Na] = 150 mM) bathing either side showed a flux ratio (influx/efflux) of 1-40+/-0-08 in twenty-seven sheep skins, which was significantly different from unity (P less than 0-001). The gestational ages ranged from 47 to 98 days (term = 147 days). Similar experiments on eight foetal pig skins at 58 days gestation (term = 114-118 days) gave a mean flux ratio of 1-10 +/- 0-03 (P less than 0-02). 2. Unidirectional sodium fluxes measured with dilute Ringer solution on the outside (mucosal) surface ([Na]0 = 100mM) gave influx to efflux ratios of 0-86 +/- 0-09 in seventeen sheep (P less than 0-05) and 1-07 +/- 0-26 in five foetal pigs; the value predicted for passive movement was 0-67. 3. Incubation with inhibitors, ouabain (10-4 M) or dinitrophenol (DNP) (10-4 M) gave a flux ratio for sodium which was not significantly different from unity in the absence of a gradient, or from 0-67 when the concentration gradient was applied. 4. Sequential measurement of unidirectional diffusional fluxes of tritiated water across foetal skin gave flux ratios of 0-98 +/- 0-02 in six sheep skins and 1-06 +/- 0-11 for four pig skins in control conditions. When the outside solution was diluted to give an osmotic gradient of 100 m-osmole. kg-1 across the skin a flux ratio of 0-95 +/- 0-07 was obtained for seven sheep and was not measured in pig skin. Hormones and inhibitors had no effect on the diffusional flux ratio for water in the presence or absence of an osmotic gradient. 5. Lysine vasopressin (ADH) (200 mu./ml.) increased influx and efflux of water in the presence and, to a lesser extent in the absence of an osmotic gradient in sheep skin. In pig skin prolactin (1 u./ml.) increased both influx and efflux, but ADH had no effect on diffusional water fluxes. 6. ADH increased sodium influx in sheep skin slightly but vasotocin (5-5 mu./ml.) was more potent, particularly in the presence of an opposing diffusion gradient. Vasotocin (55 mu./ml.) reduced sodium influx in pig skin ADH had no effect on influx or efflux and prolactin reduced sodium influx and efflux. Ouabain and DNP generally reduced permeability to both sodium and water in sheep skin but had no effect in pig skin.     

Structure and function in urinary bladder of foetal sheep

1. The structure and function of the epithelial lining of the urinary bladder of sheep foetuses was investigated by electron microscopic studies made in conjunction with a series of experiments in which the permeability of the bladder to sodium and water was measured in vitro. Measurements were made at gestational ages ranging from 50 to 141 days (term = 147 days) Osmolarity and electrolyte concentrations of urine found in the foetal bladder were also measured.2. The development of tight junctions between the bladder epithelial cells was investigated by incubating the tissue with solutions containing 1 mM-LaCl(3) on the mucosal surface. No penetration of the junctions by lanthanum was observed in foetuses of 90 days or older. In younger bladders, the epithelial layer was stripped by treatment with lanthanum, but tight junctions appeared to be fully developed in early bladders incubated without lanthanum.3. The surface structure of the luminal (mucosal) plasmalemma was fully developed at 50 days.4. Unidirectional fluxes of labelled sodium and water were measured with identical solutions bathing the two surfaces of the bladder wall. No net water movement occurred; the mean ratio of efflux to influx in nine bladders was 1.002 +/- 0.039 (S.E. of mean). Under these conditions, the flux ratio for sodium was 1.735 +/- 0.143 (S.E. of mean) in twelve bladders.5. Antidiuretic hormone (ADH) had no effect on net water movement but reduced the net efflux of sodium so that the flux ratio became 1.285 +/- 0.255 (S.E. of mean) n = 8. ADH also had a striking effect on the structure of the epithelium, causing marked swelling of the intercellular spaces. The tight junctions remained an effective barrier to lanthanum penetration under these conditions; lanthanum was not observed in the enlarged spaces.     

