Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Rectal gland tubule (RGT) segments of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PDte resistance (Rte), the PD across the basolateral membrane (PDbl), the fractional resistance of this membrane (FRbl), and intracellular activities of NA+, Cl-, K+ (apha cell) were examined. Furosemide (5 x 10(-4) mol x 1(-1)) reduced PDte from -12 +/- 0.7 to -2.3 +/- 0.2 mV (n = 63), hyperpolarized PDbl from -71 +/- 1.3 to -79 +/- 0.9 mV (n = 59), FRbl decreased from 0.2 +/- 0.03 to 0.13 +/- 0.01 (n = 21), alpha cell cl- fell from 38 +/- 4 to 11 +/- 2 mmol x 1(-1) (n = 21), alpha cell Na+ fell from 37 +/- 4 to 17 +/- 2 mmol x 1(-1) (n = 12) and alpha cell K+ was constant [113 +/- 14 vs. 117 +/- 15 mmol x 1(-1) (n = 6)]. Furosemide exerted its effects within some 20-40s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall in alpha cell cl- was approximately twice as rapid when compared to that of alpha cell Na+. Ba2+ 0.5 mmol x 1(-1) (bath) reduced PDte from -7.1 +/- 1.2 to -4.1 +/- 0.6 mV (n = 24), increased Rte from 18 +/- 2 to 22 +/- 2.5, omega cm2 (n = 14). PDbl depolarized from -75 +/- 2 to -48 +/- 2 mV (n = 42), FRbl increased from 0.2 +/- 0.02 to 0.34 +/- 0.04 (n = 14) and alpha cell K+ increased from 143 +/-28 to 188 +/- mmol x 1(-1) (n = 4). Ouabain (50 x 10(-6) mol x 1(-1), bath) reduced PDte from -12 +/-2 to -3 +/- 0.5 mV (n = 9), Rte increased from 18 +/- 3 to 21 +/- 3 omega cm2 (n = 5). PDbl depolarized from -67 +/- 4 to -26 + 3 mV (n = 14), FRbl increased from 0.23 +/- 0.04 to 0.45 +/- 0.05 (n = 6), alpha cell K+ fell only slightly from 135 +/- 15 to 112 +/- 30 mmol x 1(-1) (n = 4), but alpha cell cl- increased from 35 +/- 12 to 111 +/- 37 mmol x 1(-1) (n = 3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba2+. The ouabain effects on PDte and PDbl were completely prevented if furosemide was applied first.(ABSTRACT TRUNCATED AT 400 WORDS)     

Renal ammoniagenesis and acid excretion in the dogfish, Squalus acanthias

Renal ammoniagenesis and acid excretion were investigated in normal dogfish (Squalus acanthias) and dogfish made acidotic by HCl injection (0.65 meq X kg-1). After acid loading, renal ammonia excretion doubled, rising from 0.11 to 0.25 mueq X h-1, and titratable acid output increased from 28.6 to 44.9 mueq X h-1. Trimethylamine excretion averaged 23.2 mueq X h-1 and did not change in response to the acidosis. During the first 24 h postinjection, the increase in renal acid excretion accounted for the elimination of 15% of the acid load. In vitro studies with kidney slices demonstrated that the dogfish kidney has the capacity to synthesize ammonia from a number of amino acids including glutamine, glutamate, alanine, aspartate, and glycine, with the greatest ammonia production resulting from glutamine. The relatively high glutamine concentration in the kidney, compared with the blood, suggested a high renal capacity for glutamine synthesis. In renal homogenates, enzymatic activities for both the deamidation (glutaminase) and synthesis (glutamine synthetase) of glutamine were investigated. The Michaelis constant (Km) values for the two enzymes were found to be almost equal (4.48 mM glutamine and 4.33 mM glutamate) and at the same levels as the substrate concentrations in the kidney (3.69 mM glutamine and 3.36 mM glutamate). Subcellular localization revealed that both enzymes occur predominantly in the mitochondria. The activities of glutaminase and glutamine synthetase in the renal mitochondria suggest the presence of a substrate cycle that could be modulated to increase ammonia production during acidosis.     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PD_te), transepithelial resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of the lumen membrane (FR₁), and the cellular activities for Cl^–, Na⁺, and K⁺ (a _x ^cell ) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10^–4, adenosine 10^–4, and forskolin 10^–6 mol · l^–1) on these parameters were recorded and compared to nonstimulated state (NS). PD_te increased from –1.9±0.2 mV to –11.0±0.9 mV (n=51). PD_bI depolarized from –86±1 to –74±1.4 mV (n=52). R_te fell from 29±2.8 to 21±2 cm² (n=23), and FR₁ fell from 0.96±0.005 to 0.79±0.04 (n=9).a _K+ ^cell was constant (123±13 versus 128±17 mmol · 1^–1) (n=6), buta _Cl– ^cell -fell significantly from 48±4 to 41±3 mmol · l^–1 (n=7).a _Na+ ^cell increased from 11±2.1 to 29.5±6.6 mmol · l^–1 (n=4). In series 2 the conductivity properties were examined by rapid K⁺, and Cl^– concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K⁺ led to a hyperpolarization of PD_bI with a mean slope of 28±1.3 mV/decade (n=9) (as compared to 19 mV/decade for S-state). Reduction of lumen Cl^– led to very little depolarization of the lumen membrane PD in NS-state: 6.5±2.3 mV/decade (n=4) (as compared to 13 mV/decade for S-state). In series 3 the effects of furosemide (7 · 10^–5 mol l^–1, bath) were examined in NS and S tubules. In NS RGT segments furosemide had no effect on PD_bI or PD_te;a _Cl– ^cell fell slowly after furosemide with an initial rate of 0.33 mmol · l^–1 s^–1, as compared to 1.5 mmol · l^–1 · s^–1 for S-state. The increase ina _Cl– ^cell after removal of furosemide from NS to S-states was examined in the presence of furosemide. Despite the presence of furosemide stimulation was accompanied by a fall in R_te, FR₁, anda _Cl– ^cell . From these data we conclude that (a) stimulation by cyclic AMP increases the Cl^–-conductance of the apical cell membrane at least by a factor of 10, that (b) in the NS-state the Na⁺2Cl^–K⁺ carrier can be triggered to work at rates similar to the S state by loweringa _Cl– ^cell , and that (c) the increase in apical Cl^–-conductance is the primary event in cyclic AMP mediated stimulation of NaCl secretion.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-1, and by NIH Grant AM 34208     

