Sugar uptake, metabolism, and chloride secretion in the rectal gland of the spiny dogfish Squalus acanthias.

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.

AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

Gastric inhibitory peptide, serotonin, and glucagon are unexpected chloride secretagogues in the rectal gland of the skate (Leucoraja erinacea).

Since the discovery of the rectal gland of the dogfish shark 50 years ago, experiments with this tissue have greatly aided our understanding of secondary active chloride secretion and the secretagogues responsible for this function. In contrast, very little is known about the rectal gland of skates. In the present experiments, we performed the first studies in the perfused rectal gland of the little skate (Leucoraja erinacea), an organ weighing less than one-tenth of the shark rectal gland. Our results indicate that the skate gland can be studied by modified perfusion techniques and in primary culture monolayers, and that secretion is blocked by the inhibitors of membrane proteins required for secondary active chloride secretion. Our major finding is that three G protein-coupled receptor agonists, the incretin gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide, as well as glucagon and serotonin, are unexpected potent chloride secretagogues in the skate but not the shark. Glucagon stimulated chloride secretion to a mean value of 1,661 ± 587 µeq·h(-1)·g(-1) and serotonin stimulated to 2,893 ± 699 µeq·h(-1)·g(-1). GIP stimulated chloride secretion to 3,733 ± 679 µeq·h(-1)·g(-1) and significantly increased tissue cAMP content compared with basal conditions. This is the first report of GIP functioning as a chloride secretagogue in any species or tissue.

cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland.

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

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.

Effect of colchicine on sensitivity of duck salt gland Na,K-ATPase to Na+.

Low molecular mass proteins of the FXYD family that affect the sensitivity of Na,K-ATPase to Na+ and K+ are known to be present in Na,K-ATPases in various tissues. In particular, in Na,K-ATPase from kidney a gamma-subunit (with electrophoretic mobility corresponding to molecular mass of about 10 kD) is present, and Na,K-ATPase preparations from heart contain phospholemman (electrophoretic mobility of this protein corresponds to molecular mass of 13-14 kD), which provides for the interaction of heart Na,K-ATPase with cytoskeletal microtubules. Disruption of microtubules by colchicine removes phospholemman from heart Na,K-ATPase preparations. The goal of the present study was to reveal a low molecular mass protein (probably a member of FXYD family) in preparation of Na,K-ATPase from duck salt glands. Immunoprecipitation of solubilized duck salt gland Na,K-ATPase using antibodies against alpha1-subunit results in the coprecipitation of a 13 kD protein with the Na,K-ATPase complex. Treatment of homogenate from duck salt glands with colchicine removes this protein from the purified preparation of Na,K-ATPase. Simultaneously, we observed a decrease in the sensitivity of Na,K-ATPase to Na+ at pH 6.5. However, colchicine treatment of homogenate from rabbit kidney does not affect either the sensitivity of Na,K-ATPase obtained from this homogenate to Na+ or the content of 10 kD protein (presumably gamma-subunit). The data suggest that phospholemman (or a similar member of the FXYD family) tightly interacts with Na,K-ATPase from duck salt glands and binds it to microtubules, simultaneously participating in the regulation of the sensitivity of Na,K-ATPase to Na+.

ANG II-induced attenuation of salt gland function in Pekin ducks is not catecholamine-dependent.

Pekin ducks (Anas platyrhynchos) were bilaterally adrenalectomized (biADX), injected with 1 mg of triamcinolone (TRIAM) kg bw(-1) im and given 0.9% saline drinking water during a 24 h recovery period followed by chemical sympathectomy with 6OH DOPA 3 h before the start of experimental observations. Baseline plasma dopamine (DA) concentrations decreased from 283 +/- 88.5 pmol ml(-1) to 42.4 +/- 11.1 pmol ml(-1); epinephrine (E) from 142 +/- 46 pmol ml(-1) to 18.4 +/- 9.2 pmol ml(-1) and norepinephrine (NE) from 742 +/- 84 pmol ml(-1) to 406 +/- 38 pmol ml(-1) 1 day after biADX + TRIAM but before chemical sympathectomy. Baseline MABP increased from 132 +/- 3.2 mmHg to 209 +/- 14.3 mmHg (P < 0.05) in response to TRIAM. After chemical sympathectomy with 6OH DOPA there was an additional 90% decrease in plasma NE to 42 +/- 9.4 pmol ml(-1) and a concurrent 60% decrease in MABP to 83.4 +/- 6.9 mmHg (P < 0.05). Nasal fluid secretion was maintained by the continuous infusion of hypertonic saline (1,000 mosmol kg H(2)O(-1) at a rate of 0.3 ml kg(-1) min(-1)). Rates of nasal fluid secretion and fluid electrolyte concentrations were unchanged following biADX + TRIAM + 6OH DOPA. Angiotensin II (ANG II; dose 1 microg kg bw(-1) i.v.), attenuated nasal fluid secretion showing that the response to ANG II was not NE- dependent. Plasma NE concentrations decreased following Tyramine i.v. (33 +/- 8.5 pmol ml(-1)) there being no vasopressor response. This is the first report of the ANG II induced attenuation of duck salt gland secretion in the absence of measurable E and NE.