Antidiuretic hormone-dependent membrane capacitance and water permeability in the toad urinary bladder

Antidiuretic hormone (ADH) increased the electrical capacitance of apical membrane of the toad bladder; this effect was modulated by the osmotic gradient across the tissue. Capacitance was measured from the transepithelial voltage response to constant-current pulses using bladders depolarized with KCl-sucrose serosal solution to reduce basolateral resistance and with Na-free mucosal solution to increase apical membrane resistance. Addition of ADH (20 mU/ml) increased capacitance by 28 +/- 9% (mean +/- SD) in the absence and by 8 +/- 3% in the presence of an osmotic gradient (200 mosM, mucosal side hypotonic). With bladders stimulated in the absence of an osmotic gradient, rapidly imposing a gradient resulted in a peak rate of water flow that declined to 40% of the peak value after 15-20 min. ADH-dependent capacitance also decreased with a similar time course. Removal of ADH reversed the capacitance change (t1/2 = 10-15 min), but the reversal was slower than the decline in water flow to basal levels (t1/2 less than 5 min). Colchicine and cytochalasin B also inhibited the ADH-induced capacitance increase. The capacitance change was also inhibited when the mucosal solution was made hypertonic with raffinose. The results are interpreted within the framework of a previously proposed model of ADH-stimulated water transport in which cytoplasmic vesicular structures fuse with the apical plasma membrane.     

Activation energy for water transport in toad bladder

Activation energies (Ea) for water movement across vasopressin-(ADH) sensitive epithelia have been reported to be about 10 kcal/mol (1, 12). The present study shows that measurements of Ea for osmotic water flow across toad bladders are unreliable, because a temperature change induces marked alterations in membrane permeability to water within a 2.5-min interval. Thus bladders equilibrated with ADH either at room temperature or at 33 degrees C and then suddenly subjected to a lower temperature were found to exhibit a marked increase in membrane permeability to water. This observation suggests that there is a rapid turn-over of water permeability sites and that sudden exposure to cold inhibits the removal more than the induction of sites by ADH. To stabilize ADH-induced water channels for Ea measurements, bladders were exposed to ADH at room temperature, fixed with glutaraldehyde, and subjected to osmotic gradients at different temperatures. The Ea values for osmotic water flow across these ADH-permeabilized, glutaraldehyde-fixed bladders were 5.1 (4-12 degrees C), 4.3 (12-21 degrees C), 3.6 (21-36 degrees C), and 3.6 kcal/mol (30-38 degrees C). Ea values for shear viscosity of water in these temperature ranges were calculated to be 4.7, 4.2, 4.1, and 3.6 kcal/mol, respectively. The close correlation between Ea values for bulk water viscosity and osmotic water flow across the bladder wall suggests that an equivalent number of hydrogen bonds must be broken to achieve an increase in water flow through ADH-induced channels and an increase in fluidity of water in bulk solution.     

Effects of prolonged saline exposure on water, sodium and urea transport and on electron-microscopical characteristics of the isolated urinary bladder of the toad Bufo bufo

1. A comparison was made of various transport properties and electron-microscopical characteristics of isolated urinary bladders from toads (Bufo bufo) maintained in either tap water or 0.7% saline (0.7 g NaCl in 100 ml. H(2)O) for 10 days to 2 months.2. In the absence of Pitressin, isolated bladders from saline-adapted toads showed:(a) markedly, and significantly, lower osmotic water flow;(b) moderately, but not significantly, lower urea permeability;(c) no significant change in net sodium transport (measured as short-circuit current, I(sc)); and(d) significantly smaller intercellular space/mucosal cell ratios in electron-micrographs.3. Differences in the transport and electron-microscopical characteristics between bladders from water-exposed and saline-adapted toads became more evident in the presence of exogenous Pitressin (10 m-u./ml. serosal solution):(a) the stimulating influence of Pitressin on osmotic water flow, short-circuit current and urea permeability was considerably smaller in bladders from saline-adapted toads than in those from water-exposed toads;(b) the influence of Pitressin on short-circuit current was reduced more profoundly than that on either water flow or urea permeability;(c) the Pitressin-induced increment in intercellular space/mucosal cell ratio was significantly smaller in electron-micrographs of bladders from saline-adapted toads than in those from water exposed toads.4. The effects of saline adaptation are discussed in relation to decreased permeability of mucosal membrane barriers.     

The effect of lithium on the permeability response induced in the collecting duct by antidiuretic hormone.

