Role of the cytoskeleton in secretion of chloride by shark rectal gland

The salt gland of the spiny dogfish, Squalus acanthias, can be stimulated to secrete chloride by two different endogenous peptides: cardiac natriuretic peptide (CNP) and the neurotransmitter, vasoactive intestinal peptide (VIP). We examined the role of the actin cytoskeleton and of myosin light chains in this process by perfusing isolated rectal glands with and without an inhibitor of actin filament organization (cytochalasin D) and an inhibitor of myosin light chain kinase (ML-7). Cytochalasin D, 10(-6) M, reduced secretion stimulated by a 1-min bolus of CNP (5x10(-7) M) by 50-60%. In the presence of 10(-2) M procaine (which blocks neural release of VIP), cytochalasin D completely prevented CNP stimulation. In contrast, cytochalasin D did not inhibit stimulation of the rectal gland by VIP or by forskolin. Similarly, 5x10(-6)M ML-7 almost completely inhibited direct stimulation of rectal gland secretion by CNP, but did not alter chloride secretion induced by VIP or forskolin. Finally, the average time between hormonal injection and activation of secretion was 2 min longer for CNP than for VIP, consistent with the hypothesis that a contractile cellular function involving the cytoskeleton is important in CNP-induced chloride secretion, but less so when secretion is stimulated by VIP.     

A preliminary investigation into the roles played by the rectal gland and kidneys in the osmoregulation of the striped dogfish Poroderma africanum.

Presented here are the results of a preliminary investigation into ionic and osmotic regulation by the kidneys and rectal gland of the striped dogfish, Poroderma africanum. Fish with ligated rectal glands showed an increase in blood concentration of sodium and chloride within a short time period, reaching a maximum after four days. The blood concentration of the two ions then decreased over the following ten days. Control animals showed relatively unchanged blood-sodium and chloride levels, over the entire 14-day period. After salt loading, both control animals and those with ligated rectal glands showed initial rise in blood sodium and chloride levels, but these returned towards initial values within seven hours of injection. Comparison of the two groups indicates that the rectal gland may control blood-chloride levels more so than -sodium, although its action as a salt regulator does not seem very pronounced in either case. Urine and rectal gland fluid, were collected as a compound fluid, from normal fish, and the estimated cloacal salt loss is discussed. Urine from normal fish was also collected separately and was analysed for its contribution to salt loss. Results are discussed and compared with previous relevant findings.     

Changes in chloride secretion rate and vascular perfusion in the rectal gland of the European lesser-spotted dogfish in response to environmental and hormonal stimuli

Chloride secretion rates of rectal glands taken from the European lesser-spotted dogfish Scyliorhinus canicula adapting to 70% and 120% sea water (SW) were significantly greater and less than, respectively, those in the control 100% SW group. C-type natriuretic peptide (CNP) significantly increased chloride secretion rates above basal values in 100% SW although angiotenisn II (ANG II) had no effect. Perfusion of the secretory epithelia in rectal glands from 70% SW lesser-spotted dogfish was significantly higher than in rectal glands from 100% and 120% SW lesser-spotted dogfish. Perfusion of rectal glands with ANG II had no effect on perfusion of the secretory epithelia, although CNP perfusion induced significantly greater perfusion of the secretory epithelia than all other treatments. It remains to be determined if a reduction in environmental salinity induces an increase in plasma concentration of CNP and hence an increase in rectal gland activity.     

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.     

The shark rectal gland: a model for the active transport of chloride.

The rectal gland of the spiny dogfish, Squalus acanthias, provides an easily studied model of active chloride transport powered indirectly by Na-K-ATPase. Co-transport of sodium with chloride can be demonstrated in membrane vesicles isolated from basolateral membranes of the gland. Chloride secretion is under the hormonal control of vasoactive intestinal peptide, and possibly other agents, via adenyl cyclase and cyclic AMP. A similar mechanism is probably responsible for the active transport of chloride across other biological membranes.     

Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator.

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.     