Effect of melatonin on salt gland and kidney function of gulls, Larus glaucescens.

This study examined effects of exogenous melatonin on osmoregulatory hormones and water and sodium secretion by salt glands and excretion via the kidneys of Glaucous-winged gulls (Larus glaucescens). Six saline acclimated gulls were injected with inulin and paraminohippuric acid and then infused with 500 mM NaCl to stimulate salt gland secretion. Each bird was given infusions of NaCl alone and NaCl plus melatonin. Experiments were made one week apart in a randomized order. A large blood sample (to measure osmoregulatory hormones) was taken before infusion, at secretion, and at the end of infusion. A small blood sample was taken at the midpoint of each of six 10 min sequential collections of salt gland secretion and urine. Melatonin tended to increase plasma sodium concentration, did decrease plasma osmolality, but did not affect potassium concentration. Melatonin did not affect salt gland secretion rate or concentration nor renal plasma flow or glomerular filtration. Melatonin increased urine flow rate, tended to increase urine sodium concentration, and did decrease urine potassium concentration. Combined renal and extrarenal sodium excretion was greater during MT treatment. During NaCl infusion, angiotensin II increased, aldosterone decreased, and arginine vasotocin remained unchanged. Melatonin did not affect these responses. These data suggest an osmoregulatory role for melatonin in birds with salt glands.

A critical analysis of carbonic anhydrase function, respiratory gas exchange, and the acid-base control of secretion in the rectal gland of Squalus acanthias.

We compared in vivo responses of rectal gland secretion to carbonic anhydrase (CA) inhibition (10(-4) mol l(-1) acetazolamide) in volume-loaded dogfish with in vitro responses in an isolated-perfused gland stimulated with 5 x 10(-6) mol l(-1) forskolin and removed from systemic influences. We also measured respiratory gas exchange in the perfused gland, described the acid-base status of the secreted fluid, and determined the relative importance of various extracellular and intracellular acid-base parameters in controlling rectal gland secretion in vitro. In vivo, acetazolamide inhibited Cl(-) secretion and decreased pHi in the rectal gland, but interpretation was confounded by an accompanying systemic respiratory acidosis, which would also have contributed to the inhibition. In the perfused gland, M(CO(2)) and M(O(2)) increased in linear relation to increases in Cl(-) secretion rate. CA inhibition (10(-4) mol l(-1) acetazolamide) had no effect on Cl(-) secretion rate or pHi in the perfused gland, in contrast to in vivo, but caused a transitory 30% inhibition of M(CO(2)) (relative to stable M(O(2))) and elevation in secretion P(CO(2)) effects, which peaked at 2 h and attenuated by 3.5-4 h. Secretion was inhibited by acidosis and stimulated by alkalosis; the relationship between relative Cl(-) secretion rate and pHe was almost identical to that seen in vivo. Experimental manipulations of perfusate pH, P(CO(2)) and HCO(3)(-) concentration, together with measurements of pHi, demonstrated that these responses were most strongly correlated with changes in pHe, and were not related to changes in P(CO(2)), extracellular HCO(3)(-), or intracellular HCO(3)(-) levels, though changes in pHi may also have played a role. The acid-base status of the secreted fluid varied with that of the perfusate, secretion pH remaining about 0.3-0.5 units lower, and changing in concert with pHe rather than pHi; secretion HCO(3)(-) concentrations remained low, even in the face of greatly elevated perfusate HCO(3)(-) concentrations. We conclude that pH effects on rectal gland secretion rate are adaptive, that CA functions to catalyze the hydration of CO(2), thereby maintaining a gradient for diffusive efflux of CO(2) from the working cells, and that differences in response to CA inhibition likely reflect the higher perfusion-to-secretion ratio in vitro than in vivo.

