Phenotypic Plasticity in Animals Exposed to Osmotic Stress - Is it Always Adaptive?

Hyperplasia and hypertrophy are elements of phenotypic plasticity adjusting organ size and function. Because they are costly, we assume that they are beneficial. In this review, the authors discuss examples of tissue and organ systems that respond with plastic changes to osmotic stress to raise awareness that we do not always have sufficient experimental evidence to conclude that such processes provide fitness advantages. Changes in hydranth architecture in the hydroid Cordylophora caspia or variations in size in the anal papillae of insect larvae upon changes in medium salinity may be adaptive or not. The restructuring of salt glands in ducklings upon salt-loading is an example of phenotypic plasticity which indeed seems beneficial. As the genomes of model species are recently sequenced and the animals are easy to rear, these species are suitable study objects to investigate the biological significance of phenotypic plasticity and to study potential epigenetic and other mechanisms underlying phenotypic changes.

Immunohistochemical colocalization of G protein alpha subunits and 5-HT in the rectal gland of the cartilaginous fish Scyliorhinus canicula.

Serotonin [5-hydroxytryptamine (5-HT)] is an important neuromodulator involved in a wide range of physiological functions. The effects of serotonin are mediated by an extended family of receptors coupled to multiple heterotrimeric G-proteins, associated with cellular membrane. G proteins connect receptors to effectors and thus trigger intracellular signaling pathways. These cellular processes several regulate systemic functions such as embryonic development, gonadal development, learning and memory, and organismal homeostasis. Generally, elasmobranch fish dwell a hypersaline environment and utilize a specialized extrarenal salt secreting organ, the rectal gland, to face ionic homeostasis. In this study in addition to the morphological, histochemical and immunohistochemical description of the Scyliorhinus canicula rectal gland, for the first time, the presence of serotonin (5-HT), and distribution of different types of G protein alpha subunits (Gα o, Gα q/11, and Gα s/olf) has been investigated in the rectal gland epithelium by confocal immunofluorescence techniques. Colocalization G proteins and 5-HT in the secretory epithelium of the gland suggests serotonin acts as a hormone and involves G proteins in an autocrine-paracrine control of rectal gland homeostasis.

Molecular domains in epithelial salt cellNaCl of crustacean salt gland (Artemia).

The salt secretory cell has two distinct patterns of plasma membrane development. First, the basolateral surface forms a tubular labyrinth. It contains the subunit alpha-2 of the Na(+)-K(+)-ATPase bound together with a beta subunit for structural attachment within the lipid bilayer. Second, the apical plasma membranes form a multiple array of extending tufts. These tufts contain the subunit alpha-1 of the Na(+)-K(+)-ATPase bound together with a beta subunit for structural integrity within the lipid bilayer. The presence of an active transporter for chloride remains as an open question. It has been taken as preliminary evidence from brine shrimp cystic fibrosis toxicity that a cystic fibrosis transmembrane conductance regulator chloride channel could be present in the apical region. The presence of cytoskeletal elements being involved in the construction of a hypo-osmoregulatory apparatus is supported by the homeobox gene products derived from APH-1 m RNA found in the salt gland.

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.

Attenuation of cell cycle regulator p27(Kip1) expression in vertebrate epithelial cells mediated by extracellular signals in vivo and in vitro.

Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27(Kip1) has been implicated in this context in epithelial cells. We cloned and sequenced p27(Kip1) of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27(Kip1) downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27(Kip1) is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.

Cross-talk of phosphoinositide- and cyclic nucleotide-dependent signaling pathways in differentiating avian nasal gland cells.

In many bird species, the nasal glands secrete excess salt ingested with drinking water or food. In ducks ( Anas platyrhynchos), osmotic stress results in adaptive cell proliferation and differentiation in the gland. Using 'naive' nasal gland cells isolated from animals that had never ingested excess salt or 'differentiated' cells from animals fed with a 1% NaCl solution for 48 h, we investigated the allocation of metabolic energy to salt excretory processes and to other cellular activities. Activation of muscarinic acetylcholine receptors (carbachol) or beta-adrenergic receptors (isoproterenol) in nasal gland cells resulted in a transient peak in metabolic rate followed by an elevated plateau level that was maintained throughout the activation period. Activation of cells using vasoactive intestinal peptide, however, had only marginal effects on metabolic rate. In differentiated cells, sequential stimulation with carbachol and isoproterenol resulted in additive changes in metabolic rate during the plateau phase. Naive cells, however, developed supra-additive plateau levels in metabolic rates indicating cross-talk of both signaling pathways. Using bumetanide, TEA or barium ions to block different components of the ion transport machinery necessary for salt secretion, the relative proportion of energy needed for processes related to ion transport or other cellular processes was determined. While differentiated cells in the activated state allocated virtually all metabolic energy to processes related to salt secretion, naive cells reserved a significant amount of energy for other processes, possibly sustaining cellular signaling and regulating biosynthetic mechanisms related to adaptive growth and differentiation.

