Vasopressin V1-antagonist increases the hydroosmotic response to arginine vasopressin in frog urinary bladder

V₁-antagonist, [Mca¹, D-Phe², Sar⁷] arginine vasopressin, at a concentration 0.5 nM to 5 nM increased hydroosmotic effect of 5 nM arginine vasopressin in frog urinary bladder. 1 nM V₁-antagonist [Mca¹,O-Me-Tyr²] arginine vasopressin does not changed hydroosmotic effect of 0.1 mM dibutyryl cAMP. We suggest that — both V₁- and V₂-receptors are present on epithelial cells of frog urinary bladder and that V₁-receptors are involved in the modulation or the hydroosmotic effect of arginine vasopressin — mediated by V₂-receptors.     

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule

Previous studies have demonstrated that prostaglandin E₂ (PGE₂) inhibits arginine vasopressin-(AVP)dependent adenosine 3,5-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE₂ was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30°C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35°C). In contrast to PGE₂, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean ±SEM): 1nM AVP=47.1±6.8 fmol · mm^–1· 4 min^–1; AVP + 0.3 M PGE₂=20.1±2.7, P<0.01 versus AVP; AVP + 10 nM PMA=42.0±4.7, NS versus AVP; AVP + 50 g/ml OAG=44.1±4.8. NS versus AVP, N= 5 experiments. However, 10 nM PMA prevented PGE₂-induced inhibition: AVP + PGE₂= 44.2±3.5% of the response to AVP and 90.3±3.2% without and with PMA respectively, N= 16. Similar results were obtained with either 50 g/ml OAG or 25 g/ ml DOG (AVP + PGE₂ + OAG=92.9±6.6% of the response to AVP, N= 8; AVP + PGE₂ + DOG=94.1 ±5.3%, N= 7). OAG, DOG, PMA or PMA + PGE₂ had no intrinsic agonist activity in the rat OMCD and the addition of an inactive phorbol ester did not prevent PGE₂-induced inhibition. SSP, 50 nM or 0.1 M, did not affect the inhibition due to PGE₂ but abolished the reversion by PMA of PGE₂-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 M FK + 0.3 M PGE₂= 37.7±6.2% of the response to FK; FK + PGE₂ + 10 nM PMA=89.5±6.7%; FK + PGE₂ + PMA + 0.1 M SSP=43.1±7.9%, N= 4. The inhibition induced by an ₂-adrenergic agonist, clonidine 1 M, was not blocked by the activation of PKC. In fura-2-loaded OMCD samples, 10nM PMA decreased by 63.3±5.0% and by 57.2±7.1% the peak and plateau phases, respectively, of the increase in intracellular calcium concentration ([Ca²⁺]_i) obtained with PGE₂ when compared to control responses in the same tubules (n=12) and did not affect the increase in [Ca²⁺]_i induced by 0.1 mM carbachol. It is concluded that: (1) in the rat OMCD the activation of PKC by PMA or analogues of diacylglycerol did not reproduce PGE₂-induced inhibition of AVP- or FK-dependent cAMP accumulation, but prevented specifically this inhibitory action; and (2) this reversion might be the consequence of the effect of PKC activation which impaired the rises in [Ca²⁺]_i induced by PGE₂.     

