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     

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.     

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)     

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 ofSqualus acanthias

The present study examines the influences of pH and Ca²⁺ and several putative inhibitors on the basolateral K⁺ channel of the rectal gland ofSqualus acanthias. Excised membrane patches were examined using the patch clamp technique. It is shown that reduction of the calcium activity on the cytosolic side to less than 10^–9 mol/l has no detectable inhibitory effect on this channel. Conversely, increase in calcium activity to some 10^–3 mol/l reduced the activity of this channel. Variations in cytosolic pH had only a moderate effect on the current amplitude: alkalosis by one pH unit increased and acidosis reduced the single current amplitude by some 15%. Several inhibitors were tested in excised patches when added to the cytosolic side. Ba²⁺ (5·10^–3 mol/l), quinine (10^–3 mol/l), quinidine (10^–4 mol/l), lidocaine (1 mmol/l), tetraethylammonium (10 mmol/l), Cs⁺ (10 mmol/l), and Rb⁺ (20 mmol/l) all blocked this K⁺ channel reversibly. We conclude that the basolateral K⁺ channel of the rectal gland is distinct from other epithelial K⁺ channels inasmuch as it is not stimulated by Ca²⁺ directly, but that it is qualitatively similar to many other known K⁺ channels with respect to its sensitivity towards blockers.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the Mount Desert Island Biological Laboratory     

Cl−-channels in the apical cell membrane of the rectal gland “induced” by cAMP

Isolated rectal gland tubules (n1000) of dogfish (Squalus Acanthias) were perfused in vitro. Individual channels in the apical and basolateral cell membrane were recorded with the patch clamp method. K⁺-channels were present in excised membrane patches of the basolateral membrane in stimulated (dbcAMP + forskolin + adenosine) and in nonstimulated state. Cl^–-channels were found only in patches of the apical cell membrane when the tubule was stimulated. Cell attached recordings and simultaneous transepithelial PD measurements were obtained while the segment was stimulated. It is shown that concomitant with the increase in lumen negative PD silent membrane patches of the apical cell membrane suddenly develop Cl^–-channel activity. It is concluded that stimulation of rectal gland tubules activates Cl^–-channels in the apical cell membrane.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-2 Correspondence to first author at the above address     

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current (= transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 · 10^–7 mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from –78 to –81 mV and from –72 to –81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 · 10^–5 mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 cm², by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse.The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na⁺ 2Cl^–K⁺ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide. The weaker effect of torasemide from the peritubular side can, at least in part, be explained as an interference with chloride channels present in the basolateral membrane. Torasemide is also structurally related to chloride channel blockers such as diphenylamine-2-carboxylate.Supported by Deutsche Forschungsgemeinschaft DFG Gr 480/6-4 and 6-5     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

Mechanism of Cl− transport in eel intestinal brush-border membrane vesicles

Cl^– transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. ³⁶Cl^– uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K⁺ diffusion potential obtained by imposing a K⁺ concentration gradient (C _out>C_in) in the presence of valinomycin, (c) an inwardly directed H⁺ ion concentration gradient. The membrane-potential-driven Cl^– transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropylamino)-benzoic acid. Arachidonic acid also inhibited Cl^– uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na⁺ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl^– influx was the same in the presence of inwardly directed gradients of Li⁺, Na⁺ or K⁺, arguing against the presence of Na⁺-Cl^–, as well as K⁺-Cl^– cotransport. The absence of a significant contribution of the Na⁺-K⁺-2Cl^– cotransport mechanism to the Cl^– uptake in seawater eel intestinal BBMV was indicated from the observations that Cl^– uptake was not stimulated by the simultaneous presence of inwardly directed Na⁺ and K⁺ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na⁺ and K⁺. Furthermore, no evidence for the dependence of Cl^– uptake on the Na⁺ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K⁺ and valinomycin. The finding that the Cl^– uptake in the presence of a H⁺ gradient was not inhibited by 1 mM SITS and was significantly reduced in the presence of [K⁺]_in=[K⁺]_out and valinomycin, suggests that no anion exchanger is present in our experimental system. We conclude that Cl^– uptake in eel intestinal BBMV does not occur via an electroneutral Na⁺-dependent Cl^– transport mechanism (either cotransport or double exchange) and is realized by a Cl^– conductance.     

