Somatostatin: occurrence in urinary bladder epithelium and renal tubules of the toad, Bufo marinus.

Immunohistochemical techniques were used to detect immunoreactive somatostatin-like material in toad urinary bladder epithelium and in kidney distal tubules and collecting ducts. This material has immunological and chromatographic properties identical to those of synthetic cyclic somatostatin. The occurrence of somatostatin-like material in antidiuretic hormone-sensitive portions of the renal urinary system suggests a local regulatory or paracrine role for somatostatin.

Role of prostaglandin E2 in mediating the effects of pH on the hydroosmotic response to vasopressin in the toad urinary bladder.

Acidosis inhibits the hydroosmotic response to vasopressin. Since prostaglandins are known to modulate vasopressin-stimulated water flow we investigated the role of endogenous prostaglandin E2(PGE2) production in the pH-dependent response of the toad urinary bladder to vasopressin. Graded acidification of the serosal medial resulted in a progressive decline in vasopressin-stimulated water flow from 26.6 +/- 0.5 mg/min at pH 8.4 to 1.7 +/- 0.6 at pH 6.9. In these bladders basal PGE2 synthesis increased from 5.09 +/- 0.51 pmol/min per g hemibladder at pH 8.4 to 18.8 +/- 2.8 at pH 6.9. The addition of that concentration of PGE2 produced by the bladder at pH 7.4 (4 nM) to bladders at pH 8.4 resulted in 62-71% of the inhibition usually seen at pH 7.4; these data suggest that basal PGE2 production per se and not other products of prostaglandin synthesis or other pH-dependent events is responsible for the effect of acidosis. Preincubation with prostaglandin synthesis inhibitors reversed in major part the effect of serosal acidification on the response to submaximal concentrations of vasopressin and completely abolished the effect of pH on near maximal concentrations of the hormone. An increase in PGE2 synthesis after vasopressin was not seen at any pH. These studies establish that increased basal PGE2 synthesis plays a critical role in the pH dependence of the hydroosmotic response to vasopressin and demonstrate that factors that modulate the response to vasopressin may exert this effect by changing the basal rate of prostaglandin synthesis.

Perfusion of isolated tubules of the shark rectal gland. Electrical characteristics and response to hormones.

Both the mammalian thick ascending limb of Henle's loop and the shark rectal gland actively transport Cl against an electrochemical gradient by mechanisms involving hormone-sensitive NaCl transport. In contrast to mammalian renal tubules, individual tubules of the shark rectal gland previously have not been perfused in vitro. Using a combination of renal slice and microdissection techniques we were able to isolate and perfuse single rectal gland tubules without the use of enzyme treatment. Single tubules consistently generated lumen-negative transepithelial voltages (Vt) of -1.8 mV when perfused and bathed with identical shark Ringer's solution. The addition of cyclic AMP, vasoactive intestinal peptide (VIP), and adenosine to the bath increased Vt to -7.5, -9.0, and -4.3 mV, respectively (all P less than 0.02 compared with paired controls). Each stimulation could be reversed by addition by furosemide to the bath. The adenosine response was inhibited by theophylline, a specific inhibitor of adenosine receptors. The tubules had a low transepithelial electrical resistance of 12-26 omega X cm2 and exhibited a transepithelial permselectivity for small cations. These results indicate that tubules of the rectal gland can be perfused in vitro and have receptors for VIP and adenosine. Cyclic AMP and secretagogues hyperpolarize the membrane consistent with electrogenic chloride transport, and these effects are reversed by furosemide, an inhibitor of coupled sodium-potassium-chloride co-transport. The response of Vt to cyclic AMP and furosemide, the transepithelial electrical resistance, and the cation selective permeability of tubules are remarkably similar to measurements in perfused mammalian thick ascending limbs.

Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport.

Adenosine and adenosine analogues stimulate adenosine cyclic 3', 5'-monophosphate-dependent chloride secretion in the mammalian ileum.

Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. The purpose of these investigations was to determine the effect of adenosine on ion transport in rabbit ileum in vitro. Adenosine and some of its analogues were found to increase the short circuit current (Isc) and the order of potency was N-ethylcarboxamide-adenosine greater than or equal to 2-chloroadenosine greater than phenylisopropyladenosine greater than adenosine. Purine-intact adenosine analogues had no effect on Isc. The effect of adenosine on Isc was enhanced by deoxycoformycin, an adenosine deaminase inhibitor, and by dipyridamole, an adenosine uptake inhibitor. The increase in Isc induced by 2-chloroadenosine was partially reversed in a dose-dependent manner by 8-phenyltheophylline but not by theophylline or isobutylmethylxanthine. 2-Chloroadenosine increased cyclic AMP content, and stimulated net Cl secretion; these effects were partially blocked by 8-phenyltheophylline. These results suggest that there is an adenosine receptor on rabbit ileal mucosal cells that stimulates adenylate cyclase, which results in secondary active Cl secretion.