Differential effects of ADH on sodium,chloride, potassium,calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PD_te) and transepithelial resistance (R_te) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO ₃ ^– containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO ₃ ^– free Ringer solutions. In cTAL segments, AVP (10^–10 mol·l^–1) significantly increasedJ _Mg ²⁺ andJ _Ca ²⁺ from 0.39±0.08 to 0.58±0.10 and from 0.86±0.13 to 1.19±0.15 pmol·min^–1 mm^–1 respectively. NeitherJ _Na ⁺ norJ _Cl ^–, (J _Na ⁺: 213±30 versus 221±28 pmol·min^–1 mm^–1,J _Cl ^–: 206±30 versus 220±23 pmol·min^–1 mm^–1) nor PD_te (13.4±1.3 mV versus 14.1±1.9 mV) or R_te (24.6±6.5 cm² versus 22.6±6.4 cm²) were significantly changed by AVP. No significant effect of AVP on net K⁺ transport was observed. In mTAL segments, Mg²⁺ and Ca²⁺ net transports were close to zero and AVP (10^–10 mol·l^–1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone,J _Na ⁺ increased from 107±33 to 148±30 andJ _Cl ^– from 121±33 to 165±32 pmol·min^–1 mm^–1. The rise inJ _NaCl was accompanied by an increase in PD_te from 9.0±0.7 to 13.5±0.9 mV and a decrease in R_te from 14.4±2.0 to 11.2±1.7 cm². No K⁺ net transport was detected, either under control conditions or in the presence of AVP.To test for a possible effect of HCO ₃ ^– on transepithelial ion fluxes, mTAL segments were bathed and perfused with HCO ₃ ^– containing Ringer solutions. With the exception ofJ _Ca ²⁺ which was significantly different from zero (J _Ca ²⁺: 0.26±0.06 pmol·min^–1 mm^–1), net transepithelial fluxes of Na⁺, Cl^–, K⁺ and Mg²⁺ were unaffected by HCO ₃ ^– . In the presence of AVP,J _Mg ²⁺ andJ _Ca ²⁺ were unaltered whereasJ _NaCl was stimulated to the same extent as observed in the absence of HCO ₃ ^– . In conclusion our results indicate heterogeneity of response to AVP in cortical and medullary segments of the TAL segment, since AVP stimulates Ca²⁺ and Mg²⁺ reabsorption in the cortical part and Na⁺ and Cl^– reabsorption in the medullary part of this nephron segment.This study was supported by the Commission des communautés européennes, grant no. ST2J 00951 F(CD), and by Wissenschafts-ausschuß der Nato über den DAAD     

The effect of anti-diuretic hormone on the endolymphatic sac of the inner ear

 The anti-diuretic hormone vasopressin (AVP) regulates water excretion from the kidney by increasing the water permeability of the collecting duct. AVP binds to V₂-receptors and induces the translocation of aquaporin-2 water channels (AQP-2) into the apical plasma membrane of principal cells. By this mechanism AVP controls water reabsorption in the kidney. The effects of AVP on the endolymphatic sac (ES) of the inner ear, which is thought to mediate reabsorption of endolymph, were investigated. Both the V₂-receptor and the AQP-2 water channel were found to be expressed in the ES epithelium. In the ES AVP binds to receptors most probably of the V₂-subtype. Application of AVP to organotypically cultured ES inhibits membrane turnover in ribosomal-rich cells of the ES epithelia, which is thought to mediate translocation of AQP-2 into the surface membrane. This suggests that AVP has contrasting effects in the inner ear and kidney, which may be physiologically useful for maintaining endolymphatic pressure during severe hypovolemia. Animal experiments show that AVP causes endolymphatic hydrops after systemic application to guinea-pigs, which suggests a causal role for the increased AVP levels found in humans suffering from Ménière’s disease. Received: 3 July 1998 / Accepted: 13 July 1998     

[Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V₂ receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V₁ receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP ₃) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP ₃, which peaked at 200%–250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37°C, and declined to basal level after 5–10 min. Dose/response curves for InsP ₃, established at 30°C and 37° C using radioimmunoassay, showed a half-maximal stimulation of InsP ₃ production at about 1 nmol/l AVP and a maximal response at 10 nmol/l. Similar values were obtained for the response to AVP in terms of cAMP accumulation. InsP ₃ content in the presence of higher concentrations of AVP (1 mol/l) was significantly lower (P<0.001) than in the presence of 10 nmol/l AVP, giving a bell-shaped appearance to the dose/response curve at 37° C but not at 30° C. The V₂ receptor agonist, 1-deamino-[8DArg]vasopressin (dAVP) did not stimulate the formation of InsP ₃, and the V₁ receptor antagonist d(CH₂)₅[Tyr(Me)²]AVP inhibited AVP-induced InsP ₃ formation, which therefore appeared to be mediated by V₁ receptor occupancy. Under the same conditions, AVP also induced the formation of diradylglycerol via V₁ receptor activation, with an analogous dose/response curve. These results show that AVP, in addition to its well-known action through V₂ receptors, also acts on the mouse mTAL through a V₁-mediated stimulation of phospholipase C. Cyclic AMP controls this transduction pathway: dAVP (10 nmol/l), dibutyryl-cAMP (1 mmol/l and 0.1 mmol/l) and forskolin (1 mol/l) decreased the InsP ₃ formation induced by AVP. Dibutyryl-cAMP itself at 37°C also reduced the diglyceride content.     