Revealing of proteins interacting with Na,K-ATPase.

Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.

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.

Salt gland blood flow in the hatchling green turtle, Chelonia mydas.

Microsphere and morphometric techniques were used to investigate any circulatory changes that accompany secretion by the salt glands of hatchling Chelonia mydas. Salt glands were activated by a salt load of 27.0 mmol NaCl x kg body mass (BM)(-1), resulting in a mean sodium secretion rate of 4.14 +/- 0.11 mmol Na x kg BM(-1) x h(-1) for a single gland. Microsphere entrapment was approximately 160-180 times greater in the active salt gland than the inactive gland, inferring a similar change in blood flow through salt gland capillaries. The concentration of microspheres trapped in the salt gland was significantly correlated with the rate of tear production (ml x kg BM(-1) x h(-1)) and the total rate of sodium secretion (mmol Na x kg BM(-1) x h(-1)) but not with tear sodium concentration (mmol Na x l(-1)). Adrenaline (500 microg x kg BM(-1)) inhibited tear production within 2 min and reduced microsphere entrapment by approximately 95% compared with active glands. The volume of filled blood vessels increased from 0.03 +/- 0.01% of secretory lobe volume in inactive salt gland sections to 0.70 +/- 0.11% in active gland sections. The results demonstrate that capillary blood flow in the salt gland of C. mydas can regulate the activity of the gland as a whole.

Mode of activation of salt secretion by C-type natriuretic peptide in the shark rectal gland.

We studied the modes of activation of the salt-secreting rectal gland of the spiny dogfish, Squalus acanthias, by the native cardiac peptide CNP. The stimulatory action of CNP in isolated perfused glands is inhibited by 10 mM procaine, presumably by blocking release of vasoactive intestinal peptide (VIP) from nerves. Procaine reduces the slope of the dose-response curve of human CNP and that of shark CNP (each P < 0.0001). CNP increases short-circuit current in cultured rectal gland cells from 4.8 +/- 1.6 to 27.0 +/- 7.8 microA/cm2. It also stimulates the secretion of chloride in isolated perfused glands in the presence of 10 mM procaine from 72 +/- 31 to 652 +/- 173 microeq. h(-1). g(-1). These results suggest that CNP has a direct cellular action not mediated by the neural release of VIP. The residual stimulation of perfused glands in the presence of procaine was almost completely inhibited by staurosporine [10 nM; an inhibitor of protein kinase C (PKC)] from 652 +/- 173 to 237 +/- 61 microeq. h(-1). g(-1). Although CNP stimulates guanylyl cyclase in shark rectal gland, chloride secretion of perfused glands was not elicited by 8-bromoadenosine-cGMP (8-BrcGMP) alone nor by the activator of PKC phorbol ester. The combination of PKC activation and 8-BrcGMP infusion, however, stimulated chloride secretion in perfused glands from 94 +/- 30 to 506 +/- 61 microeq. h(-1). g(-1), a level comparable to that observed in glands blocked with procaine. Several parallel pathways appear to be synergistic in activating chloride secretion stimulated by CNP in the rectal gland.

Hepatic toxicity and persistence of ser/thr protein phosphatase inhibition by microcystin in the little skate Raja erinacea.

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 microg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 microg/kg microcystin. Mortality occurred at doses of 500 microg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 microg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

The Na+2Cl-K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage.

Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.

The synthesis of a small heat shock/alpha-crystallin protein in Artemia and its relationship to stress tolerance during development.