Demonstration of nerve fibers immunoreactive to peptide N-terminal histidine C-terminal isoleucine (PHI) and vasoactive intestinal peptide (VIP) in the pig pineal gland

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.     

Cyclic AMP regulation of active chloride transport in the rectal gland of marine elasmobranchs.

Active transport of chloride in the salt secreting rectal gland of the dogfish Squalus acanthias is markedly stimulated by the addition of theophylline (0.01 to 5 mM) or dibutyryl cyclic AMP (0.05 mM) during in vitro perfusion. The effect occurs promptly and may persist for two hours. Specific hormonal effectors have not yet been identified. The isolated perfused rectal gland thus offers a unique opportunity to examine the cellular mechanisms of active chloride secretion.     

Innervation of the cat pineal gland by neuropeptide Y-immunoreactive nerve fibers: an experimental immunohistochemical study

An immunohistochemical study of the cat pineal gland was performed using a rabbit polyclonal antibody directed against neuropeptide Y (NPY) and an antibody directed against the C-terminal flanking peptide of neuropeptide Y (CPON). Numerous NPY- and CPON-immunoreactive (IR) nerve fibers were demonstrated throughout the gland and in the pineal capsule. The number of IR nerve fibers in the capsule was high and from this location fibers were observed to penetrate into the gland proper via the pineal connective tissue septa, often following the blood vessels. From the connective tissue septa IR fibers intruded into the parenchyma between the pinealocytes. Many IR nerve fibers were observed in the pineal stalk and in the habenular as well as the posterior commissural areas. The number of NPY/CPON-IR nerve fibers in pineal glands from animals bilaterally ganglionectomized two weeks before sacrifice was low. The source of most of the extrasympathetic NPY/CPONergic nerve fibers is probably the brain from where they enter the pineal via the pineal stalk. However, an origin of some of the fibers from parasympathetic ganglia cannot be excluded due to the presence of a few IR fibers in the pineal capsule of ganglionectomized animals. It is concluded that the cat pineal is richly innervated with NPYergic nerve fibers mostly of sympathetic origin. The posttranslational processing of the NPY promolecule results in the presence of both NPY and CPON in intrapineal nerve fibers.     

Immunohistochemical studies on peptide- and amine-containing endocrine cells and nerves in the gut and the rectal gland of the ratfish Chimaera monstrosa

Summary The occurrence and distribution of endocrine cells and nerves were immunohistochemically demonstrated in the gut and rectal gland of the ratfish Chimaera monstrosa (Holocephala). The epithelium of the gut mucosa revealed open-type endocrine cells exhibiting immunoreactivity for serotonin (5HT), gastrin/cholecystokinin (CCK), pancreatic polypeptide (PP)/FMRFamide, somatostatin, glucagon, substance P or gastrin-releasing peptide (GRP). The rectum contained a large number of closed-type endocrine cells in the basal layer of its stratified epithelium; the majority contained 5HT- and GRP-like immunoreactivity in the same cytoplasm, whereas others were immunoreactive for substance P. The rectal gland revealed closed-type endocrine cells located in the collecting duct epithelium. Most of these contained substance P-like immunoreactivity, although some reacted either to antibody against somatostatin or against 5HT. Four types of nerves were identified in the gut and the rectal gland. The nerve cells and fibers that were immunoreactive for vasoactive intestinal peptide (VIP) and GRP formed dense plexuses in the lamina propria, submucosa and muscular layer of the gut and rectal gland. A sparse network of gastrin- and 5HT-immunoreactive nerve fibers was found in the mucosa and the muscular layer of the gut. The present study demonstrated for the first time the occurrence of the closed-type endocrine cells in the mucosa of the rectum and rectal gland of the ratfish. These abundant cells presumably secrete 5HT and/or peptides in response to mechanical stimuli in the gut and the rectal gland. The peptide-containing nerves may be involved in the regulation of secretion by the rectal gland.     