Chloride and potassium conductances of cultured human sweat ducts

The purpose of this study was to characterize the ion conductances, in particular those for Cl^– and K⁺, of human sweat duct cells grown in primary culture. Sweat duct cells from healthy individuals were grown to confluence on a dialysis membrane, which was then mounted in a mini-Ussing chamber and transepithelial and intracellular potentials were measured under open-circuit conditions. Under control conditions the epithelia developed mucosa-negative transepithelial potentials, V _te, of about –10mV. The apical membrane potential, V _a, was –25 mV to –30 mV (n=97) in most cells, but several cells had a higher potential of about –55 mV (n=29). Mucosal amiloride (10 mol/l) hyperpolarized V _a from –31±1 mV to a new sustained level of –46±2 mV (n=36). These changes were accompanied by increase in the fractional resistance of the apical membrane, fR _a, and decreases of V _te and the equivalent short-circuit current, I _sc. In amiloride-treated tissues an increase in mucosal K⁺ concentration (5 mmol/l to 25 mmol/l) depolarized V _a by 5±1 mV (n=8), while the same step on the serosal side depolarized V _a by 20±2 mV (n=8). A Cl^– channel blocker 3,5-dichloro-diphenylamine-2-carboxylate DCl-DPC; 10 mol/l) depolarized V _a by 5±1 mV (n=6), an effect that was lost after amiloride application. The blocker had no effect from the serosal side. Reduction of mucosal Cl^– (from 120 to 30 or 10 mmol/l) depolarized V _a by 9–11 mV (n=35), an effect that was often followed by a secondary hyperpolarization of 10–30 mV (n=27). Isoproterenol (5 mol/l) increased the V _a responses to low Cl^– such that the depolarizing response was increased from 10±1 mV to 19±2 mV (n=8); the hyperpolarizing response seemed to be reduced. With changes in Cl^– concentration on the serosal side, V _a remained relatively constant at –25 mV, while V _te decreased from –8 mV to–3 mV; hence, V _bl depolarized by about 5 mV. Taken together, our results show that the human sweat duct epithelium possesses Na⁺, K⁺ and Cl^– conductances on the luminal membrane and Cl^– and K⁺ conductances on the basolateral membrane. The Cl^– conductances on the luminal membrane is sensitive to DCl-DPC, and can be activated by isoproterenol. The small K⁺ conductance on the luminal membrane could account for some K⁺ secretion in sweat glands.     

Effect of secretin and inhibitors of HCO3 −/H+ transport on the membrane voltage of rat pancreatic duct cells