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.

ANG II-induced attenuation of duck salt gland secretion does not depend upon the release of adrenal catecholamines.

Pekin ducks (Anas platyrhynchos) were bilaterally adrenalectomized (ADX) using a two-stage procedure and given daily i.m. injections of 1 mg kg bw(-1) of dexamethasone (DEXA), a steroid lacking mineralocorticoid activity, and 0.9% saline drinking water ad libitum to counterbalance renal losses of salt and water. Mean arterial blood pressure (mmHg) fell from 161+/- 3.7 (intact controls) to 116 +/- 6.9 (bilateral ADX+DEXA), a decrease of 27%, but heart rates (HR) were unchanged. The nasal salt glands were fully active after ADX + DEXA. Rates of fluid secretion and electrolyte and osmolal concentrations were unchanged. Secretion stopped, then rebounded several minutes later if ADX + DEXA ducks were injected i.v. with 1 microg of [Asn(1),Val(5)]-angiotensin II (ANG II) kg bw(-1) which showed that attenuation was not adrenal catecholamine-dependent.

Effects of cadmium ingestion on plasma and osmoregulatory hormone concentrations in male and female Pekin ducks.

Effects of ingested cadmium (Cd) on body mass and plasma, urine, salt-gland secretion, and osmoregulatory hormone concentrations were assessed in male and female Pekin ducks, Anas platyrhynchos, acclimated to 450 mM NaCl over 6 wk and then held an additional 13 wk on 300 mM NaCl (prolonged saline exposure). Groups of six birds ate diets containing 0 (control), 50 (low-Cd diet), or 300 (high Cd diet) micrograms Cd/g food. Ducks that ingested Cd, especially females, lost body mass. Cadmium ingestion did not affect salt-gland secretion concentration. Control males had higher plasma osmolality and lower relative plasma volume. These increased in both sexes during saline acclimation. The high-Cd diet suppressed the increase in plasma osmolality in both sexes, but a rise in relative plasma volume occurred only in females. Following prolonged exposure to saline, plasma osmolality and relative plasma volume were reduced in control ducks but further elevated in ducks on the high-Cd diet. Cadmium ingestion suppressed the increase in urine osmolality that occurred in control ducks during saline acclimation. Neither saline acclimation nor Cd ingestion affected plasma concentrations of arginine vasotocin or prolactin. Arginine vasotocin was not correlated with plasma osmolality; prolactin was negatively correlated with plasma osmolality, but only in males. Cadmium suppressed the increase in angiotensin II that occurred at higher salinities in control and low-Cd males. This study examined the effects of gradually increasing body cadmium content on osmotic homeostasis. Cadmium affected plasma and urine, but not salt-gland secretion, concentrations and some of these responses were sexually disparate. Cadmium did not affect osmoregulatory hormones (arginine vasotocin and prolactin) by which observed changes in plasma concentration might have been influenced.

Intracellular accumulation of mercury enhances P450 CYP1A1 expression and Cl- currents in cultured shark rectal gland cells.

The effects of acute and subchronic exposure to mercury on the Cl- current (ICl) were investigated in cultured shark rectal gland (SRG) cells. The effects of intracellular accumulation of mercury on cytochrome P450 (P450) were also assessed. Bath perfusion of a cocktail solution containing forskolin, 1-isobutyl-3-methylxanthine, and 8-bromoadenosine monophosphate enhanced ICl. Addition of 10 microM HgCl2 significantly inhibited the cAMP-activated ICl (p < 0.05, n = 11). Intracellular dialysis with ATP gamma S did not prevent the inhibitory effect of mercury on ICl. In contrast, incubation of SRG cells with 10 microM HgCl2 for 48 hrs markedly increased ICl (p < 0.01, n = 12). Dephosphorylation of the channel by intracellular dialysis with phosphatase I and II abolished the mercury-incubated increase in ICl. The P450-mediated metabolite of arachidonic acid, 11,12-epoxyeicosatrienoic acid (11,12-EET), significantly increased ICl. However, application of 11,12-dihydroxyeicosatrienoic acid (11,12-DHT) did not alter ICl. Mercury incubation for 48 hrs did not alter the protein expression of Cl- channels, but caused an induction of CYP1A1 in cultured SRG cells. In addition, co-incubation of SRG cells with mercury and the P450 inhibitor clotrimazole prevented the mercury-incubated increase in ICl. Our results demonstrate that acute and subchronic application of mercury has opposing effects on ICl in cultured SRG cells. The acute effect of mercury on ICl may result from mercury blockade of Cl- channels. The subchronic effect of mercury on ICl may be due to an induction of P450 CYP1A1 and its mediated metabolites, but not due to an over-expression of Cl- channels.

