Hormone-dependent dissociation of blood flow and secretion rate in the lingual salt glands of the estuarine crocodile, Crocodylus porosus

Salt and water balance in the estuarine crocodile, Crocodylus porosus, involves the coordinated action of both renal and extra-renal tissues. The highly vascularised, lingual salt glands of C. porosus excrete a concentrated sodium chloride solution. In the present study, we examined the in vivo actions of vasoactive intestinal peptide (VIP), B-type natriuretic peptide (BNP) and angiotensin II (ANG II) on the secretion rate and blood perfusion of the lingual salt glands. These peptides were selected for their vasoactive properties in addition to their reported actions on salt gland activity in birds and turtles and rectal gland activity in elasmobranchs. The femoral artery was cannulated in seven juvenile crocodiles for delivery of peptides and measurement of mean blood pressure and heart rate. In addition, secretion rate of, and blood flow to, the salt glands were recorded simultaneously using laser Doppler flowmetry. VIP stimulated salt secretion was coupled to an increase in blood flow and vascular conductance of the lingual salt glands. BNP was a potent stimulant of salt gland secretion, resulting in a maximal secretion rate of more than 15-fold higher than baseline; however, this was not coupled to an increase in perfusion rate, which remained unchanged. ANG II failed to stimulate salt gland secretion and there was a transient decrease in salt gland blood flow and vascular conductance. It is evident from this study that blood flow to, and secretion rate from, the lingual salt glands of C. porosus are regulated independently; indeed, it is apparent that maximal secretion from the salt glands may not require maximal blood flow.     

Ion secretion by salt glands of desert iguanas (Dipsosaurus dorsalis)

Unlike the NaCl-secreting salt glands of many birds and reptiles, the nasal salt glands of lizards can secrete potassium as well as sodium, with either chloride or bicarbonate as the accompanying anion. The factors responsible for initiating secretion by the gland and the rates of cation and anion secretion were studied in the desert iguana, Dipsosaurus dorsalis. Lizards were given combinations of ions for several days, and secreted salt was collected daily and analyzed for sodium, potassium, chloride, and bicarbonate. Maximum total cation secretion rate was 4.4+/-0.38 micromol/g/d. Cation secretion ranged from 24% to 100% potassium; even high NaCl loads did not abolish potassium secretion. Maximum bicarbonate secretion was about 0.5 micromol/g/d; chloride was the predominant anion. Secretion rate increased only in response to those treatments that included potassium and/or chloride; sodium ions and other osmotic loads (e.g., sucrose) did not increase secretion. This is in contrast to birds and some other reptiles with salt glands, which initiate NaCl secretion in response to any osmotic load. The specificity of the response of the salt gland of Dipsosaurus may be related to the ecological importance of dietary potassium and chloride for herbivorous desert lizards.     

Interrelationships between heat acclimation and salivary cooling mechanism in conscious rats

1. Adaptation of salivary cooling mechanism during acclimation to heat (34 degrees C) and its role in thermoregulation of the rats was studied on conscious rats with either one submaxillary gland chronically cannulated or both submaxillaries ligated. 2. During heat stress (40 degrees C) acclimated rats showed a decrease both in rectal temperature threshold for salivation (Tre-TS), in salivary flow rate and in Tre (hyperthermic plateau). Animals survived for extended periods and rats with ligated glands survived 40% less than non-ligated rats. 3. For both cannulated and ligated rats short term acclimation (5 days) was the most effective. 4. It is suggested that earlier activation of salivation mechanism is associated with the decreased hyperthermic plateau and that the decreased salivary flow rate allows better control of water balance of the animals. Consequently, survival period during heat stress is extended.     