The diffusional and osmotic water permeability of collecting ducts in isolated papillae of rats' kidneys were measured in papillae taken from normal and lithium pretreated rats. The diffusional water permeability of collecting ducts in papillae from normal rats in the absence of ADH was 4.1 +/- 0.2 (S.E.M.) (n = 18) muM s-1 increasing to 7.2 +/- 0.6 mum s-1 with ADH. Values obtained with lithium (10 mM) in the medium, perfusate or both and in papillae taken from lithium pretreated rats did not differ significantly from the above. The cyclic AMP content of the papillae taken from normal rats was 83 +/- 6 pm mg protein in the absence of ADH and increased to 196 +/- 12 (n = 13) with 500 mu units ml-1 ADH. Lithium 10 mM in the medium did not alter this response. Papillae from lithium pretreated rats had a similar basal level of cyclic AMP but the increment in a lithium (10 mM) medium after ADH was significantly less. These results indicate that the impaired water handling of lithium treated rats is probably not due to a failure of the membrane to increase its permeability to water after ADH. Though lithium does alter the production of cyclic AMP this is not believed to be important regarding any alteration in water permeability. We believe it is probable that lithium interferes with sodium chloride transport at some more proximal nephron segment thereby producing the syndrome of polyuria.     

Sodium nitroprusside induced cGMP accumulation in isolated frog skin epithelium. Effect on cAMP, hydroosmotic and natriferic response to antidiuretic horomone

The cGMP content of isolated frog skin epithelia has been measured, and the basal level was found to be 14.3 +/- 1.7 fmol/mg dry lweight. 0.1 mM sodium nitroprusside induced a 10-fold increase in the cGNP level within 5 min after which it rose more slowly. The maximum increase in cGMP level was obtained with 1 mM sodium nitroprusside, giving a 20-50-fold increase. 1 mM sodium nitroprusside per se had no effect on osmotic water flow or active sodium transport. On the other hand, the osmotic water flow response to arginine vasotocin was somewhat enhanced in skins which had been pretreated with 1 mM sodium nitroprusside; thus the water flow responses to 1 and 31 ng/ml arginine vasotocin were on the average 31 and 14% higher in skins exposed to sodium nitroprusside than in control skins. Sodium nitroprusside had no effect on the increment in sodium transport rate elicited by arginine vasotocin. Sodium nitroprusside alone increased the cAMP level slightly; the enhanced cAMP level, reached after 30 min incubation with 40 ng/ml arginine vasotocin, was 20% higher in the presence of 1 mM sodium nitroprusside. In conclusion: cGMP has no effect on osmotic water flow nor on active sodium transport and is not involved in the regulation of sodium transport by antidiuretic hormone. However, cGMP (or sodium nitroprusside) has a moderate effect on the hormone-stimulated osmotic water flow.     

Does the gradual hydroosmotic response to antidiuretic hormone depend on intracellular cAMP accumulation or on the formation of intramembrane particle aggregates?

In experiments on frog urinary bladder the mechanisms behind the gradual development of a hydroosmotic reaction to antidiuretic hormone (ADH) were investigated. It was suggested that the velocity of hydroosmotic reaction may be limited by (a) formation and insertion of particle aggregates into the apical membrane or (b) by velocity of cAMP formation. The urinary bladders were exposed to 23 nM ADH for different times (from 1 to 20 min) and water flow was measured over a period of 40 min. It was found that the value of the full hydroosmotic response increased progressively with the time of exposure to the hormone; however, the enhancement of water flow was equal during each time interval before reaching the reaction maximum. A direct correlation between the value of ADH-stimulated water flow, cAMP content in bladder tissue and frequency of particle aggregates in the granular cell apical membrane was observed. The content of cAMP in ADH-treated bladders was higher by 80% in the absence than in the presence of an osmotic gradient. Pretreatment of urinary bladders with 50 M cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, significantly accelerated the development of the hydroosmotic reaction and increased the magnitude of water flow in comparison with the effect of ADH only. No changes in cyclic AMP phosphodiesterase activity were found in the urinary bladder homogenates under the action of ADH, so it seems likely that accumulation of cAMP depends only on the increase of adenylate cyclase activity. The data obtained allow one to conclude that the gradual hydroosmotic response to ADH depends on the accumulation of cAMP, which may be considered as the main limiting factor of the velocity of the hydroosmotic reaction.     