Secretion and stabilization of the layers of the egg capsule of the dogfish Scyuorhinus canicula

The egg capsule of the dogfish is a unique, collagcnous, layered structure secreted by the nidamental gland which has nine, remarkably discrete, transverse zones of tubular glands. The present paper traces the origin of the four layers of the capsule to particular zones within the gland. Evidence is presented for the existence of DOPA, DOPA oxidase. protein(s) rich in tyrosyl residues and a peroxidase within the same storage granules within the secretory cells of the C and E zones. It is suggested that these interact when secreted to cross-link the inner and outer surfaces of the egg capsule. Evidence is presented that the middle layer which forms the bulk of the thickness of the egg capsule and has the highest collagen content may be partly stabilized by the peroxidation of tyrosyl residues. The mechanical significance of crosslinking in this system and the possible cytological mechanisms involved in the secretion of the tanning agents and enzymes are discussed.     

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.     

Innervation of the parathyroid in the european starling (Sturnus vulgaris)

Anatomical studies were conducted to characterize the source, type, and distribution of parathyroid gland innervation in European starlings. Denervation experiments demonstrated that the parathyroid glands and adjacent carotid bodies are innervated by nerve fibers originating in the nodose ganglion of the vagus nerve. In the parathyroid parenchyma, these fibers terminate adjacent to chief cells or near vascular smooth muscle. Vagal fibers also form synapses with catecholamine-containing glomus cells of the carotid body. Blood that first perfuses the carotid body subsequently perfuses the parathyroid parenchyma. These observations suggest that vagal innervation may influence parathyroid function in starlings either through direct chief cell innervation or through alteration of vascular perfusion. A neurohemal relationship also may exist between the carotid body and parathyroids.     

Tyrosine hydroxylase and neuropeptide-Y immunoreactivity in pineal glands developing in situ and in pineal grafts

Summary Postnatal development of the innervation of the pineal gland in situ as well as the reinnervation of pineal grafts by tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined using the avidin-biotin-peroxidase immunohistochemical technique. TH-immunoreactive nerve fibers appeared in the pineal gland on the second postnatal day (P2) in both hamsters and gerbils. NPY-immunoreactive nerve fibers first appeared in the pineal gland of gerbils on P2 and in the hamsters on P3. By the seventh postnatal day (P7), the pineal glands of both hamsters and gerbils were richly innervated by TH- and NPY-fibers that appeared as smooth fibers or fibers with sporadic varicosities. By the age of 4 weeks, the innervation of the pineal glands of hamsters and gerbils by TH-and NPY-fibers was fully developed. Abundant TH- and NPY-fibers formed a dense meshwork in the parenchyma of the superficial and deep pineals. The great majority of the fibers bore a large number of varicosities. More NPY-fibers were found in the pineal glands of gerbils than hamsters. NPY fibers were distributed evenly throughout the pineal glands of the gerbil, but they were more often located in the central region of the superficial pineal of the hamster. For the pineal grafts, superficial pineals from neonatal and 4-week-old hamsters were transplanted to different sites in the third cerebral ventricle (infundibular recess, posterior third ventricle) or beneath the renal capsule. The pineal grafts from 4-week-old donors appeared to undergo severe degeneration and eventually disappeared. The pineal grafts from neonatal hamsters, however, successfully survived and became well integrated into their new locations. Abundant TH-and NPY-fibers in the host brain were found surrounding the pineal grafts placed in the third cerebral ventricle, but were only rarely seen entering the parenchyma of the grafts. A few TH-fibers were demonstrated in the renal grafts 4 weeks after transplantation. These studies describe the postnatal development of the innervation of the pineal glands in situ by TH-and NPY-nerve fibers, and demonstrate a lack of reinnervation of cerebroventricular pineal grafts by TH and NPY fibers from adjacent host brain.Portions of the results of this paper were previously reported in abstract form at the 1990 Meeting of The American Association of Anatomists (Anat Rec 226:57A)     