Regulation of salt gland, gut and kidney interactions

Marine birds can drink seawater because their cephalic 'salt' glands secrete a sodium chloride (NaCl) solution more concentrated than seawater. Salt gland secretion generates osmotically free water that sustains their other physiological processes. Acclimation to saline induces interstitial water and Na move into cells. When the bird drinks seawater, Na enters the plasma from the gut and plasma osmolality (Osm(pl)) increases. This induces water to move out cells expanding the extracellular fluid volume (ECFV). Both increases in Osm(pl) and ECFV stimulate salt gland secretion. The augmented intracellular fluid content should allow more rapid expansion of ECFV in response to elevated Osm(pl) and facilitate activation of salt gland secretion. To fully utilize the potential of the salt glands, intestinally absorbed NaCl must be reabsorbed by the kidneys. Thus, Na uptake at gut and renal levels may constrain extrarenal NaCl secretion. High NaCl intake elevates plasma aldosterone concentration of Pekin ducks and aldosterone stimulates intestinal and renal water and sodium uptake. High NaCl intake induces lengthening of the small intestine of adult Mallards, especially males. High NaCl intake has little effect on glomerular filtration rate or tubular sodium Na uptake of birds with competent salt glands. Relative to body mass, kidney mass and glomerular filtration rate (GFR) are greater in birds with salt glands than in birds that do not have them. Birds with salt glands do not change GFR, when they drink saline. Thus, their renal filtrate contains excess Na that is, in some species, almost completely renally reabsorbed and excreted in a more concentrated salt gland secretion. Na reabsorption by kidneys of other species, like mallards is less complete and their salt glands make less concentrated secretion. Such species may reflux urine into the hindgut, where additional Na may also be reabsorbed for extrarenal secretion. During exposure to saline, marine birds maintain elevated aldosterone levels despite high Na intake. Marine birds are excellent examples of physiological plasticity.     

Coping with excess salt: adaptive functions of extrarenal osmoregulatory organs in vertebrates

In all organisms, changing environmental conditions require appropriate regulatory measures to physiologically adjust to the altered situation. Uptake of excess salt in non-mammalian vertebrates having limited or no access to freshwater is balanced by extrarenal salt excretion through specialized structures called ‘salt glands’. Nasal salt glands of marine birds are usually fully developed in very early stages of their lives since individuals of these species are exposed to salt soon after hatching. In individuals of other bird species, salt uptake may occur infrequently. In these animals, glands are usually quiescent and glandular cells are kept in a fairly undifferentiated state. This is the situation in ‘naive’ ducklings, Anas platyrhynchos, which have never been exposed to excess salt. When these animals become initially osmotically stressed, the nasal glands start to secrete a moderately hypertonic sodium chloride solution but secretory performance is meager. Within 48 h after the initial stimulus, however, the number of cells per gland is elevated by a factor of 2–3, the secretory cells differentiate and acquire full secretory capacity. During this differentiation process, extensive surface specializations are formed. The number of mitochondria is increased and metabolic enzymes and transporters are upregulated. These adaptive growth and differentiation processes result in a much higher efficiency of salt excretion in acclimated ducklings compared with naive animals. Receptors and signal transduction pathways in salt gland cells controling the adaptive processes seem to be the same as those controling salt secretion, namely muscarinic acetylcholine receptors and receptors for vasoactive intestinal peptide. This review focusses on signal transduction pathways activated by muscarinic receptors which seem to fine-tune salt secretion in salt-adapted ducklings and may control adaptive growth and differentiation processes in the nasal gland of naive animals.     

The dynamics of the sodium, potassium, calcium, magnesium contents in the fresh water mollusc zebra musselDreissenia polymorpha during stress

The sodium and magnesium concentration in the hemolymph of the zebra musselDreissenia polymorpha decreased by 25.1 and 25.6%, respectively, in 7.5 h after catching and transportation. One day later, the content of all four cations studied in the molluscan body was significantly reduced. After 7 days, the decrease in the tissue concentration, as compared to initial levels, was 2.7, 2.5, 4, and 4.4 times for sodium, potassium, calcium, and magnesium, respectively. After 18 days of acclimation, the concentrations of cations in the hemolymph and molluscan body did not differ from the initial values. Comparative analysis shows that bivalve molluscs and fish have a common negative mechanism connected with a loss of salt from the organism during the initial period of stress. Possible causes are discussed of the decrease in the salt content in the body of the marine bivalve molluscs to minimal values during their migration to the fresh water in the course of evolution.     

The dynamics of the sodium, potassium, calcium, magnesium contents in the fresh water mollusc zebra musselDreissenia polymorpha during stress

The sodium and magnesium concentration in the hemolymph of the zebra musselDreissenia polymorpha decreased by 25.1 and 25.6%, respectively, in 7.5 h after catching and transportation. One day later, the content of all four cations studied in the molluscan body was significantly reduced. After 7 days, the decrease in the tissue concentration, as compared to initial levels, was 2.7, 2.5, 4, and 4.4 times for sodium, potassium, calcium, and magnesium, respectively. After 18 days of acclimation, the concentrations of cations in the hemolymph and molluscan body did not differ from the initial values. Comparative analysis shows that bivalve molluscs and fish have a common negative mechanism connected with a loss of salt from the organism during the initial period of stress. Possible causes are discussed of the decrease in the salt content in the body of the marine bivalve molluscs to minimal values during their migration to the fresh water in the course of evolution.     