Foetal placental blood flow in the lamb

1. Fifteen sheep foetuses of 1.5-5.2 kg body weight were prepared with indwelling arterial and venous catheters for experimentation one to six days later.2. Unanaesthetized foetuses were found to have mean arterial and central venous blood pressures of 40 +/- 1.5 (S.E. of mean) and 2.0 +/- 0.3 (S.E. of mean) mm Hg respectively, compared to intra-uterine pressure. Intra-uterine pressure was 16 +/- 0.8 (S.E. of mean) mm Hg with respect to atmospheric pressure at mid-uterine level.3. Mean placental blood flow of the foetuses was 199 +/- 20 (S.E. of mean) ml./(min.kg body wt.). Mean cardiac output in eleven of the foetuses was 658 +/- 102 (S.E. of mean) ml./(min.kg).4. Mean foetal and maternal colloid osmotic pressures were 17.5 +/- 0.7 (S.E. of mean) and 20.5 +/- 0.6 (S.E. of mean) mm Hg respectively at 38 degrees C.5. Intravenous infusions into six ewes of 1.8 mole of mannitol and 0.4 mole of NaCl resulted in significant increases in foetal plasma osmolarity, sodium, potassium, and haemoglobin concentrations, without detectable transfer of mannitol to the foetal circulation.6. In the sheep placenta there is osmotic and hydrostatic equilibration of water. As a consequence, there should be an interaction between foetal placental blood flow and foetal water exchange with the maternal circulation. It was concluded that this interaction tends to stabilize foetal placental blood flow.     

Development of intra-renal solute gradients in foetal and post-natal life

Summary Tissue from three regions of the kidneys of foetal, newborn and mature pigs, sheep and cows was excissed and the intrarenal gradients for sodium, potassium and urea were measured. Gradients were slight in pig and calf foetuses. Sheep foetuses showed a steep intra-renal gradient for sodium which did not change in early post-natal life. The gradient for sodium and urea increased in all species after birth but there were species differences in the rate of increase. In newborn and weaned pigs administration of sodium chloride or urea did not affect the intra-renal gradient for either substance; both acted as osmotic diuretics. Injection of lysinevasopressin increased urinary osmolarity but without change in the intra-renal gradient. It is suggested that maturation of renal concentrating ability runs parallel with the development of the intra-renal solute gradient.Part of this work was reported at the 2nd International Symposium of Pediatric Nephrology, Paris, August 1971.     

Sodium nitroprusside induced cGMP accumulation in isolated frog skin epithelium. Effect on CAMP,hydroosmotic and natriferic response to antidiuretic hormone

The cGMP content of isolated frog skin epithelia has been measured, and the basal level was found to be 14.3±1.7 fmol/mg dry weight. 0.1 mM sodium nitroprusside induced a 10-fold increase in the cGNP level within 5 min after which it rose more slowly. The maximum increase in cGMP level was obtained with 1 mM sodium nitroprusside, giving a 20–50-fold increase. 1 mM sodium nitroprusside per se had no effect on osmotic water flow or active sodium transport. On the other hand, the osmotic water flow response to arginine vasotocin was somewhat enhanced in skins which had been pretreated with 1 mM sodium nitroprusside; thus the water flow responses to 1 and 31 ng/ml arginine vasotocin were on the average 31 and 14% higher in skins exposed to sodium nitroprusside than in control skins. Sodium nitroprusside had no effect on the increment in sodium transport rate elicited by arginine vasotocin. Sodium nitroprusside alone increased the cAMP level slightly; the enhanced cAMP level, reached after 30 min incubation with 40 ng/ml arginine vasotocin, was 20 % higher in the presence of 1 mM sodium nitroprusside. In conclusion: cGMP has no effect on osmotic water flow nor on active sodium transport and is not involved in the regulation of sodium transport by antidiuretic hormone. However, cGMP (or sodium nitroprusside) has a moderate effect on the hormone-stimulated osmotic water flow.     

