Rectal Gland Cells Research Articles

Expression of the odd-2 Gene in C. elegans.

The odd-2 gene in C. elegans is an orthologue of the odd-skipped gene in Drosophila and the odd-skipped related genes in mammals. The mammalian genes have been shown to be expressed in a variety of tissues and cancers. It was previously reported that ODD-2 is expressed in the intestine and shows some expression outside of the intestine in the tail region. Using a partial ODD-2 ::GFP fusion, we hypothesize that the expression outside of the intestine may be in rectal gland cells, and we also report that ODD-2 may be expressed in the germline sheath cells.

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.

HdfR is a regulator in Photorhabdus luminescens that modulates metabolism and symbiosis with the nematode Heterorhabditis

The bacterium, Photorhabdus luminescens, is an insect pathogen that also maintains a mutualistic interaction with nematodes from the family Heterorhabditis. Photorhabdus luminescens is carried in the gut of the infective juvenile (IJ), a nematode stage that infects soft-cuticled insect larvae in the soil. Photorhabdus luminescens is released into the insect blood to convert the insect into a biomass that is able to support nematode growth and development. Nematode reproduction continues for 2-3 generations before the nematodes enter an alternative developmental pathway leading to a new generation of IJs that are efficiently colonized by P. luminescens in a process called transmission. Transmission begins with the adherence of P. luminescens to the rectal gland cells (RGC) located in the gut of the hermaphrodite. The bacteria enter, and replicate within, the RGC resulting in vacuolization, and ultimately lysis, of the RGC. In this way P. luminescens are released throughout the body cavity of the hermaphrodite to encounter, and colonize, the developing IJs. In this study we show that the LysR-type regulator, HdfR, is required for normal transmission in P. luminescens TTO1. Transcriptome analysis revealed that HdfR regulates the expression of 124 genes, including genes involved in arginine metabolism, hydroxyphenylacetate catabolism and pigment production. Using fluorescence microscopy we show that the ΔhdfR mutant is able to attach to, and grow within, the hermaphrodite as well as wild-type bacteria but subsequent steps in transmission are delayed. Therefore, HdfR plays an important role in coordinating the interaction between P. luminescens and its nematode partner during transmission.

Studies on Acanthocheilonema viteae cystatin: Genomic organization, promoter studies and expression in Caenorhabditis elegans

Cystatins are reversible, tightly binding inhibitors of cysteine proteases. Filarial cystatins have been ascribed immunomodulatory properties and have been implicated in protective immunity. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cystatin gene locus of A. viteae. The gene is composed of 4 exons separated by 3 introns and spans ~2 kb of genomic DNA. The upstream genomic sequence contains transcriptional factor binding sites such as AP-1 and NF-Y, an inverted CCAAT sequence, and a TATA box. To investigate sites of cystatin expression, Caenorhabditis elegans worms were transformed by microinjection with the putative promoter region and the first exon of the A. viteae cystatin gene fused to the reporter GFP. In transgenic worms fluorescence was observed in the pharyngeal and rectal gland cells suggesting that cystatin is secreted. Additionally, A. viteae cystatin was expressed in C. elegans to explore its potential as an expression system for filarial genes.

Interaction of FXYD10 (PLMS) with Na,K-ATPase from Shark Rectal Glands: CLOSE PROXIMITY OF Cys74 OF FXYD10 TO Cys254 IN THE A DOMAIN OF THE α-SUBUNIT REVEALED BY INTERMOLECULAR THIOL CROSS-LINKING

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark: MOLECULAR CLONING, SEQUENCE, EXPRESSION, CELLULAR DISTRIBUTION, AND FUNCTIONAL EFFECTS OF PLMS

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase alpha-subunit and PLMS showed the presence of alpha and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1 approximately P --> E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) --> E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase alpha-subunit is important for the modulation of shark Na,K-ATPase activity.

Hypotonic induction of SGK1 and Na+ transport in A6 cells.