Fertilized oocytes of the brine shrimp Artemia franciscana undergo either ovoviviparous or oviparous development, yielding free-swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Encystment is followed by diapause, wherein metabolism is greatly reduced; the resulting cysts are very resistant to extreme stress, including desiccation and long-term anoxia. The synthesis of p26, a small heat shock/alpha-crystallin protein produced only in oviparously developing Artemia, is shown in this paper to be transcriptionally regulated. A p26 mRNA of about 0.7 kb was detected on Northern blots in the second day after oocyte fertilization. It peaked as embryos encysted and declined rapidly when activated cysts resumed development. The appearance of p26 protein, as indicated by immunoprobing of Western blots, followed mRNA by 1 day; it also increased as encystment occurred but remained constant during postgastrula development of cysts. However, p26 underwent a marked reduction during emergence of nauplii and could not be detected in cell-free extracts of second-instar larvae. p26 entered nuclei of encysting embryos soon after synthesis and was localized therein as late as instar II, when it was restricted to a small set of salt gland nuclei. First-instar larvae derived from cysts were more thermotolerant than larvae that had developed ovoviviparously, but synthesis of p26 was not induced by heat under the experimental conditions employed. Additionally, transformed bacteria synthesizing p26 were more thermotolerant than bacteria that lacked the protein. The results support the proposal that p26, a developmentally regulated protein synthesized during embryo encystment, has chaperone activity in vivo and protects the proteins of encysted Artemia from stress-induced denaturation.

In vivo and in vitro induction of c-fos in avian exocrine salt gland cells.

Osmotic stress in ducklings (Anas platyrhynchos) results in salt secretion and adaptive cell proliferation and differentiation in the nasal glands. We investigated whether osmotic stress in vivo or muscarinic ACh receptor activation in vitro changed the expression levels of the cellular protooncogene products Fos and Jun, which may play a role in the initiation of the adaptive processes. Using Fos- and Jun-specific polyclonal antisera in Western blot experiments, we demonstrated that Jun is constitutively expressed in nasal gland tissue, whereas Fos is not detectable in tissue from unstressed (naive) animals. Under conditions of osmotic stress imposed by replacing the drinking water of the animals with a 1% NaCl solution, Jun protein remains constant in nasal gland tissue, whereas Fos protein is transiently upregulated. Treatment of cultured nasal gland tissue with muscarinic agonists results in a transcriptionally regulated expression of Fos in an atropine-sensitive manner. Immunohistochemical experiments show that Fos accumulation occurs in the nuclei of the secretory cells. These results indicate that the activation of the c-fos gene induced by muscarinic ACh receptor-mediated signaling pathways may play an important role in the initiation of adaptive growth and differentiation processes in nasal glands of osmotically stressed ducklings.

Confocal microscopic observation of cytoskeletal reorganizations in cultured shark rectal gland cells following treatment with hypotonic shock and high external K+.

The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between the actin cytoskeleton and cell volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ and hypotonically-induced cell swelling displayed a fading of F-actin staining intensity, particularly at the basolateral cell borders. However, spectroscopic measurement of the F-actin present in similarly treated rectal gland slices failed to demonstrate a net change in F-actin amount. In an effort to resolve the structural reorganizations of F-actin which may be occurring during high K+ and hypotonic shock treatments, we have used cultured SRG cells in conjunction with confocal microscopic immunocytochemical localization techniques to examine actin filament, microtubule, and cytokeratin filament dynamics under these two experimental conditions. The results reveal that F-actin in control cells exists in an array of parallel linear bundles (which do not appear to be stress fiber-like given their lack of staining for myosin II or alpha-actinin) that is reorganized to a punctate pattern in hypotonic shock and a dense meshwork in high K+. The linear bundle pattern of F-actin returns in cells undergoing regulatory volume decrease. Quantitative western blotting of F-actin in SRG cell detergent extracted cytoskeletons indicates no significant difference in the relative amounts of F-actin present in control, hypotonic shocked, or high K+ cells. Anti-tubulin and anti-cytokeratin labeling of the treated SRG cells suggest that these other major cytoskeletal elements are not significantly altered by the treatments. Taken together, our results reinforce the concept that there is an association between the structural organization of the actin cytoskeleton and cell volume regulation in the SRG epithelial cells.

cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells.

The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 microM) and glibenclamide (400 microM). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents in excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.

Electrophysiological properties of different cell types in the shark rectal gland.