Effects of digitoxin and lithium, used as a marker of passive Na transport, on secretin-dependent bile flow in the pig

The present study was performed in anaesthetized pigs, and the first aim was to assess the role of Na,K-ATPase in secretin-dependent biliary HCO3 secretion (JbHCO3). Intra-arterial administration of the cardiac glycoside digitoxin (0.2 mg/kg-1) reduced hepatic Na K-ATPase activity, JbHCO3 and secretin-dependent bile flow by 24, 55 and 34% respectively. In the second part of this study lithium (Li) was used as a marker of passive Na transport to assess the electrochemical gradient for Na flux into bile duct lumen during secretin-stimulated bile flow and impeded biliary osmotic water flow by i.v. infusion of glucose. At plasma glucose 85 (73-96) mmol l-1, bile [Na] and [Li] exceeded their concentrations in plasma by 57 and 47% respectively. By using the Nernst equation, transepithelial potential difference (PD) during hyperglycaemia was estimated to be -6.2 (0 to -10.8) mV (ductal lumen negative), which corresponds to a [Li]bile/[Li]plasma ratio of 1.3 (1.0-1.5). The ratio was not significantly different from the observed [Li]bile/[Li]plasma ratio of 1.4 (1.3-1.5). It is concluded (1) that Na, K-ATPase is necessary for JbHCO3, probably by sustaining the cell membrane PD (cell interior negative) which is a driving force for apical electrogenic HCO3 secretion, and (2) transepithelial Li (and hence Na) flux is driven solely by the negative transcellular PD during secretin-stimulated bile flow in the pig.     

Effect of hydrostatic pressure on ADH induced osmotic water flow in toad bladder

The effect of hydrostatic pressure (HP) on antidiuretic hormone (ADH) stimulated osmotic water flow (Jv) across the toad urinary bladder was evaluated. Jv for ADH-stimulated bladders was significantly reduced by an elevation of the serosal HP gradient to 1 cm H₂O. Subsequent elimination of the HP gradient resulted in a recovery of Jv. Serosal HP also caused a reversible increase in sucrose permeability (P sucrose). For ADH-treated bladders fixed with glutaraldehyde during serosal HP exposure, subsequent exposure to a mucosal or serosal HP gradient caused acceleration or inhibition of Jv, respectively. The reduction in ADH-associated Jv with serosal HP was apparently caused by a back-flux of water through a paracellular pathway. Jv and P sucrose were not affected by mucosal HP during ADH stimulation. The results suggest a specific sensitivity of a paracellular pathway to a small serosal HP gradient in bladders with ADH-stimulated water flow. The reversibility of this effect on P sucrose suggests that the elements comprising the apical junctions are dynamic structures capable of recovering at least some of their permeability properties.     

Localization of K-NPPase and Li+ secretion in the exocrine pancreas of the pig

To study the mode of transepithelial Na+ transport into pancreatic ducts during secretin-dependent NaHCO3 secretion, Na, K-ATPase was first localized within the exocrine pancreas of the pig using a cytochemical reaction for K-dependent p-nitrophenylphosphatase (K-NPPase). K-NPPase staining was confined to the lateral cell membrane bordering the intercellular spaces between ductal cells, negating the possibility of primary active, transcellular Na+ transport into pancreatic ducts. To assess how transepithelial Na+ transport may be coupled to HCO-3 secretion, net flux of Li+ into pancreatic juice was measured following intravenous systemic Li+ loading of 12 secretin infused, anaesthetized pigs. At plasma Li+ 32 (23-35) mmol l-1, Li+ displaced Na+ as accompanying cation to secreted HCO-3, and Li+/Na+ in pancreatic juice matched Li+/Na+ in arterial plasma. During superimposed inhibition of pancreatic water flux by hyperglycaemia, Li+ and Na+ were both transported against a transepithelial concentration gradient. Li+ reduced pancreatic HCO-3 secretion rate by 14 (-2 to -20)%, as well as Na,K-ATPase activity in a separate in vitro assay. The finding that Li+ substituted for Na+ in the secretion even during reduced osmotic water flow suggests that Na+ and Li+ are transported together with secreted HCO-3 into pancreatic juice by an electrogenic mechanism in addition to solvent drag and diffusion.     