Rectal gland morphology of freshwater and seawater acclimated bull sharks Carcharhinus leucas

To compare rectal gland morphology of bull sharks Carcharhinus leucas , animals captured in the freshwater reaches of the Brisbane River, Australia, were acclimated to sea water over 17 days with 1 week in the final salinity. A control group was left in fresh water for 17 days. Animals in fresh water and sea water were strongly hyper- and hypo-ionic with respect to plasma Na⁺ and Cl^–, respectively. This difference necessitates NaCl secretion by the rectal gland in sea water and conservation of NaCl in fresh water. Structural differences in the rectal gland of freshwater and seawater acclimated bull sharks were limited. There was no difference in rectal gland cross-sectional area, lumen area, rectal gland vein area, number of secretory tubules or secretory cells per secretory tubule in freshwater and seawater acclimated animals. At a cellular level, there was no difference between the degree of basolateral and lateral folding, number of mitochondria or number of desmosomes per tight junction. Tight junction width was significantly greater in seawater acclimated animals. The number of red blood cells in the interstitial tissue was also significantly higher in seawater acclimated animals, possibly as a result of increased blood perfusion of the secretory epithelia. The lack of major structural changes in the rectal glands of bull sharks acclimated to fresh water and sea water most likely represents the salinity gradient in the Brisbane River where animals are found throughout the river and can experience large fluctuations in salinity over short distances. Differences in rectal gland morphology of bull sharks in fresh water and sea water are discussed in terms of their relevance to osmoregulation in elasmobranchs.     

Ionic composition of the rectal contents and excreta of the reduviid bug Triatoma infestans

We have investigated the chemical composition of the rectal contents, faeces and urine of the blood-sucking bug Triatoma infestans. This is the environment in which the important disease-causing organism, Trypanosoma cruzi, lives. Directly after feeding of Triatoma infestans, the pH of the excreta switched from an acidic to an alkaline pH and, 1 day later, back to a slightly acidic pH. The osmolality varied in the initial excreta and in the rectal contents on the day following the meal between 300 and 460 mosmol/kg H(2)O, but after an additional day it increased to 350-970 mosmol/kg H(2)O. Determinations by ion capillary electrophoresis showed that sulphate and phosphate dominated the rectal contents in unfed bugs. After feeding, the first four drops of fluid excreta were mainly a sodium chloride solution (>150 mM for each). One to 10 days after feeding strong individual variations in the concentrations of individual ions were evident, especially for potassium and sodium. Mean concentrations of chloride remained at about 70 mM; sulphate and phosphate showed an increase within the first 1 or 2 days and then reached a level of about 160 and 210 mM, respectively. The rectal contents of long-term starved bugs contained high concentrations of phosphate and potassium; sulphate and sodium were slightly lower.     

Lithium in marine elasmobranchs as a natural marker of rectal gland contribution in sodium balance

Natural concentrations of lithium in blood plasma and urine of several species of elasmobranchs and teleosts from the Black Sea and in rectal gland fluid of the former were determined by mass-spectrometric isotope dilution techniques. Unlike the teleosts, the elasmobranchs showed a prominent shift of Li/Na ratio in blood plasma with respect to the surrounding water, the plasma Li/Na ratio being five times lower than that of sea-water. Li-Na selectivity was found to be high in the kidneys and negligible in the rectal gland. Differences in Li-Na selectivity between kidneys and rectal gland are used as a basis for the method of estimation of relative contributions of these excretory organs in sodium excretion. Permanent contributions of the kidneys and rectal gland in sodium excretion of the ray Dasyatis pastinaca were found to be nearly equal.     