GFAP-immunopositive structures in spiny dogfish, Squalus acanthias, and little skate, Raia erinacea, brains: differences have evolutionary implications

GFAP expression patterns were compared between the brains of a spiny dogfish (Squalus acanthias) and a little skate (Raia erinacea). After anesthesia, the animals were perfused with paraformaldehyde. Serial vibratome sections were immunostained against GFAP using the avidin-biotin method. Spiny dogfish brain contained mainly uniformly-distributed, radially arranged ependymoglia. From GFAP distribution, the layered organization in both the telencephalon and the tectum were visible. In the cerebellum, the molecular and granular layers displayed conspicuously different glial structures; in the former a Bergmann glia-like population was found. No true astrocytes (i.e., stellate-shaped cells) were found. Radial glial endfeet lined all meningeal surfaces. Radial fibers also seemed to form endfeet and en passant contacts on the vessels. Plexuses of fine perivascular glial fibers also contributed to the perivascular glia. Compared with spiny dogfish brain, GFAP expression in the little skate brain was confined. Radial glia were limited to a few areas, e.g., segments of the ventricular surface of the telencephalon, and the midline of the diencephalon and mesencephalon. Scarce astrocytes occurred in every brain part, but only the optic chiasm, and the junction of the tegmentum and optic tectum contained large numbers of astrocytes. Astrocytes formed the meningeal glia limitans and the perivascular glia. No GFAP-immunopositive Bergmann glia-like structure was found. Astrocytes seen in the little skate were clearly different from the mammalian and avian ones; they had a different process system – extra large forms were frequently seen, and the meningeal and perivascular cells were spread along the surface instead of forming endfeet by processes. The differences between Squalus and Raia astroglia were much like those found between reptiles versus mammals and birds. It suggests independent and parallel glial evolutionary processes in amniotes and chondrichthyans, seemingly correlated with the thickening of the brain wall, and the growing complexity of the brain. There is no strict correlation, however, between the replacement of radial ependymoglia with astrocytes, and the local thickness of the brain wall. Accepted: 6 March 2001     

GFAP-immunopositive structures in spiny dogfish, Squalus acanthias, and little skate, Raia erinacea, brains: differences have evolutionary implications