The aim of the present study was to study the effect of secretin on the electrophysiological response of pancreatic ducts. Furthermore, we investigated the effects of lipid-soluble buffers and inhibitors of HCO₃ ^–/H⁺ transport. Ducts obtained from fresh rat pancreas were perfused in vitro. Secretin depolarized the basolateral membrane voltage, V _bl, by up to 35 mV (n=37); a halfmaximal response was obtained at 3×10^–11 mol/l. In unstimulated ducts a decrease in the luminal Cl^– concentration (120 to 37 mmol/l) had a marginal effect on V _bl, but after maximal secretin stimulation it evoked a 14±2 mV depolarization (n=6), showing that a luminal Cl^– conductance G _Cl- was activated. The depolarizing effect of secretin on V _bl was often preceded by about a 6 mV hyperpolarization, most likely due to an increase in the basolateral G _K+. Perfusion of ducts with DIDS (4,4 — diisothiocyanatostilbene — 2,2 — disulphonic acid, 0.01 mmol/l) or addition of ethoxzolamide (0.1 mmol/l) to the bath medium diminished the effect of secretin. Acetate or pre-treatment of ducts with NH₄ ⁺/NH₃ (10 mmol/l in the bath) depolarized the resting V _bl of –65±2 mV by 16±4 mV (n=7) and 19±3 mV (n=10), respectively. The fractional resistance of the basolateral membrane (FR _bl) doubled, and the depolarizing responses to changes in bath K⁺ concentrations (5 to 20 mmol/l) decreased from 22±1 to 11±2 mV. The Na⁺/H⁺ antiporter blocker EIPA (5-[N-ethyl-N-isopropyl]-amiloride, 0.1 mmol/l) also depolarized V _bl by 10±1 mV, FR_bl increased and the response to K⁺ concentration changes decreased (n=7). Effects of EIPA and ethoxzolamide on V _bl were greater in ducts deprived of exogenous HCO₃ ^–/CO₂. Taken together, the present study shows that secretin increased the basolateral G _K+ and the luminal G _Cl-. The depolarizing effect of secretin was diminished following inhibition of HCO₃ ^– transport (DIDS), or HCO₃ ^–/H⁺ generation (ethoxzolamide). Manoeuvres that presumably led to lowered intracellular pH (NH₄ ⁺/NH₃ removal, acetate, EIPA) decreased the basolateral G _K+. The present data support our previously published model for pancreatic HCO₃ ^– secretion, and indicate that the basolateral membrane possesses a pH-sensitive G _K+.     

Electrophysiological investigation of microdissected gastric glands of bullfrog II. Basolateral membrane properties in the presence of histamine

Following the technical approach described in the preceding publication we have investigated if, and how, stimulation of gastric HCl secretion affects the basolateral ion transport properties of oxyntopeptic cells of Rana catesbeiana stomach. To this end microdissected gastric glands were punctured with conventional or H⁺-sensitive glass microelectrodes and the effects of changing bath ion concentrations on the cell membrane potential (V _b) and cell pH (pH_i) were determined. Except for a transient alkalinization, histamine (0.5 mmol/l) did not significantly affect V _b or pH_i. The latter averaged 7.18±0.03 (mean±SEM, n=5) under resting conditions (0.1 mmol/l cimetidine) and 7.21±0.07 (n=5) in the presence of histamine. In addition, neither the initial velocity nor the final steady-state value of the cell alkalinization following a 101 reduction of bath Cl^– concentration changed in the presence of histamine, and the same holds true for the cell acidification following a 101 reduction of bath HCO₃ ^– concentration. These observations indicate that the basolateral Cl^–/HCO₃ ^– exchanger was not stimulated by histamine, and that no other base transporters were activated. By contrast, the V _b response to elevation of bath K ⁺ concentration decreased, and so did the initial depolarizing V _b response to bath Cl^– substitution, while the secondary hyperpolarizing response increased. The latter observations are compatible with the notion that stimulation by histamine reduced a pH-insensitive part of the basolateral K⁺ conductance and reduced also the basolateral Cl^– conductance.     

Isolated perfused rabbit colon crypts: stimulation of Cl− secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (V _b1) was –65±1 mV (n=240). Bath Ba²⁺ (1 mmol/ l) and verapamil (0.1 mmol/l) depolarized V _b1 by 21±2 mV (n=7) and 31±1 (n=4), respectively. Lowering of bath Cl^– concentration hyperpolarized V _b1 from –69±3 to –75±3 mV (n=9). Lowering of luminal Cl^– concentration did not change V _b1. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on V _b1 in non-stimulated crypts. Forskolin (10^–6 mol/l) in the bath depolarized V _b1 by 29±2 mV (n=54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of V _b1 was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized V _b1 significantly and concentration dependently with a potency sequence of bumetanide > piretanide furosemide. Lowering bath Cl^– concentration hyperpolarized V _b1. Lowering of luminal Cl^– concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of V_b1 by 7±2 mV (n=10). We conclude that V_b1 of rabbit colonic crypt cells is dominated by a K⁺ conductance. Stimulation of the cells by forskolin opens a luminal Cl^– conductance. Basolateral uptake of Cl^– occurs via a basolateral Na⁺ : 2Cl^– : K⁺ cotransport system.     