Membrane potential measurements of transitional cells from the crista ampullaris of the Gerbil

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was –87±1 mV (n=103). This value is not significantly different from –83±2 mV (n=24) measured in Cl^– free solution. [Cl^–] steps from 150 to 15 mmol/l (n=24) depolarized the membrane by about 2 mV, indicating a minor Cl^– conductance. The transference number for K⁺ was 0.75±0.01 (n=79) obtained from the PD responses to K⁺ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K⁺] steps was reduced by Ba²⁺ (2·10^–6 to 10^–3 mol/l), quinidine (10^–3 mol/l), quinine (10^–3 mol/l), Rb⁺ (20 mmol/l), Cs⁺ (20 mmol/l), NH₄ ⁺ (20 mmol/l) and Tl⁺ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba²⁺ in the presence of 3.6 mmol/l K⁺ was shifted to the right by approximately three decades in the presence of 25 mmol/l K⁺ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl^–. Transitional cells were depolarized by ouabain, suggesting the presence of (Na⁺+K⁺-ATPase.This work was supported by grants from the Deafness Research Foundation to PhW and the National Institute of Health (NS 19490) to DCM     

Mechanisms of chloride influx during KCl-induced swelling in the chicken retina

An increase in extracellular KCl ([KCl]_o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl_o-induced retinal swelling were investigated in the present study, with emphasis on the Cl^– transport mechanisms. Increasing [KCl]_o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]_o was biphasic, with one component from 5 to 35 mM [KCl]_o and another from 35 to 100 mM. As Cl^– omission prevented swelling in all conditions, the effect of cotransporter or Cl^– channel blockers was examined to investigate the mechanisms of Cl^– influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]_o 25 mM (K25), and by 14–17% at [KCl]_o 54 mM (K54). The Cl^– channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90–95% at K54 (NPPB IC₅₀ 60.7 µM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC₅₀ 173.9 µM). Na⁺ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl^– influx at K25 and of Cl^– channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Müller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

 NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl^– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl^– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl^– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 μmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G _m). The membrane capacitance (C _m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C _m correlated with the increase in G _m with a slope of 51 fF/nS. Next the effect of furosemide (500 μmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na⁺2Cl^–K⁺ uptake and to reduce cell volume but not membrane trafficking of Cl^– channels. Furosemide reduced G _m slightly (due to the fall in cytosolic Cl^– concentration) and C _m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC _m/ΔG _m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 μmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G _m or C _m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C _m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl^– channels in these cells. Received: 11 March 1998 / Received after revision 15 April 1998 / Accepted: 20 April 1998     