Zur Cytologie der Rectaldrüsen von Knorpelfischen

The stratified epithelium of the central collecting duct of the elasmobranch(Scylliorhinus canicula, Galeorhinus galeus andRaja batis) rectal gland consists of 3 to 6 layers of cells: one superficial, and several basal cell layers. In the superficial layer normally three different types of cells can be distinguished (a) goblet cells, (b) cells with apical secretory granules and (c) flask-shaped cells. The superficial layer ofScylliorhinus canicula reveals a further cell type, so-called mitochondria-rich cells. The epithelial areas built by these cells are always single-layered. The goblet-cells are very similar to goblet cells found in the intestine of vertebrates. Their dominant structures are a well developed ergastoplasm, a large Golgi-apparatus and mucous granules compactly filling the apical cell region. The cells with apical secretory granules are columnar or dumbbell shaped. They contain a rough-surfaced endoplasmic reticulum and a well developed Golgi-apparatus. The secretory granules are loosely distributed within the Golgi-field and are arranged in one or more rows just below the cell apex. The flask shaped cells are characterized by a cytoplasm rich in small vesicles. They posses few dictyosomes and several small mitochondria. There is some evidence for endocytotic activity. The mitochondria-rich cells are characterized by lateral cell interdigitations, by a basal labyrinth and by numerous mitochondria. They are similar to the excretory cells of rectal gland parenchyma. The cells of the basal epithelium layers are differenciated only to a small extent. They are joined in a loose formation with white blood cells often found in the intercellular spaces. The function of the elasmobranch rectal gland is not restricted to the excretion of concentrated salt solutions. There is also a significant secretion of mucous substances. The tubule glands are primarily excretory, the epithelium cells of the central collecting duct mainly secretory in function.     

Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes

Summary To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands ofSqualus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na₂SO₄, 25 mM K₂SO₄ gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide. 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na₂SO₄, 50 mM K₂SO₄ gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.Abbreviations SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TALH thick ascending limb of Henle's loop     

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.     

Functional vasoactive intestinal polypeptide (VIP)-system in salt glands of the Pekin duck

Summary In saltwater-acclimated ducks with fully specialized supraorbital salt glands, intracarotid application of acetylcholine (5 nmoles/min/kg b.w.) or porcine vasoactive intestinal polypeptide (pVIP) (240 pmoles/min/kg b.w.) induced secretion from the salt glands at threshold conditions of secretory activity. pVIP-like immunoreactivity could be localized in fibers of the postganglionic secretory nerve ramifying throughout the glandular parenchyma. Both middle-sized arterioles and secretory tubules were innervated, and pVIP-immunoreactive varicose fibers formed peritubular baskets around the basal region of secretory tubules indicating direct innervation of the secretory tissue. pVIP-specific staining could be abolished by preabsorption of the antiserum with peptide extracts of salt-gland tissue. Synthetic pVIP and endogenous VIP from salt glands of the duck co-eluted on the HPLC system, suggesting structural similarity of the peptides. Membrane-binding studies with radioiodinated pVIP revealed the presence of high-affinity binding sites in salt-gland tissue. Affinities of unlabeled pVIP analogues to compete for these binding sites were as follows: pVIP > PHI > pVIP antagonist > secretin > pVIP (10–28) > chicken VIP (16–28). Peptide extracts of salt glands had affinities similar to pVIP. Binding sites could be localized mainly at the apical end of the radially arranged secretory tubules, as demonstrated by receptor autoradiography.It is concluded that, in addition to the classical parasympathetic transmitter acetycholine, VIP serves as neuromodulator/transmitter in cranial parasympathetic control of avian salt-gland secretion by acting on both the arteriolar network and the secretory tubules of the gland.     

Organ-related distribution of phospholemman in the spiny dogfish Squalus acanthias

The distribution of phospholemman among nine different organs of the spiny dogfish (Squalus acanthias) has been determined on the basis of Western blotting of microsomal material. Only rectal gland (100%), brain (43%), heart (18%), and kidney (19%) (abundancies as percent of the concentration in rectal gland) contained the protein, but not gill and colon. The relative abundance in the brain makes this organ a preferential test system for phospholemman in fishes that lack a rectal gland like teleosts.