Effect of NH4 +/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K⁺. In excised membrane patches the luminal K⁺ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (V _m) in slow-whole-cell (SWC) recordings and the open probability (P _o) of K⁺ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of V _m can be recorded. The pipette solution contained K⁺ 125 mmol/l and Cl^– 32 mmol/l. Intracellular pH was determined by 2,7 bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH₄ ⁺/NH₃ to the bath. In the presence of NH₄ ⁺/NH₃ intracellular pH acidified by 0.53±0.11 units (n=7). Inhibition of the Na⁺2Cl^– K⁺ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62±0.14 units (n=7). In SWC experiments V _m of TAL cells was -72±1 mV (n=70). NH₄ ⁺/NH₃ depolarised V _m by 22±2 mV (n=25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH₄ ⁺ from 24±3 mV to 7±3 mV. Under control conditions the single-channel conductance of TAL K⁺ channels in CAN experiments was 66±5 pS and the reversal voltage for K⁺ currents was 70±2 mV (n=35). The P _o of K⁺ channels in CAN patches was reduced by NH₄ ⁺/NH₃ from 0.45±0.15 to 0.09±0.07 (n=7). NH₄ ⁺/NH₃ exposure depolarised the zero current voltage of the permeabilised patches by-9.7±3.6 mV (n=5). The results show that TAL K⁺ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH₄ ⁺/NH₃ exposure at concentrations which are physiologically relevant because Na⁺2Cl^–K⁺(NH₄ ⁺) cotransporter-mediated import of NH₄ ⁺ exceeds the rate of NH₃ diffusion into the TAL. K⁺ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH₄ ⁺ uptake occurs by the Na⁺2Cl^–K⁺ cotransporter. The findings that, in the presence of NH₄ ⁺/NH₃ and furosemide, V _m is not completely repolarised and that K⁺ channels are not activated suggest that the respective K⁺ channels may in addition to their pH regulation be inhibited directly by NH₄ ⁺/NH₃.     

Swelling of rat mesangial cells induces a Ca2+-dependent Cl− conductance

Membrane voltage (V _m) and ion currents of rat mesangial cells in primary culture were measured with the patch-clamp technique in the fast whole-cell configuration.V _m was –44 ± 1 mV (_n = 138). A reduction of the osmolality from 290 to 190 mosmol/kg depolarizedV _m from –44 ± 1 to –29 ± 1 mV (_n = 118) and increased the inward and outward conductances (Gm) from 14±2 to 39 ± 4 nS and 13±2 to 37 ± 4 nS (_n = 84), respectively. During the hypotonicity-induced depolarization the cell capacitance increased significantly from 33 ± 3 to 42 ± 4 pF (_n = 40). The effect of hypotonic cell swelling onV _m was increased in a bath with a reduced extracellular Cl^– of 32 mmol/l (by 71 ± 4%,_n = 23), indicating that a Cl^– conductance was activated. The permselectivity of this conductance was I^– Br^– > Cl^–. TheV _m response was not affected in the presence of a reduced extracellular Na⁺ of 5 mmol/l (n = 13) and was inhibited in a solution with reduced extracellular Ca²⁺ concentration (by 63 ± 9%,n = 14). In microfluorescence measurements with the Ca²⁺-sensitive dye fura-2 hypotonic cell swelling induced a sustained increase of the intracellular Ca²⁺ activity, [Ca²⁺]_i (_n = 19). The increase of [Ca²⁺]_i was completely inhibited when the extracellular solution was free of Ca²⁺. TheV _m response to hypotonic cell swelling was not attenuated in the presence of the L-type Ca²⁺ channel blockers nicardipine (n = 5), nifedipine (n = 5) and verapamil (n = 5) (all at 1 mol/l). The data indicate that in rat mesangial cells, osmotic swelling induces a Ca²⁺ influx from extracellular space. This Ca²⁺ influx activates a Cl^– conductance resulting in a depolarization ofV _m. The enhanced Cl^– conductance may lead to KCl extrusion and hence regulatory volume decrease.     