Copper homeostasis and toxicity in the elasmobranch Raja erinacea and the teleost Myoxocephalus octodecemspinosus during exposure to elevated water-borne copper

Clear nosed skate, Raja erinacea were exposed to 0.10 (control), 0.52 or 1.73 microM copper and sculpin, Myoxocephalus octodecemspinosus were exposed to 0.10 or 1.73 microM copper (as CuSO4) in Salisbury Cove seawater for up to seven days. Skate gill copper concentrations increased 40-50 fold over background in response to copper exposure at both concentrations. In comparison, sculpin gill levels only increased 3-fold. While there was no evidence for internalized copper in the skate arising from the water-borne exposure, sculpin kidneys, but not livers, exhibited elevated copper concentrations after the seven days of exposure. The marked difference in branchial copper accumulation between the skate and the sculpin likely explains why elasmobranchs appear to be more sensitive to metal exposure than most marine teleost fish. Brain tissue from both species and the skate rectal gland contained relatively high background copper concentrations. Copper exposure caused an initial transient reduction in skate plasma total ammonia (Tamm), but eventually led to elevated plasma Tamm. Despite the marked branchial copper accumulation in the skate, there was no reduction in gill Na/K-ATPase activity. Similarly, Na/K-ATPase activity in skate rectal gland and intestine, as well as in sculpin gill and intestine were not affected by copper exposure. Plasma sodium, magnesium and chloride were not affected by copper exposure in either the skate or the sculpin.     

Osmoregulatory responses of glaucous-winged gulls (Larus glaucescens) to dehydration and hemorrhage

The effects of dehydration and hemorrhage on plasma ionic, osmotic, and antidiuretic hormone (arginine vasotocin) concentrations and of hemorrhage on salt gland secretion and glomerular filtration rate were evaluated in glaucous-winged gulls, Larus glaucescens. Dehydration for 24 h did not affect plasma ionic, osmotic or arginine vasotocin concentrations; 72 h dehydration significantly elevated plasma osmolality, plasma sodium and chloride concentrations, and plasma arginine vasotocin concentration, but did not affect plasma potassium concentration. Constant infusion of 0.8 mol·l^-1 NaCl increased plasma arginine vasotocin concentration and produced salt gland secretion in seven gulls; four secreted well, while three secreted less well. Removal of 20% blood volume during saline infusion immediately reduced (P<0.001) salt gland secretion rate in all gulls. After bleeding, good secretors maintained glomerular filtration rate and urine flow rate; the poorer secretors increased glomerular filtration rate and became diuretic. Blood replacement returned salt gland secretion rate to the prebleeding level (P<0.05) without affecting salt gland secretions sodium concentration in gulls which secreted well, but did not restimulate salt gland secretion in gulls which secreted poorly. Reinfusion of blood had no effect on glomerular filtration rate. Bleeding and blood replacement did not affect plasma arginine vasotocin concentration.Abbreviations AVT arginine vasotocin - ECF extracellular fluid - ECFV extracellular fluid volume - EDTA ethylenediaminetetra-acetate - EWL evaporative water loss - GFR glomerular filtration rate - Hct hematocrit - LB large blood sample - [Na⁺]_pl plasma sodium concentration - Osm_pl plasma osmolality - PEG polyethylene glycol - RH relative humidity - RIA radioimmunoassay - SB small blood sample - SGS salt gland secretion - T _a ambient temperature - TFA trifluoroacetic acid - UFR urine flow rate     

Plasma osmolyte concentrations and rectal gland mass of bull sharks Carcharhinus leucas, captured along a salinity gradient

Bull sharks (Carcharhinus leucas) were captured across a salinity gradient from freshwater (FW) to seawater (SW). Across all salinities, C. leucas were hyperosmotic to the environment. Plasma osmolarity in FW-captured animals (642 +/- 7 mosM) was significantly reduced compared to SW-captured animals (1067 +/- 21 mosM). In FW animals, sodium, chloride and urea were 208 +/- 3, 203 +/- 3 and 192 +/- 2 mmol l(-1), respectively. Plasma sodium, chloride and urea in SW-captured C. leucas were 289 +/- 3, 296 +/- 6 and 370 +/- 10 mmol l(-1), respectively. The increase in plasma osmolarity between FW and SW was not linear. Between FW (3 mosM) and 24 per thousand SW (676 mosM), plasma osmolarity increased by 22% or 0.92% per 1 per thousand rise in salinity. Between 24 per thousand and 33 per thousand, plasma osmolarity increased by 33% or 4.7% per 1 per thousand rise in salinity, largely due to a sharp increase in plasma urea between 28 per thousand and 33 per thousand. C. leucas moving between FW and SW appear to be faced with three major osmoregulatory challenges, these occur between 0-10 per thousand, 11-20 per thousand and 21-33 per thousand. A comparison between C. leucas captured in FW and estuarine environments (20-28 per thousand ) in the Brisbane River revealed no difference in the mass of rectal glands between these animals. However, a comparison of rectal gland mass between FW animals captured in the Brisbane River and Rio San Juan/Lake Nicaragua showed that animals in the latter system had a significantly smaller rectal gland mass at a given length than animals in the Brisbane River. The physiological challenges and mechanisms required for C. leucas moving between FW and SW, as well as the ecological implications of these data are discussed.     