Serum and glucocorticoid-regulated kinase-1 (SGK1) is a serine-threonine kinase that is regulated at the transcriptional level by numerous regulatory inputs, including mineralocorticoids, glucocorticoids, follicle-stimulating hormone, and osmotic stress. In the distal nephron, SGK1 is induced by aldosterone and regulates epithelial Na+ channel-mediated transepithelial Na+ transport. In other tissues, including liver and shark rectal gland, SGK1 is regulated by hypertonic stress and is thought to modulate epithelial Na+ channel- and Na+-K+-2Cl- cotransporter-mediated Na+ transport. In this report, we examined the regulation of SGK1 mRNA and protein expression and Na+ currents in response to osmotic stress in A6 cells, a cultured cell line derived from Xenopus laevis distal nephron. We found that in contrast to hepatocytes and rectal gland cells, hypotonic conditions stimulated SGK1 expression and Na+ transport in A6 cells. Moreover, a correlation was found between SGK1 induction and the later phase of activation of Na+ transport in response to hypotonic treatment. When A6 cells were pretreated with an inhibitor of phosphatidylinositol 3-kinase (PI3K), Na+ transport was blunted and only inactive forms of SGK1 were expressed. Surprisingly, these results demonstrate that both hypertonic and hypotonic stimuli can induce SGK1 gene expression in a cell type-dependent fashion. Moreover, these data lend support to the view that SGK1 contributes to the defense of extracellular fluid volume and tonicity in amphibia by mediating a component of the hypotonic induction of distal nephron Na+ transport.

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 (I Cl) 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 I Cl. Addition of 10 μM HgCl 2 significantly inhibited the cAMP-activated I Cl ( p < 0.05, n = 11). Intracellular dialysis with ATPγS did not prevent the inhibitory effect of mercury on I Cl. In contrast, incubation of SRG cells with 10 μM HgCl 2 for 48 hrs markedly increased I Cl ( p < 0.01, n = 12). Dephosphorylation of the channel by intracellular dialysis with phosphatase I and II abolished the mercury-incubated increase in I Cl. The P450-mediated metabolite of arachidonic acid, 11,12-epoxyeicosatrienoic acid (11,12-EET), significantly increased I Cl. However, application of 11,12-dihydroxyeicosatrienoic acid (11,12-DHT) did not alter I Cl. 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 I Cl. Our results demonstrate that acute and subchronic application of mercury has opposing effects on I Cl in cultured SRG cells. The acute effect of mercury on I Cl may result from mercury blockade of Cl − channels. The subchronic effect of mercury on I Cl may be due to an induction of P450 CYP1A1 and its mediated metabolites, but not due to an over-expression of Cl − channels.

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.

Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

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, Cl–secretion and protein phosphorylation state in shark rectal gland cells.

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

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

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.

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 c1) and the inwardly rectifying K + channel (I k1). Type II and III cells only had I c1. 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 c1. In sharp contrast, 11% had I k1 and I c1, and 89% had only I c1 in cultured SRG cells. Extracellular application of 10 μM forskolin significantly enhanced I c1 in primary cultured SRG cells. This enhancement was influenced by intracellular Ca 2+ and blocked by 50 μM Ni 2+. Other compounds, such as vasoactive intestinal peptide(VIP) and 8-(4-chlorophenylthio)-adenosine3′:5′-cyclic monophosphate (cpt-cAMP) also enhanced I c1. Interestingly, cAMP and forskolin significantly inhibited I k1 in cultured and fresh SRG cells. I K1 was blocked by micromolar concentrations of Ba 2+ 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 c1 and I k1 in SRG cells are strongly modulated by cAMP, forskolin, and VIP, as well as Ca 2+, K +, and Na + ions.

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.

Ultrastructural and cytochemical characterization of cultured dogfish shark rectal gland cells.