Stimulation of colonic anion secretion by monochloramine: action sites

During inflammatory bowel disease, reactive oxygen metabolites are released by phagocytes reacting with intraluminal NH₃ to produce monochloramine (NH₂Cl). NH₂Cl is assumed to play role in the pathogenesis of inflammation-associated diarrhoea, as it is able to induce intestinal secretion. The aim of the present study was to determine the action sites of NH₂Cl in rat colonic epithelium with Ussing chamber and fura-2 experiments. In intact mucosa, NH₂Cl (5·10^–6–10^–4 mol·l^–1) evoked a concentration-dependent increase in short-circuit current (I_sc), consistent with the induction of anion secretion, as demonstrated by anion substitution and transport blocker experiments. When the apical membrane was permeabilised by the ionophore nystatin, two basolateral action sites of NH₂Cl (5·10^–5 mol·l^–1) could be identified, i.e. an increase in the K⁺ conductance and a stimulation of the Na⁺–K⁺ pump. When tissues were basolaterally depolarised by a high K⁺ concentration, the stimulation of an apical Cl^– conductance by NH₂Cl was observed. In isolated colonic crypts loaded with the Ca²⁺-sensitive fluorescent dye fura-2, NH₂Cl (5·10^–5 mol·l^–1) evoked an increase in the intracellular Ca²⁺ concentration. This increase was independent from the presence of Ca²⁺ in the extracellular medium, but was inhibited by blockade of intracellular sarcoplasmatic, endoplasmatic Ca²⁺-ATPases with cyclopiazonic acid (10^–5 mol·l^–1). The NH₂Cl-evoked Ca²⁺ release was sensitive against inhibition of ryanodine receptors with ruthenium red (5·10^–5 mol·l^–1) and against inhibition of inositol-1,4,5-trisphosphate (IP₃) receptors with 2-aminoethoxydiphenylborate (10^–4 mol·l^–1). Both blockers also inhibited the NH₂Cl-induced increase in I_sc. These results indicate that an intracellular Ca²⁺ release via ryanodine and/or IP₃ receptors is involved in oxidant stimulation of anion secretion in rat colon.     

The “small” conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

Besides the larger Cl^– channel with a single channel conductance of about 45 pS, a small channel was observed in the luminal membrane of the dogfish rectal gland [9]. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11±1 pS (n=21), and its current-voltage relationship was linear in the voltage range+90 to –90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl^– over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na⁺. The Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this small conductance Cl^– channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl^– channels, which differ markedly in their characteristics.Supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the MDIBL     

Evidence from O2 uptake measurements for Na+−K+−2 Cl− co-transport in the rabbit submandibular gland

There is evidence that the production of primary saliva by acinar cells is a consequence of Na⁺–Cl^– co-transport but more recently it has been proposed that in fact Na⁺–K⁺–2 Cl^– co-transport is responsible. The latter would be energetically more efficient and the present experiments were designed to measure the stoichiometry of acinar secretion in order to distinguish between these two mechanisms.Submandibular salivary glands from anaesthetised rabbits were isolated vascularly and oxygen consumption measured from the oxygen content of arterial inflow and venous effluent blood and the total flow through the gland. Measurements were made in the steady-state at rest and during different secretion rates induced by parasympathetic nerve stimulation. The rate of sodium transport across the acinar and ductal epithelium was determined from plasma and salivary sodium concentration and salivary flow rate.Multiple regression analysis of this data showed that 22.1 mol Na⁺ was secreted per mol O₂ consumed while 11.9 mol Na⁺ was reabsorbed per mol O₂ consumed. Since acinar secretion is energetically about twice as efficient as ductal absorption, a mechanism for Na⁺ transport other than that for tight epithelia must be involved. Na⁺K⁺–2 Cl^– co-transport is thus more likely than Na⁺–Cl^– and it is suggested that Na⁺–K⁺–2 Cl^– co-transport is the main mechanism involved in salivary acinar secretion.     

The role of cytosolic Ca2+ in the secretion of NaCl in isolated in vitro perfused rectal gland tubules of Squalus acanthias