Ionic strength and the polyvalent cation receptor of shark rectal gland and artery

The dogfish shark Squalus acanthias regulates plasma osmolality and extracellular volume by secreting a fluid from its rectal gland which has a higher NaCl and lower urea concentration than plasma. We have previously identified the presence of a calcium-sensing receptor or polyvalent cation sensing receptor (CaSR) on vascular smooth muscle of the rectal gland artery (RGA) and rectal gland tubules (RGT). Activity of the CaSR is influenced by changes in ionic strength. This led us to speculate that the ingestion of invertebrate sea animals increased plasma ionic strength, resulting in inhibition of the receptor, relaxation of RGA, and reversal of inhibition of chloride secretion by the RGT. In contrast, ingestion of fish could diminish ionic strength and have the opposite effect. To study the effect of changes in extracellular ionic strength, shark Ringers solutions were adjusted to three different ionic strengths with NaCl, but the osmolarities were kept constant by varying the concentration of urea. High ionic strength inhibited and low ionic strength enhanced the response to increasing external Ca2+ from 2.5 to 4.7 mM in RGT. The increase in cytosolic Ca2+ ([Ca2+]i) of cells in low, normal, and high ionic strength Ringers solution was 344 +/- 60, 201 +/- 26, and 114 +/- 15 nmol/L, respectively. The [Ca2+]i responses of RGA to external Ca2+ in Ringers of three different ionic strengths were 323 +/- 43, 231 +/- 14, and 56 +/- 11 nmol/L, respectively. Activation of the CaSR by spermine was reduced by more than 50% by high ionic strength in both RGT and RGA. Whether the small changes in shark plasma ionic strength that occur after a shark ingests marine animals with lower and higher ionic strength modulates salt secretion by the rectal gland is not yet known.     

Effects of salt loading on salt gland function in the euryhaline turtle,Malaclemys terrapin

Summary The estuarine turtle,Malaclemys terrapin is able to ionregulate when acclimated to fresh water, 55% sea water or 100% (full strength) sea water, but when in 100% sea water it does not volume regulate successfully. Orbital gland secretions collected by a new eye cup method are very low in animals from all three salinities without salt load. After salt loading the animals from all three groups produce an orbital gland secretion with a sodium concentration greater than sea water. The concentration of ions and kinetics of the response are similar in all three groups. Orbital gland secretion returns to control preload levels well before the injected load is excreted. There is no correlation between the plasma sodium concentration and any of the parameters of the orbital gland response. There is also no correlation between the concentration of sodium in the tear fluid or the rate of sodium excretion and the level of K⁺-stimulatedp-nitrophenylphosphatase activity in the gland. Some of these unexpected results may relate to the estuarine habitat occupied byMalaclemys.Abbreviations K ⁺–NPPase potasium stimulated p-nitrophenylphosphatase - Na–K-ATPase sodium, potassium stimulated adenosine triphosphatase     

The free-running circadian rhythms of two schizophrenics

Two chronic schizophrenic patients and a psychiatrist spent 21 days in an isolation unit. For the first 4 days they lived on normal time but thereafter the clock was removed and they were free-running. The psychiatrist followed the schedule set by the schizophrenics, one of whom spontaneously decided the times of retiring and rising while the other followed passively. The psychiatrist commonly retired some time later but without disturbing the schizophrenics, the mean duration of whose days was 23.7 h, distinctly shorter than is usual in healthy subjects. This was made up of an activity period of 11.77 h and a rest period of 11.94 h. Pulse rate and temperature were measured frequently throughout the waking hours and rectal temperature was monitored during sleep. Urine samples were also collected throughout the 24 h and were analysed for potassium, sodium, chloride, creatinine, phosphate, calcium and uric acid. Urinary and temperature rhythms followed approximately the activity rhythm, in both healthy and schizophrenic subjects. Pulse rate in the schizophrenics followed a rhythm with a period slightly less than that of activity, and in one schizophrenic showed a consistently early phasing. 11-hydroxycorticosteriods at the end of the observation showed a very early phasing corresponding to that of activity. The findings suggest that schizophrenics may have an abnormally short circadian period.     