The dogfish shark rectal gland (SRG) is histologically complex, containing connective, nerve, and smooth muscle tissue and at least three types of epithelial cells: secretory tubule, central duct, and endothelial. This cellular heterogeneity precludes studies of the intact SRG from distinguishing direct tubular effects of mediators that modulate chloride secretion from their indirect effects on nonepithelial cells such as neurons. Primary SRG cultures express high levels of secretagogue-stimulated chloride secretion, suggesting that SRG cells retain a significant level of cytodifferentiation in vitro. However, because nontubular cells could contaminate these cultures, the question of whether secretagogues activate chloride secretion through direct or indirect effects on tubular epithelial cells remains unresolved. To address this issue, detailed ultrastructural and cytochemical analyses of SRG cultures were carried out to assess the level of cellular heterogeneity and the degree of cytodifferentiation expressed by SRG cells in vitro. The results demonstrate that, after 15 days, primary SRG monolayer cultures are composed exclusively of tubular epithelial cells with no detectable contamination by central duct cells, fibroblasts, smooth muscle cells, endothelial cells, or neurons. Tubular epithelial cells express most of the structural features of native SRG cells, including numerous mitochondria, massive basolateral surface amplification, complex tight junctions, and an extensive tubulovesicular system in the apical cytoplasm. Cultured SRG cells also display a striking level of polarization of cytoplasmic organelles and plasma membrane secretagogue receptors. These results account for the exceptionally high rates of electrogenic chloride secretion by SRG tubular epithelial cells in vitro and confirm that the effects of secretagogues on transport activity reflect their direct interaction with tubular epithelial cells.

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.

CAMP-activated Cl- channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland

Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.

Evidence for the role of actin filaments in regulating cell swelling

Actin filaments could play a role in regulation of cell swelling by two distinct mechanisms. One is by a tensile mechanism involving the coordinated interaction of actin and actin-associated proteins with all plasma membrane domains. The actin-membrane linkage would restrain cell swelling in the event of the influx of water. In shark rectal gland cells, conditions that cause massive cell swelling (i.e., high K medium, exposure to mercurials) are associated with disruption of membrane-associated actin filaments. Under conditions that result in only moderate swelling (Na-pump inhibition, Li substitution) the actin filaments remain associated with the cell membrane. Thus, in this cell type, disruption of the actin-membrane organization is correlated with increased swelling. Another mechanism by which actin could limit cell swelling is via regulation of ion transport proteins that are activated by cell swelling. This could be accomplished by a vesicle transport and insertion mechanism that delivers ion transport units to the cell membrane or by interaction with transport proteins already present in the membrane. Cell-attached patch clamp studies of RCCT-28A cells exposed to hypotonic medium demonstrated the activation of Cl-channel activity coincident with an alteration in actin. Activation of the channel was mimicked by stretching the membrane. Exposure of inside-out patches to cytochalasins also increased Cl-channel activity. Treatment of isolated patches with phalloidin inhibited stretch-induced activation. Thus, regulation of a volume-sensitive Cl-channel appears to be directly related to the state of organization of actin filaments.

Neuropeptide Y inhibits chloride secretion in the shark rectal gland

We studied the effects of the 36-amino acid peptide, neuropeptide Y (NPY), on salt secretion by the rectal gland of Squalus acanthias. We used three preparations: whole isolated perfused glands, freshly prepared separated rectal gland tubules, and confluent monolayers of cultured rectal gland cells. In perfused glands NPY inhibited secretion stimulated by vasoactive intestinal peptide (VIP), forskolin, or adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline. Maximal inhibition of 63 +/- 3.4% was seen at 3 x 10(-8) M NPY, with half-maximal effect at 3 x 10(-9) M. NPY did not inhibit the basal activity of rectal gland adenylate cyclase or that stimulated by VIP. The inhibitory action of NPY was not prevented by procaine, nifedipine, or diltiazem, suggesting that it was not secondary to the release of somatostatin or other unknown neurotransmitters from rectal gland nerves. In confirmation, somatostatin was not detected in the venous effluent after administration of NPY. NPY also inhibited transport-related oxygen consumption in separated rectal gland tubules and inhibited short-circuit current generated by confluent monolayers of primary cultures of rectal gland cells. The results indicate that NPY inhibits chloride secretion by a direct action on cells of the shark rectal gland at a site distal to the generation of cAMP.