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.     

Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca²⁺ ([Ca²⁺]_i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca²⁺]_i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca²⁺]_i in a dose-dependent manner. [Ca²⁺]_i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10^–6M, and to 766±162 nmol/l with AVP, 10^–7 M. The increment in [Ca²⁺]_i by both hormones was by intracellular Ca²⁺ release and entry through plasma membrane Ca²⁺ channels. 8-Bromoadenosine-3, 5-cyclic monophosphate (8-BrcAMP), 10^–4M, increased [Ca²⁺]_i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 mol), ([Ca²⁺]_i changes: 86±4 to 390±29 nmol/l) and removal of bath Ca _o ²⁺ , ([Ca²⁺]_ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca²⁺]_i. Thapsigargin, 1.5 mol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca²⁺ transients, and partially inhibited PTH-mediated Ca²⁺ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca²⁺ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca²⁺]_i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca²⁺ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca²⁺ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca²⁺ and be modulated by different pathways which may have diverse effects on cTAL function.     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.     

Tolerance to haemorrhage during vasopressin antagonism and/or captopril treatment in conscious sheep

The effect of separate and combined blockade of vasopressin (AVP) V₁-receptors and angiotensin II formation on resistance to a slow venous haemorrhage (0.7 ml kg^-1 min^-1) was studied in six conscious adult sheep by bleeding to the point of an abrupt fall in the mean systemic arterial pressure (MSAP). Intravenous administration of the V₁-receptor antagonist [d(CH₂)₅Tyr(Me)AVP] (10 μg kg^-1) and/or the angiotensin I converting enzyme inhibitor captopril (20 mg+1 mg h^-1) did not cause any significant haemodynamic changes in the normovolaemic animal. The volume of haemorrhage necessary to induce acute hypotension (MSAP < 50 mmHg) was significantly smaller after AVP blockade alone (13.8±0.7 ml kg^-1; P < 0.01) but not after captopril treatment (14.7±1.6 ml kg^-1; n.s.) compared to control animals receiving no drug treatment (16.8±0.6 ml kg^-1). The combined treatment with the AVP antagonist and captopril caused a further decrease in tolerance to haemorrhage (9.4±1.2 ml kg^-1; P < 0.001). Blockade of AVP V₁-receptors was associated with an attenuated increase in systemic vascular resistance immediately after the end of haemorrhage, concomitant with an accentuated lowering of the central venous pressure. In contrast, captopril treatment decreased the degree of vasoconstriction mainly during the second half of the post-haemorrhage observation period of 1 hour. It is concluded that both AVP and angiotensin II contribute to the maintenance of the MSAP during haemorrhage in conscious sheep. During the spontaneous recovery after hypotensive blood loss, a vasoconstrictor effect of AVP is evident mainly during the initial 15 min, whereas at later stages angiotensin II appears to be of relatively greater importance.     

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig)

The effect of arginine-vasopressin (AVP, 0.1 M) on elementary Ca²⁺ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl₂ as the charge carrier. At a constant potential of –30 mV, bath applied AVP increased the channel openness (NP _o) by a factor of 4.7±3.0 (mean±SD, n=9), the effect resulted from an increase in the frequency of opening (factor 2.5±0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (_o1=0.8±0.4 ms), a double exponential distribution was obtained during AVP (_o1=0.8±0.5 ms, _o2=7.5±0.7 ms). The Ca²⁺ agonist BAYk8644 (1 M) changed the open time distribution similarly to AVP (_o1=1.0±0.5 ms, _o2=9±2.8 ms). With 1 M BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0±1 (n=6). The results are compatible with the idea that AVP can change the gating of L-type Ca²⁺ channels from mode 1 to mode 2.     

Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonie epithelial cells (HT-29/B6)

We investigated the inhibitory action of K⁺ channel blockers on carbachol-stimulated Ca²⁺ entry into human Cl^–-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K⁺ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 M) caused a clearly biphasic intracellular calcium (Ca_i response: Ca_i was stimulated from resting levels (85±3 nM, n=100) to a sudden transient peak (821±44 nM) followed by a sustained plateau (317±12 nM). The maintained elevation was dependent on external Ca²⁺ and represented a new steady state between Ca²⁺ entry and exit across the plasma membrane. A monophasic Ca²⁺ response was induced in the absence of external Ca²⁺ and after the initial peak Ca_i returned to baseline. The Ca_i plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 M) or the inorganic Ca²⁺ channel blocker lanthanum (effective concentration for 50% inhibition of Ca₁ plateau EC₅₀=68±18 nM), but it was unaffected by the organic Ca²⁺ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K⁺ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca²⁺ entry. The Ca_i plateau was calculated to be 50% inhibited by barium (96±2 M), lidocaine (74±3 M) and NPPB (27±10 M). The Ca_i plateau was transiently increased by 1 M and 10 M NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl^– channels (so that the membrane potential approached V _K). The resting Ca_i was transiently increased by 50 M and 300 M NPPB to 308±13 nM and 447±153 nM, respectively, suggesting that NPPB induced a Ca²⁺ release from internal Ca stores. We conclude that carbachol-stimulated Ca²⁺ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Ca_i levels to K⁺ channel blockers indicates that there are feedback relationships among rates of Ca²⁺ entry, activity of Ca²⁺-activated K⁺ and Cl^– channels and membrane potential.     

Involvement of arginine vasopressin in endogenous central histamine-induced reversal of critical haemorrhagic hypotension in rats

Objective and design:Histamine is a potent stimulator of arginine vasopressin (AVP) release and therefore, the role of AVP was studied in the reversal of critical haemorrhagic hypotension induced by endogenous central histamine after inhibition of histamine N-methyltransferase (HNMT) activity in rats. Material:In 48 ethylurethane-anaesthetised male Wistar rats cardiovascular parameters and plasma hormone concentrations were measured. Treatment:Haemorrhage-shocked rats with mean arterial pressure (MAP) 20–25 mmHg were injected intracerebroventricularly (icv) with HNMT inhibitor metoprine (20 g) after pre-treatment with V_1a, V_1b and V₂ receptor antagonists – [-mercapto-,cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg⁸]AVP (10 g/kg; iv), SSR149415 (10 mg/kg; ip) and [adamantaneacetyl¹,O-Et-D-Tyr²,Val⁴, aminobutyryl⁶,Arg^8,9]AVP (10 g/kg; iv), respectively, or saline. Methods:MAP, heart rate (HR) and regional haemodynamics were monitored within 2 h after treatment or to death if it occurred earlier. Plasma hormone concentrations were measured using enzyme immunoassays. ANOVA followed by Neuman-Keules test, and Fishers exact test were used to compare the results. Results:Metoprine produced a long-lasting increase in MAP, HR, renal, hindquarters and mesenteric blood flows, and a 100% survival at 2 h (P < 0.05 vs. the control group). The action was associated with increased plasma AVP concentration (587.5 ± 98.9 vs. 387.3 ± 125.2 pg/ml; P < 0.05) in comparison to the control group as measured at 20 min after treatment. V_1a, but not V_1b and V₂, receptor antagonist inhibited metoprine-induced haemodynamic effects, with no influence on survival at 2 h. SSR149415 did not influence ACTH and adrenaline plasma concentrations in the metoprine-treated group. Conclusion:AVP, acting via V_1a receptors, is involved in endogenous central histamine-induced reversal of critical haemorrhagic hypotension in rats.Received 9 October 2003; returned for revision 2 December 2003; accepted by A. Falus 23 December 2003