Role of plasma ANG II in the excretion of acute sodium load in a bird with salt glands (Anas platyrhynchos)

This study was designed to further examine the role of plasma ANG II in the excretion of sodium in the Pekin duck, a bird with salt glands. Renal and extrarenal (salt gland) excretion of an intravenously administered isotonic saline load was monitored over a 4-h period in a group of eight birds under two conditions: the control condition, in which isotonic saline infusion decreased endogenous plasma ANG II from 102.6 to 16.5 pg/ml, and the experimental condition, in which ANG II suppression was prevented by intravenous infusion of a 3.5 ng. kg(-1). min(-1) dose of synthetic ANG II. ANG II infusion significantly decreased the total sodium excretion (by 15%), primarily via an inhibition of salt gland output. The results suggest that ANG II suppression facilitates the excretion of an administered sodium load in birds with salt glands.     

Role of the renin-angiotensin system in the adaptation to high salt intake in immature rats

The response of the renin-angiotensin system, extracellular fluid volume, plasma volume, plasma sodium and mean arterial blood pressure to an increase in salt intake (8% NaCl in the diet for 10 days) was compared in immature (20 days) and adult (80 days) rats which were either sham-operated or uninephrectomised. Salt feeding induced a significant increase in plasma sodium in immature animals, and a greater suppression of the renin-angiotensin system in immature than in adult rats, although extracellular fluid volume, plasma volume and blood pressure remained unchanged. Following uninephrectomy, however, the renin-angiotensin system was maximally suppressed in both age groups and in younger animals extracellular fluid volume, plasma volume and blood pressure were significantly increased. It is concluded that (i) the renin-angiotensin system in immature rats is more responsive to a chronically increased salt intake, (ii) this greater responsiveness partly compensates for the lower natriuretic efficiency of the kidneys of immature rats, which becomes evident after reduction of renal mass, and (iii) these events bear a relation to the higher susceptibility of prepubertal rats to the hypertensive effect of a chronically increased salt intake.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

Plasma membrane biogenesis in the avian salt gland: a biochemical and quantitative electron microscopic autoradiographic study

The avian salt gland provides an ideal system for the study of plasma membrane (PM) biogenesis. Feeding ducklings 1% sodium chloride (salt stress) induces the secretory cells of the gland to synthesize large amounts of PM, which forms an extensive basolateral PM domain after 7-9 days of treatment. In the present study, the initial biosynthetic events following salt stress were investigated. In vivo studies using 3H-uridine indicated that increased rates of RNA synthesis could be detected by 2 hr after the beginning of salt stress and continued through at least 12 hr. Under in vitro conditions, increased rates of protein and glycoprotein synthesis (as monitored by 3H-leucine and 3H-fucose incorporation, respectively) were also detected after 2 hr and continued through 7-9 days. Increased levels of Na,K-ATPase, a specific secretory cell PM marker, were detected after 8 hr of treatment as monitored by specific activity and 3H-ouabain binding. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis coupled with fluorography indicated that both 3H-leucine and 3H-fucose were incorporated into partially purified preparations of Na,K-ATPase isolated after 12 hr. Light microscopic autoradiographic analysis of pulse-chase experiments indicated that in secretory cells of 12-hr salt-stressed glands, 3H-leucine- and 3H-fucose-labelled products reached the cell periphery by 1-2 hr after the initial pulse. The incorporation of both tritiated precursors was predominantly associated with the secretory cells. Quantitative electron microscopic autoradiography indicated that 3H-leucine is initially taken up by elements of the rough endoplasmic reticulum (RER) and cytoplasm (5 min postpulse), subsequently transported to and concentrated within components of the Golgi apparatus (10 min of chase), and ultimately incorporated into all domains of the plasma membrane of secretory cells by 1-2 hr of chase. The data is consistent with a flow of newly synthesized membrane components from RER to Golgi to plasma membrane and is analogous to the pattern previously found for the synthesis and processing of PM proteins in a wide variety of cell types.