The composition of maternal plasma and foetal urine after feeding and drinking in chronically catheterized ewes during the last two months of pregnancy

1. The fluid sacs and bladders of sixteen foetuses in fourteen ewes were catheterized between 81 and 92 days gestational age and the rumens of four ewes were also catheterized.2. Between 95 and 145 days gestational age in forty-six 24 hr experiments hourly samples of maternal plasma and foetal urine were obtained and in fifteen experiments foetal fluid samples were also taken at 4- to 6-hr intervals.3. The osmolality, pH, and concentrations of sodium, potassium, chloride, glucose, fructose and urea were measured on all samples.4. During experiment there was no significant variation in the composition of amniotic or allantoic fluid. Marked changes in osmolality occurred in maternal plasma and foetal urine when ewes drank after feeding, but not in ewes that received water intrarumenally via catheter while feeding or in fasting ewes. Post-prandial changes in maternal plasma osmolality may have altered transplacental water fluxes and as a result foetal plasma volume and osmolality.5. The results suggest that the foetus alters renal water retention by varying antidiuretic hormone (ADH) secretion in response to changes in blood volume and at later gestational ages plasma osmolality as well.6. Post-prandial changes in the [Na(+)]/[K(+)] ratio of foetal urine suggested that foetal adrenocorticotrophin (ACTH) secretion is influenced by variations in foetal blood volume and glucose concentrations.7. The post-prandial changes in foetal urine composition observed here support previous suggestions (Mellor & Slater, 1972) about the role of foetal urine in foetal fluid formation which were based on gestational changes in the composition of foetal fluids and urine sampled once daily during the post-absorptive state.     

Permeability changes in Necturus proximal tubule during volume expansion

The permeability of Necturus proximal tubule to hydrophilic nonelectrolytes of varying molecular size was studied under control conditions and during isotonic expansion of the animal's extracellular volume. Transepithelial permeability was measured in perfused tubular segments under conditions of zero net water flux. During volume expansion, tubular permeability to urea increased slightly, whereas mannitol decreased slightly and permeability to sucrose was significantly decreased. Volume expansion had a greater effect on osmotic flow parameters; the NaCl reflection coefficient decreased from 0.64 to 0.47 (summer animals) and from 0.41 to 0.27 (winter animals). Osmotic water flux and hydraulic conductivity increased but only in the lumen-to-capillary direction. Reflection coefficients of nonelectrolytes measured at the apical surface were reduced during volume expansion for probing molecules greater than 3 A in radius and were unchanged for smaller molecules, less than 3 A, suggesting two pore populations. We propose that an increase in tight-junction permeability can account for modification of osmotic flow parameters, whereas the whole thickness of the epithelium, particularly the intercellular space, plays the dominant role in regulation of diffusional permeability.     

Pressure effects on the ADH-induced initiation of water flow in toad bladder

Earlier studies employed colchicine to demonstrate the need for microtubules in the ADH-induced initiation of increased water permeability in toad bladder. We have used colchicine and hydrostatic pressure together to determine whether formed or growing microtubules are required for initiation of the ADH response in Bufo marinus. When ADH and 8,000 psi were administered simultaneously, the ADH-induced increase in water flow was inhibited while under pressure by 107 +/- 7% (n = 6). Application of 8,000 psi for 10 min before ADH administration resulted in an increased initiation of the ADH osmotic response over the non-pressure-treated control (average acceleration, rate of water flow increase during first 3 min after ADH stimulation, 1.25 +/- 0.27 vs. 0.43 +/- 0.12 mg X cm-2 X min-2, n = 6). In addition, the inhibition of the ADH response brought about by colchicine incubation was overcome with pretreatment of the colchicine-incubated bladders with 8,000 psi for 10 min (average 3-min acceleration, 0.18 +/- 0.04 vs. 1.13 +/- 0.06 mg X cm-2 X min-2, respectively). Repeating the experiments with dibutyryl cAMP gave similar results. We interpret these data as suggesting that growing microtubules are required for initiation. The proposed model is as follows. Pressure removes colchicine inhibition by introducing, through disassembly of formed microtubules, more colchicine-free tubulin subunits. These subunits are then available following decompression to reassemble when the tissue is challenged with hormone.     