Electrophysiological properties of different cell types were studied in single rectal gland cells of Squalus acanthias by the whole-cell voltage clamp technique. Based on electrophysiological characteristics and primary morphological observations (light microscope, X400), three cell types (named as I, II, and III) were found in isolated fresh cells and two cell types (I and II) in primary cultured cells of the shark rectal gland (SRG). Type I cells had both Cl- (I(Cl)) and the inwardly rectifying K+ channel (I(K1)). Type II and III cells only had I(Cl) Under X400 light microscope granular materials in the cytoplasm were found in Type I and II cells, but not in Type III cells. The data from this study show that 65 % of isolated fresh SRG cells strongly expressed the K+ channel with much less amount of the Cl- channel and 35% had only I(Cl). In sharp contrast, 11% had I(K1) and I(Cl), and 89% had only I(Cl) in cultured SRG cells. Extracellular application of 10 microM forskolin significantly enhanced I(Cl) in primary cultured SRG cells. This enhancement was influenced by intracellular Ca2+ and blocked by 50 microM Ni2+. Other compounds, such as vasoactive intestinal peptide (VIP) and 8-(4-chlorophenylthio)-adenosine3':5'-cyclic monophosphate (cpt-cAMP) also enhanced I(Cl). Interestingly, cAMP and forskolin significantly inhibited I(K1) in cultured and fresh SRG cells. I(K1) was blocked by micromolar concentrations of Ba2+ and significantly altered by extracellular K+ concentrations. The present data suggest that 1) the shark rectal gland contains different cell types which may play various roles in the process of salt secretion; 2) I(Cl) and I(K1) in SRG cells are strongly modulated by cAMP, forskolin, and VIP, as well as Ca2+, K+, and Na+ ions.

Clustered distribution of calcium sensitivities: an indication of hetero-tetrameric gating components in Ca2+-activated K+ channels reconstituted from avian nasal gland cells.

Calcium-activated potassium channels (maxi K+ channels) isolated from avian nasal salt gland cells were reconstituted into lipid bilayers and characterized. The 266 pS channel is blocked discretely by charybdotoxin from the external solution at nanomolar concentrations and by Ba2+ from the cytosolic side at micromolar concentrations. Fast tetraethylammonium (TEA) block is seen as apparent reductions in amplitude of the unitary currents. From the extent of the reductions, TEA binding affinity was calculated to be 0.16 mM from the external solution and 37 mm from internal solution. The overall channel properties conform to those of maxi K+ channels in other epithelial tissues. The calcium sensitivity of the channel was found to be variable from channel to channel, extending over a wide range of concentrations from 1 to 1,000 microM. Examination of the pooled calcium titration curves, revealed that these curves are grouped into five clusters, and the probability distribution of the clusters matches a binomial distribution. The Hill coefficient derived from the titration curves varies from 1 to 5 and is linearly correlated to calcium binding with a slope of 1 per 10-fold change in Kd. Clustered titration curves with such a characteristic suggest that the gating components and the calcium binding sites of the maxi K+ channels in the avian nasal gland are hetero-tetrameric and may result from random mixing of two distinct subunits possessing high and low calcium sensitivities, respectively.

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.

Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.

The effect of cytoplasmic Cl concentration ([Cl]i) on the activation state ([3H]benzmetanide binding rate) and phosphorylation state (32P incorporation) of the Na-K-Cl cotransporter was evaluated in secretory tubules isolated from the dogfish shark rectal gland. Reduction of [Cl]i at relatively constant cell volume (by removal of extracellular Cl or Na or by addition of bumetanide) increased cotransporter activation and phosphorylation. Raising extracellular K concentration ([K]o) from 4 to 80 mM, a maneuver that elevated [Cl]i above normal, reduced basal cotransport activity and rendered it entirely refractory to forskolin. High [K]o also blocked activation and phosphorylation in response to cell shrinkage, despite the fact that [Cl]i was already greatly elevated as a consequence of osmotic water loss. The phosphatase inhibitor calyculin A also promoted activation, but not in cells preexposed briefly to high [K]o. In summary, maneuvers than lower [Cl]i activate the cotransporter, whereas those that elevate [Cl]i (or prevent it from decreasing) block activation in response to secretory stimuli. Cell Cl appears to govern its own rate of entry via Na-K-Cl cotransport by impeding regulatory phosphorylation of the Na-K-Cl cotransport protein.