Salt and water regulation by the leatherback sea turtle Dermochelys coriacea.

We measured the salt and water balance of hatchling leatherback sea turtles, Dermochelys coriacea, during their first few days of life to investigate how they maintain homeostasis under the osmoregulatory challenge of a highly desiccating terrestrial environment and then a hyperosmotic marine environment. Hatchlings desiccated rapidly when denied access to sea water, with their hematocrit increasing significantly from 30.32+/-0.54 % to 38.51+/-1.35 % and plasma Na(+) concentration increasing significantly from 138.2+/-3.3 to 166.2+/-11.2 mmol l(-1) in 12 h. When hatchlings were subsequently put into sea water, hematocrit decreased and plasma Na(+) concentration was unchanged but both were significantly elevated above pretreatment values. In other hatchlings kept in sea water for 48 h, body mass and plasma Na(+) concentration increased significantly, but hematocrit did not increase. These data show that hatchlings were able to osmoregulate effectively and gain mass by drinking sea water. We stimulated hatchlings to secrete salt from the salt glands by injecting a salt load of 27 mmol kg(-1). The time taken for secretion to begin in newly hatched turtles was longer than that in 4-day-old hatchlings, but the secretory response was identical at 4.15+/-0.40 and 4.13+/-0.59 mmol Na(+) kg(-1) h(-1) respectively. Adrenaline and methacholine were both potent inhibitors of salt gland secretion in a dose-dependent manner, although methacholine administered simultaneously with a subthreshold salt load elicited a transient secretory response. The results showed that hatchling leatherbacks are able to tolerate significant changes in internal composition and efficiently use their salt glands to establish internal ionic and water balance when in sea water.

A Ca(2+)-sensing receptor modulates shark rectal gland function.

The elasmobranch Squalus acanthias controls plasma osmolality and extracellular fluid volume by secreting a hypertonic fluid from its rectal gland. Because we found a correlation between extracellular Ca(2+) concentration and changes in cytosolic Ca(2+) ([Ca(2+)](i)), we sought the possible presence of a calcium-sensing receptor in rectal gland artery and tubules. Cytosolic Ca(2+) of both tissues responded to the addition of external Ca(2+) (0.8-5.3 mmol l(-1)) in a linear fashion. Spermine, Gd(3+) and Ni(2+), known agonists of the calcium-sensing receptor, increased [Ca(2+)](i). To assess the participation of inositol triphosphate (IP(3)) generation, sarcoplasmic/endoplasmic reticulum (SR/ER) Ca(2+) depletion, and activation of store-operated Ca(2+) entry, we utilized thapsigargin and ryanodine to deplete Ca(2+) SR/ER stores and the inhibitory reagents TMB-8 and 2-APB to block IP(3) receptors. In each case, these agents inhibited the [Ca(2+)](i) response to agonist stimulation by approximately 50 %. Blockade of L-channels with nifedipine had no significant effect. Increases in ionic strength are known to inhibit the calcium-sensing receptor. We postulate that the CaSR stimulates Ca(2+)-mediated constriction of the rectal gland artery and diminishes cyclic AMP-mediated salt secretion in rectal gland tubules during non-feeding conditions. When the shark ingests sea water and fish, an increase in blood and interstitial fluid ionic strength inhibits the activity of the calcium-sensing receptor, relaxing the rectal gland artery and permitting salt secretion by the rectal gland tubules.

The effects of dietary sodium loading on the activity and expression of Na, K-ATPase in the rectal gland of the European dogfish (Scyliorhinus canicula).

cDNA fragments of both the alpha- and beta-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na-K-Cl cotransporter from the European dogfish (Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for alpha- and beta-subunits of the Na, K-ATPase, the Na-K-Cl cotransporter and CFTR chloride channel were detected but not until 1-2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of alpha- and beta-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.

Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias.

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.

Morphological and pathological effects of cadmium ingestion on Pekin ducks exposed to saline.

This study examined the effects of simultaneous exposure to saline and cadmium (Cd) on organ mass and histology of a bird with salt glands, the Pekin duck, Anas platyrhynchos. Three mixed-sex groups, each containing 6 birds, ate duck pellets containing 0, 50, or 300 microg Cd/g, respectively, for 4 1/2 mo and drank 300 mM NaCl. Only females on the high-Cd diet lost body mass. Ingestion of Cd reduced heart mass in females. There was increased mass of Harderian and salt glands in both sexes. Mass of kidneys and liver increased only in males, and the gut mass (also length) increased more in males. Cadmium ingestion also induced (1) inflammation of renal interstitium and degenerative tubular changes, (2) marked degenerative changes in testes, (3) increased heart water content, (4) decreased cytoplasmic volume of liver cells, (5) reduced proportion of basophilic granular cells in chromaffin tissue of the adrenal glands, and (6) in the ileum, increased heterophilia in the lamina propria and, only in females, the apoptosis to mitosis ratio in crypt cells of the epithelium. The ducks' outward appearance gave no indication that ingesting large amounts of cadmium for 4 1/2 mo produced deleterious effects, but the physiological consequences were profound. Both sexes had greatly reduced gonadal mass and the males produced no sperm. The higher dietary level greatly hypertrophied the liver, kidneys, and gut only in males. The cadmium-induced changes in organs, particularly in the gonads, kidneys, and adrenal glands, should greatly impair the health and reproductive capacity of these ducks.

Ca(2+) and p38 MAP kinase regulate mAChR-mediated c-Fos expression in avian exocrine cells.

Muscarinic acetylcholine receptors (mAChRs) in exocrine tissue from the avian nasal salt gland are coupled to phospholipase C and generate inositol phosphate and Ca(2+) signals upon activation. An early effect of receptor activation in the secretory cells is a transient accumulation of c-Fos protein. In cooperation with constitutively expressed Jun, Fos presumably serves as a transcription factor altering gene expression during cell growth and differentiation processes in the gland associated with adaptation to osmotic stress in animals. Nothing is known, however, about the mAChR-dependent signaling pathways leading to Fos expression in these cells. By incubation of isolated nasal gland tissue in short-term culture with activators or inhibitors of signaling pathways and quantitative Western blot analysis of Fos abundance, we have now identified the sustained elevation of the intracellular Ca(2+) concentration and the activation of the p38 mitogen-activated protein (MAP) kinase as intermediate signaling elements for the regulation of c-Fos by muscarinic receptor activation. It is suggested that p38 MAP kinase, rather than exclusively mediating stress responses, is involved in the regulation of cellular growth and differentiation controlled by G protein-coupled receptors.

Control of salt gland activity in the hatchling green sea turtle, Chelonia mydas.

We studied the control of salt gland secretion in hatchling Chelonia mydas. The threshold salt load to activate salt secretion was between 400 mumol NaCl 100 g bodymass (BM)-1 and 600 mumol NaCl 100 g BM-1, which caused an increase in plasma sodium concentration of 13% to 19%. Following a salt load of 2700 mumol NaCl 100 g BM-1, salt gland secretion commenced in 12 +/- 1.3 min and reached maximal secretory concentration within 2-7 min. Maximal secretory rate of a single gland averaged 415 mumol Na 100 g BM-1 h-1. Plasma sodium concentration and total osmotic concentration after salt loading were significantly higher than pretreatment values within 2 min. Adrenalin (25 micrograms kg BM-1) and the cholinergic agonist methacholine (1 mg kg BM-1) inhibited salt gland activity. Atropine (10 mg kg BM-1) reversed methacholine inhibition and stimulated salt gland secretion when administered with a subthreshold salt load. Arginine vasotocin produced a transient reduction in sodium secretion by the active gland, while atrial natriuretic factor, vasoactive intestinal peptide and neuropeptide Y had no measurable effect on any aspect of salt gland secretion. Our results demonstrated that secretion of the salt gland in C. mydas can be modified by neural and hormonal chemicals in vivo and that the cholinergic and adrenergic stimulation of an exocrine gland do not appear to have the typical, antagonist actions on the chelonian salt gland.