A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP.

In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system.

On the mechanism of lithium-induced diabetes insipidus in man and the rat.

The mechanism of lithium-induced diabetes insipidus was investigated in 96 patients and in a rat model. Polydipsia was reported by 40% and polyuria (more than 3 liter/day) by 12% of patients receiving lithium. Maximum concentrating ability after dehydration and vasopressin was markedly impaired in 10 polyuric patients and was reduced in 7 of 10 nonpolyuric patients studied before and during lithium therapy. Severe polyuria (more than 6 liter/day) was unresponsive to trials of vasopressin and chlorpropamide, but improved on chlorothiazide. Rats receiving lithium (3-4 meq/kg/day) developed massive polyuria that was resistant to vasopressin, in comparison to rats with comparable polyuria induced by drinking glucose. Analysis of renal tissue in rats with lithium polyuria showed progressive increase in the concentration of lithium from cortex to papilla with a 2.9-fold corticopapillary gradient for lithium. The normal corticopapillary gradient for sodium was not reduced by lithium treatment. The polyuria was not interrupted by brief intravenous doses of vasopressin (5-10 mU/kg) or dibutyryl cyclic AMP (10-15 mg/kg) capable of reversing water diuresis in normal and hypothalamic diabetes insipidus rats (Brattleboro strain). The present studies suggest that nephrogenic diabetes insipidus is a common finding after lithium treatment and results in part from interference with the mediation of vasopressin at a step distal to the formation of 3',5' cyclic AMP.

Vasoactive intestinal peptide, forskolin, and genistein increase apical CFTR trafficking in the rectal gland of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR trafficking in an intact epithelium.

Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.

Cloning of sgk serine-threonine protein kinase from shark rectal gland ­ a gene induced by hypertonicity and secretagogues.

Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl­secretion and protein phosphorylation state in shark rectal gland cells.

Treatment of severe lithium-induced polyuria with amiloride.

One of the major side effects of lithium is nephrogenic diabetes insipidus. The established treatment for the disorder is thiazide diuretics, which are associated with hypokalemia and reduced lithium excretion, predisposing the patient to lithium toxicity. Amiloride is a new diuretic that reduces lithium-induced polyuria in animals without affecting lithium or potassium levels. The authors found that 10-20 mg/day of amiloride given to eight patients who had become hypokalemic while being treated with hydrochlorothiazide for lithium-induced nephrogenic diabetes insipidus increased renal concentrating ability and reduced polyuria. They conclude that amiloride can be useful in treating lithium-induced nephrogenic diabetes insipidus and merits future randomized clinical trials.

The Comparative Toxicogenomics Database (CTD): a resource for comparative toxicological studies.

The etiology of most chronic diseases involves interactions between environmental factors and genes that modulate important biological processes (Olden and Wilson, 2000. Nat Rev Genet 1(2):149-153). We are developing the publicly available Comparative Toxicogenomics Database (CTD) to promote understanding about the effects of environmental chemicals on human health. CTD identifies interactions between chemicals and genes and facilitates cross-species comparative studies of these genes. The use of diverse animal models and cross-species comparative sequence studies has been critical for understanding basic physiological mechanisms and gene and protein functions. Similarly, these approaches will be valuable for exploring the molecular mechanisms of action of environmental chemicals and the genetic basis of differential susceptibility.

Cellular and molecular biology of chloride secretion in the shark rectal gland: regulation by adenosine receptors.

The rectal gland of the dogfish shark (Squalus acanthias) is a sodium chloride secreting epithelial organ whose function was discovered in 1959 by Wendell Burger. The gland, composed of homogenous tubules of a single cell type, is an important model for secondary active chloride transport. Hormonal stimulation of chloride secretion in this system activates asymetrically arranged transport proteins (apical cAMP-activated CFTR-like Cl- channels, basolateral Na/K/2Cl cotransporters, Na/K-ATPase activity, and K+ channels). Five receptors, hormones, and membrane proteins of the shark rectal gland involved in chloride secretion have been cloned recently. Because the intact gland can be perfused via a single artery and vein, it has been possible to examine precisely the metabolic regulation of chloride transport by endogenous adenosine. Rectal gland cells have a high density of both stimulatory A2 type and inhibitory A1 type adenosine receptors. When stimulated by secretagogues, chloride secretion and venous adenosine concentrations increase in parallel, with chloride secretion increasing from approximately 150 to 2100 microEq/hr/g, and adenosine concentrations increasing from approximately 5 to approximately 890 nM. This work of ion transport is accompanied by a marked fall in intracellular ATP activity and a rise in both intracellular AMP and adenosine activity. Agents that prevent the interaction of endogenous adenosine with extracellular receptors significantly increase the chloride transport response to secretagogues. When chloride transport is inhibited by blocking the Na/K/2Cl cotransporter with bumetanide, both adenosine release and chloride secretion fall to basal values. We recently cloned a unique adenosine receptor subtype that is distinct from previously cloned mammalian adenosine receptors. Because of its highly specialized function, single cell type, and simple vascular system, the shark rectal gland is an ideal model system for examining the metabolic regulation of chloride secretion by adenosine receptors.

Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland.

We used the specific tyrosine kinase inhibitor genistein to define the involvement of tyrosine phosphorylation in the regulation of chloride transport in the rectal gland of the dogfish shark, a model for chloride secretion via a cystic fibrosis transmembrane conductance regulator (CFTR)-like channel. In the perfused gland, genistein (100 microM) promptly increased chloride secretion from basal values of 159 +/- 36 to 966 +/- 49 mueq.h-1.g-1 (P < 0.0001). Bumentanide fully reversed genistein-induced secretion. In primary culture monolayers of rectal gland tubular cells, genistein, but not the inactive 7-glucoside form, genistin, increased short-circuit current in a dose-dependent manner, from basal values of 2.7 +/- 4.3 to 104 +/- 10 microA/cm2 (P < 0.0001). Apically applied genistein induced significantly greater chloride secretion than basolateral addition. Genistein did not increase the adenosine 3',5'-cyclic monophosphate (cAMP) content of either perfused glands or cultured monolayers. Using an anti-phosphotyrosine antibody, we observed phosphorylation of multiple proteins. Four peptides, with molecular masses of 250, 210, 55, and 53 kDa, responded to genistein treatment with a decrease in tyrosine phosphorylation. These data demonstrate the following: 1) genistein induces bumetanide-sensitive chloride secretion in both perfused rectal glands and cultured tubular cells; 2) these effects are not accompanied by an elevation of tissue cAMP, indicating that genistein-induced secretion is not mediated by the cAMP-protein kinase A pathway; and 3) genistein-sensitive peptides are present in the rectal gland cell and are candidates for involvement in the regulation of chloride secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cl- secretion by cultured shark rectal gland cells. I. Transepithelial transport.

To facilitate analysis of the regulation of epithelial Cl- transport by hormones, neurotransmitters, and autocrine mediators, we have developed a primary monolayer culture system for shark rectal gland (SRG) epithelial cells. Cultures exhibit vigorous transcellular Cl- secretion which can be measured using short-circuit current or 36Cl flux methods. Transport is markedly reduced by bumetanide or barium, inhibitors of Na(+)-K(+)-2Cl- cotransport and K+ channels, respectively. This indicates that Cl- secretion by SRG monolayers occurs by a mechanism similar to that described in numerous native Cl- secretory epithelia. Forskolin, 10 microM 2-chloroadenosine, or vasoactive intestinal peptide, potent secretagogues in the isolated perfused SRG, stimulate Cl- secretion by SRG cultures. Submicromolar concentrations of 2-chloroadenosine, which inhibit Cl- secretion in the native SRG, reduce forskolin-stimulated short-circuit current in SRG cultures. Somatostatin, another inhibitor of Cl- secretion by the native SRG, reduces forskolin-stimulated adenosine 3',5'-cyclic monophosphate accumulation in SRG cultures. These results demonstrate that SRG cultures are fully responsive to mediators which activate or inhibit secretion by the native epithelium.

Protein kinase C zeta is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland.

Protein kinase C zeta (PKC zeta) is an atypical PKC isoform that has recently been implicated in cell division and cell growth. However, there has been no morphologic evidence for the involvement of PKC zeta in mitogenic signal transduction. Here we use immunocytochemistry to demonstrate that PKC zeta co-localizes with microtubules in both interphase and metaphase cells of the shark rectal gland in primary culture. This co-localization is present after non-ionic detergent treatment and is disrupted by nocodazole. During mitosis, PKC zeta is associated with the mitotic apparatus and co-localizes with beta-tubulin in spindle microtubules, while entirely sparing astral microtubules. These findings provide the first evidence that PKC zeta is associated with the mitotic apparatus. The striking presence of PKC zeta in the central portion of the mitotic apparatus suggests a functional role for this kinase isoform in cell division.

Kidney composition and renal concentration ability in young rabbits.

1. The water, sodium, urea, potassium and ammonia concentrations of the kidney cortex and inner medulla of adult rabbits, and of rabbits 1, 4, 14 and 21 days old, were measured after mild dehydration. The urine osmolarity and serum and urine urea concentrations were also measured.2. Potassium and ammonia showed no consistent variation in the different parts of the kidney or at the different ages. Sodium concentrations expressed as m-equiv/100 g fresh weight were about twice as high in the medulla as in the cortex at all ages.3. The medulla/cortex ratio of urea concentration increased markedly between the fourteenth and twenty-first day after birth. So also did the urine osmolarity of the mildly dehydrated animals.4. The rise in the medulla/cortex urea ratio between birth and 21 days, and the even higher ratio found in adult animals, was due to a decrease in the concentration of urea in the cortex rather than to an increase in its concentration in the medulla.