GFAP expression patterns were compared between the brains of a spiny dogfish (Squalus acanthias) and a little skate (Raia erinacea). After anesthesia, the animals were perfused with paraformaldehyde. Serial vibratome sections were immunostained against GFAP using the avidin-biotin method. Spiny dogfish brain contained mainly uniformly-distributed, radially arranged ependymoglia. From GFAP distribution, the layered organization in both the telencephalon and the tectum were visible. In the cerebellum, the molecular and granular layers displayed conspicuously different glial structures; in the former a Bergmann glia-like population was found. No true astrocytes (i.e., stellate-shaped cells) were found. Radial glial endfeet lined all meningeal surfaces. Radial fibers also seemed to form endfeet and en passant contacts on the vessels. Plexuses of fine perivascular glial fibers also contributed to the perivascular glia. Compared with spiny dogfish brain, GFAP expression in the little skate brain was confined. Radial glia were limited to a few areas, e.g., segments of the ventricular surface of the telencephalon, and the midline of the diencephalon and mesencephalon. Scarce astrocytes occurred in every brain part, but only the optic chiasm, and the junction of the tegmentum and optic tectum contained large numbers of astrocytes. Astrocytes formed the meningeal glia limitans and the perivascular glia. No GFAP-immunopositive Bergmann glia-like structure was found. Astrocytes seen in the little skate were clearly different from the mammalian and avian ones; they had a different process system - extra large forms were frequently seen, and the meningeal and perivascular cells were spread along the surface instead of forming endfeet by processes. The differences between Squalus and Raia astroglia were much like those found between reptiles versus mammals and birds. It suggests independent and parallel glial evolutionary processes in amniotes and chondrichthyans, seemingly correlated with the thickening of the brain wall, and the growing complexity of the brain. There is no strict correlation, however, between the replacement of radial ependymoglia with astrocytes, and the local thickness of the brain wall.     

Dual mechanism for catecholamine secretion in the dogfish shark Squalus acanthias

Both 1,1-dimethyl-4-phenylpiperazinium iodide, a ganglionic stimulating drug (DMPP), and potassium ion (K+) cause a pressor response when injected into Squalus acanthias, an elasmobranch. The pressor responses are due to increased secretion of epinephrine and norepinephrine. The pressor response to DMPP can be blocked by prior infusion of hexamethonium, a ganglionic blocking drug. However, ganglionic blockade does not inhibit the pressor response to K+. Plasma catecholamine concentrations do not increase significantly in response to challenge with DMPP after hexamethonium infusion, but exceedingly high levels of plasma catecholamines quickly appear after K+ injection following hexamethonium infusion. It is concluded that there are at least two mechanisms controlling catecholamine secretion in the dogfish, one of which involves the ganglion cells that are intimately associated with chromaffin cells in the chromophil bodies that are so characteristic of this species and elasmobranchs in general.     

Identification of Leydig-like cells in the testis of the dogfish Squalus acanthias

The site of hormone synthesis in the testis of elasmobranchs has been the subject of much controversy. This is primarily due to the problem of whether Leydig cells are present or absent in the testes of many species of elasmobranchs. In previous studies we have shown that key enzymes associated with the biosynthesis of androgen increase in activity during the spermatogenetic cycle of Squalus acanthias (Canick et al., 1983). To determine the site of this activity we undertook an electron microscope study of Squalus testes to identify cells that possessed the structural correlates of steroid production. This report describes cells present in the interstitial tissue that are morphologically analogous to Leydig cells occurring in the testes of higher vertebrates. Although these cells possessed an agranular reticulum, tubulovesicular mitochondria, and lipid droplets, they were mesenchymal in appearance. We have, therefore, preferred to describe these cells as Leydig-like.     

The alkaline tide goes out and the nitrogen stays in after feeding in the dogfish shark, Squalus acanthias

In light of previous work showing a marked metabolic alkalosis ("alkaline tide") in the bloodstream after feeding in the dogfish shark (Squalus acanthias), we evaluated whether there was a corresponding net base excretion to the water at this time. In the 48 h after a natural voluntary meal (teleost tissue, averaging 5.5% of body weight), dogfish excreted 10,470 micromol kg(-1) more base (i.e. HCO3- equivalents) than the fasted control animals (which exhibited a negative base excretion of -2160 micromol kg(-1)). This large activation of branchial base excretion after feeding thereby prevented a potentially fatal alkalinization of the body fluids by the alkaline tide. The rate peaked at 330 micromol kg(-1) h(-1) at 12.5-24 h after the meal. Despite a prolonged 1.7-fold elevation in MO2 after feeding ("specific dynamic action"), urea-N excretion decreased by 39% in the same 48 h period relative to fasted controls. In contrast, ammonia-N excretion did not change appreciably. The N/O2 ratio declined from 0.51 in fasted animals to 0.19 in fed sharks, indicating a stimulation of N-anabolic processes at this time. These results, which differ greatly from those in teleost fish, are interpreted in terms of the fundamentally different ureotelic osmoregulatory strategy of elasmobranchs, and recent discoveries on base excretion and urea-retention mechanisms in elasmobranch gills.     

Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

The rectal gland of the dogfish (Squalus acanthias) secretes chloride via a chloride channel present in the apical cell membrane. Using the patch clamp technique in isolated perfused rectal gland tubules [7], two types of chloride channels are demonstrable in the apical membrane of cyclic AMP treated tubule segments. A small channel of about 11 pS and another channel of 40–50 pS are present. The small channel is described in the succeeding report. With NaCl on both sides (excised patches) the current amplitude of the larger channel is an almost linear function of the voltage (±50 mV). However, the open probability of this channel is grossly reduced at negative clamp potentials (corresponding to cell hyperpolarization). Therefore, the macroscopic Cl^– current through this channel is reduced with hyperpolarization on the cytosolic side. An analysis of time constants of this channel reveals that at depolarized voltages two open and two closed time constants of about 1 ms and of about 10 ms, respectively, are demonstrable. With hyperpolarized voltages the larger open state time constant is reduced significantly. This type of chloride channel is blocked reversibly by diphenylamine-2-carboxylate (10^–4 mol/l) and by 5-nitro-2-(3-phenylpropylamino)-benzoate (10^–5 mol/l). The channel is selective for Cl^– over Na^– and K^– as well as over Br^–. It is, however, permeable for NO ₃ ^- . Since this channel is very rare or absent in nonstimulated rectal gland tubules, it is very likely that this type of channel is responsible for hormone and cAMP dependent chloride secretion in this organ.Supported by Deutsche Forschungsgemeinschaft Gr 480 and by NSF and NIH grants to the MDIBL     

Potassium channels in the basolateral membrane of the rectal gland of the dogfish (Squalus acanthias)

Previous studies in isolated, in vitro perfused rectal gland tubules (RGT) have revealed that the basolateral membrane possesses a K⁺ conductive pathway. In the present study, we have utilized the patch clamp technique in RGT segments to characterize this pathway. The basolateral membrane was approached with patch pipettes at the open end of in vitro perfused segments [5]. Recordings were obtained in cell-attached as well as in excised inside-out patches. In cell-attached patches with the pipette filled with a KCl solution (274 mmol/l) and the bath containing NaCl shark Ringer (275 mmol/l), inward K⁺ currents (from pipette into cell) with a mean slope conductance of 123±26 pS (n=3) were observed. We were unable to generate outward K⁺ currents at high depolarizing (cell more positive) clamp voltages. This indicates inward rectification of this channel. To examine the rectification properties further, excised (inside out) patches were exposed to K⁺ concentration gradients, directed out of, as well as into the pipette. With NaCl in the pipette and KCl in the bath, K⁺ outward currents were observed. The current-voltage (IV) relation revealed Goldman-type rectification, with a mean single channel conductance of 185±28 pS (n=7) at high positive voltages (linear range of the IV curve). The single-channel permeability coefficient for K⁺ was 0.26±0.04 ·10^–12 cm³/s (n=7). In the reversed experiment (pipette KCl, bath NaCl), inward currents of similar kinetics and amplitude were obtained. The single channel conductance was 146±21 pS (n=7) at high negative voltages (linear range of the IV curve). The single channel permeability coefficient for K⁺ was 0.21±0.03·10^–12 cm³/s (n=7). We were not able to reverse the currents in any of these experiments, indicating that this channel is highly selective for K⁺ over Na⁺. In all three series of experiments, the kinetic appearance of the channels was similar. Bursts of activity were followed by interburst pauses. The open state was described by a single time constant of 3.0±0.2 ms, whereas the closed state was described by two time constants of 0.7±0.2 ms and 2.8±0.5 ms (n=8). It can be concluded that these channels permit K⁺ inward and outward currents. They are probably the equivalent of the basolateral K⁺ conductance as observed in a previous study [12]. Under physiological conditions a single channel conductance of some 20 pS is predicted from the present data. In cell-attached patches, with a high K⁺ concentration in the pipette, the channel behaves as an inward rectifier.Supported by Deutsche Forschungsgemeinschaft Gr 4808 and by NSF and NIH grants to the MDIBL. Parts of this study have been published in the Mount Desert Island Biol. Bulletin 1984, 1985.     

Development of agranular reticulum in Sertoli cells of the testis of the dogfish Squalus acanthias during spermatogenesis

Biochemical analyses of Squalus testis indicates that key enzymes involved with androgen production increase progressively from immature regions containing spermatogonia to mature regions in the late spermatid stage of maturation (Canick et al., 1983). In an effort to identify cells possessing the cytological characteristics of steroid production and to determine the structural correlates of the observed functional changes, we have carried out an electron microscopic study of Squalus testis. This report demonstrates that Sertoli cells contain a well-developed agranular reticulum, mitochondria with tubulovesicular cristae, and numerous lipid droplets. Moreover, as germ cells mature, there is an increase in abundance of agranular reticulum in the adjacent Sertoli cells. By the time of spermatid elongation, this has reached dramatic proportions and fills the Sertoli cell as a mass of tubules. These results lead us to conclude that the Sertoli cell is responsible for secretion of the increasing amounts of androgen during the spermatogenetic cycle in Squalus.