Interdependence of albumin and sodium transport in the foetal and new-born pig intestine

1. Everted sacs of new-born pig intestines incubated in bicarbonate saline at 37 degrees C, transferred bovine plasma albumin across the mucosa into fluid bathing the serosa, the amount transferred increasing as the concentration of albumin in the mucosal fluid was raised from 0.5 to 16 g/100 ml.2. The rate of albumin transfer across the foetal pig intestine showed an apparent maximum, about 400 mug/g intestine/hr, 2 weeks before birth. The transfer at birth, about 200 mug/g intestine/hr, fell sharply during the next 2 days but later returned to that previously found at birth.3. When sacs were prepared from the intestines of 1 to 7-day-old pigs part of the recovered albumin was degraded. No digestion was found when the intestines of new-born or foetal pigs were used.4. The transfer of water and sodium, but not glucose, measured across the foetal and new-born pig intestine, was consistently higher when albumin was present in the mucosal fluid: the transmural potential difference was lowered by the presence of albumin. These differences disappeared during the first 2 days of life.5. Both the total and ouabain-sensitive adenosine triphosphatase (ATPase) activities of the pig intestinal epithelium fell within 24 hr of birth. There was some increase in total ATPase activity in older pigs but the ouabain-sensitive activity remained low.6. The relation between albumin and sodium transport, seen at a time when albumin is not being metabolized, suggests that the transfers are closely coupled. The movement of sodium into a mucosal cell down its own concentration gradient may provide energy for the translocation of albumin.     

Comparison of the direct antiglobulin rosetting reaction (DARR) and direct immunofluorescence (DIF) for demonstration of sIg-bearing lymphocytes in pigs, sheep and cattle.

Tests with untreated and trypsin-treated red cells (rbc) from a variety of species showed that anti-Ig-coupled pig RBC are good indicator cells for the study of ruminant blood sIg + lymphocytes by the DARR test; coupled donkey and rabbit RBC are suitable for investigating pig lymphocytes. The different species showed the following percentages of sIg + lymphocytes (M +/- SE) by direct immunofluorescence (DIF) and the direct antiglobulin rosetting reaction (DARR) respectively:pigs 9.2 +/- 0.7% and 16.3 +/- 1.2%; sheep 20.2 +/- 1.2% and 33.1 +/- 1.6%; Cattle 13.5 +/- 1.4% and 28.9 +/- 3.5%. The mean ratio of sIg + lymphocytes shown by the two tests (DARR/DIF) for each species was 1.80 +/- 0.08 for pigs, 1.73 +/- 0.7 for sheep and 2.15 +/- 0.18 for cattle. Preincubation of pig and sheep lymphocytes at 37 degrees for 1 h did not alter the proportion of sIg + lymphocytes detected by either test. Thus the DARR test reveals a further population of sIg + lymphocytes in addition to that detected by immunofluorescence, whose number is proportional to the B population as measured by DIF and whose sIg is intimately associated with the membrane.     

Contamination of prolactin preparations by antidiuretic hormone and oxytocin

Since impurities consisting of neurohypophysical hormones in prolactin powder may be responsible for the vascular and renal effects attributed to prolactin, rat (NIH-RP-1), ovine (NIH-P-S-10, S-12), and bovine (NIH-P-B4) prolactin preparations were examined for their content of ADH and oxytocin by rat antidiuresis, milk-ejection, and blood pressure assays. Activities were identified as due to ADH or oxytocin by incubation of prolactin solutions with antisera against ADH, oxytocin, and prolactin, or with pregnancy plasma. The ADH content of rat, ovine (P-S-10, P-S-12) and bovine prolactin was found to be 104.5 +/- 7.1 (means +/- SE), 2.5 +/- 0.2, 1.6 +/- 0.1, and 1.6 +/- 0.5 mU/mg powder, respectively; the corresponding values for oxytocin content were 155.3 +/- 3.5, 1.2 +/- 0.1, 0.5 +/- 0.1, and 1.2 +/- 0.01 mU/mg powder, respectively. Because antidiuretic, milk-ejection, and blood pressure activities of the various prolactins were eliminated after incubation with antisera against ADH and oxytocin, or with pregnancy plasma, but not with prolactin antisera, it is concluded that the reported vascular and renal prolactin effects are attributable to ADH contamination of the prolactin preparation rather than to the prolactin molecule itself. These findings have implications for renal and vascular prolactin research.