 In many exocrine glands cytosolic Ca²⁺ ([Ca²⁺]_i) plays a pivotal role in stimulation-secretion coupling. In the rectal gland of the dogfish Squalus acanthias this appears not to be the case and it is believed that secretion is mainly controlled by the Cl^– conductance of the luminal membrane. We have examined this question in a study of isolated in vitro perfused rectal gland tubules (RGT). Three types of measurements were performed: (1) measurements of [Ca²⁺]_i by the fura-2 technique; (2) measurements of transepithelial electrical parameters, i.e. transepithelial voltage (V _te), transepithelial resistance (R _te), the equivalent short-circuit current (I _sc) and the voltage across the basolateral membrane (V _bl), and (3) whole-cell patch-clamp measurements of cellular voltage (V _m), conductance (G _m) and membrane capacitance (C _m). The data indicates that carbachol (CCH) increases [Ca²⁺]_i by increasing store release and Ca²⁺ influx. Other agonists, producing cytosolic cAMP, also increased [Ca²⁺] by enhancing Ca²⁺ influx. CCH hyperpolarized these cells and enhanced G _m significantly. The effect of CCH on V _te and I _sc was most marked under control conditions and disappeared in RGT otherwise stimulated by agonists that lead to cAMP production. It is concluded that [Ca²⁺]_i plays a major role in the stimulation of NaCl secretion in RGT by enhancing the basolateral K⁺ conductance. cAMP-producing agonists enhance [Ca²⁺]_i by increased Ca²⁺ influx. CCH releases Ca²⁺ from respective stores. CCH, unlike the cAMP-producing agonists, only increases basolateral K⁺ conductance. It modulates secretion especially under conditions in which the cAMP pathway is not fully activated. Received: 25 November 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Water and solute secretion by the choroid plexus

The cerebrospinal fluid (CSF) provides mechanical and chemical protection of the brain and spinal cord. This review focusses on the contribution of the choroid plexus epithelium to the water and salt homeostasis of the CSF, i.e. the secretory processes involved in CSF formation. The choroid plexus epithelium is situated in the ventricular system and is believed to be the major site of CSF production. Numerous studies have identified transport processes involved in this secretion, and recently, the underlying molecular background for some of the mechanisms have emerged. The nascent CSF consists mainly of NaCl and NaHCO₃, and the production rate is strictly coupled to the rate of Na⁺ secretion. In contrast to other secreting epithelia, Na⁺ is actively pumped across the luminal surface by the Na⁺,K⁺-ATPase with possible contributions by other Na⁺ transporters, e.g. the luminal Na⁺,K⁺,2Cl⁻ cotransporter. The Cl⁻ and HCO₃ ⁻ ions are likely transported by a luminal cAMP activated inward rectified anion conductance, although the responsible proteins have not been identified. Whereas Cl⁻ most likely enters the cells through anion exchange, the functional as well as the molecular basis for the basolateral Na⁺ entry are not yet well-defined. Water molecules follow across the epithelium mainly through the water channel, AQP1, driven by the created ionic gradient. In this article, the implications of the recent findings for the current model of CSF secretion are discussed. Finally, the clinical implications and the prospects of future advances in understanding CSF production are briefly outlined.     

Control of rectal gland secretion by blood acid-base status in the intact dogfish shark (Squalus acanthias)

In order to address the possible role of blood acid-base status in controlling the rectal gland, dogfish were fitted with indwelling arterial catheters for blood sampling and rectal gland catheters for secretion collection. In intact, unanaesthetized animals, isosmotic volume loading with 500 mmol L-1 NaCl at a rate of 15 mL kg-1 h-1 produced a brisk, stable rectal gland secretion flow of about 4 mL kg-1 h-1. Secretion composition (500 mmol L-1 Na+ and Cl-; 5 mmol L-1 K+; <1 mmol L-1 Ca2+, Mg2+, SO(4)2-, or phosphate) was almost identical to that of the infusate with a pH of about 7.2, HCO3- mmol L-1<1 mmol L-1 and a PCO2 (1 Torr) close to PaCO2. Experimental treatments superimposed on the infusion caused the expected disturbances in systemic acid-base status: respiratory acidosis by exposure to high environmental PCO2, metabolic acidosis by infusion of HCl, and metabolic alkalosis by infusion of NaHCO3. Secretion flow decreased markedly with acidosis and increased with alkalosis, in a linear relationship with extracellular pH. Secretion composition did not change, apart from alterations in its acid-base status, and made negligible contribution to overall acid-base balance. An adaptive control of rectal gland secretion by systemic acid-base status is postulated-stimulation by the "alkaline tide" accompanying the volume load of feeding and inhibition by the metabolic acidosis accompanying the volume contraction of exercise.