Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart

 We have previously shown activation of α₁-adrenergic receptors increases Na⁺-K⁺ pump current (I _p) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on I _p. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased I _P at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K _0.5) was 6 μM at 15 nM cytosolic [Ca²⁺] ([Ca²⁺]_i) and13 nM at 314 nM [Ca²⁺]_i. The maximal increase at either [Ca²⁺]_i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC₈), increased I _p similarly. The effect of PMA on I _P was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α₁-adrenergic agonist effects both were sensitive to [Ca²⁺]_i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased I _p uniformly at all voltages. However, they differed in that α₁-activation caused a maximum increase of 15% vs 30% via PMA, and α₁-effects were less sensitive to [Ca²⁺]_i than PMA effects. These results demonstrate that activation of PKC causes an increase in I _p in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α₁-adrenergic activation to I _p is entirely through PKC, however α₁-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist. Received: 21 August 1998 / Received after revision: 7 December 1998 / Accepted: 11 December 1998     

Direct effects of tolbutamide on mitochondrial function, intracellular Ca2+ and exocytosis in pancreatic β-cells

 Using the whole-cell voltage-clamp method to measure ATP-sensitive K⁺(K_ATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca²⁺ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic β-cells. Tolbutamide (100 μM) and 100 nM 4-β-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms found in β-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our data support the idea that tolbutamide can potentiate secretion from β-cells via a PKC-dependent pathway. Because PKC and sulphonylureas can modulate the activity of K_ATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell K_ATP currents but did not affect their sensitivity to tolbutamide. Down-regulation of PKC affected neither the magnitude nor the tolbutamide sensitivity of the K_ATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 μM) mobilized intracellular Ca²⁺ and prolonged Ca²⁺ transients elicited by cholinergic mobilization of intracellular Ca²⁺ stores. Tolbutamide (0.1–0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated K_ATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca²⁺ handling, which ultimately leads to activation of Ca²⁺-dependent enzymes such as PKC. Received: 1 September 1998 / Received after revision: 9 November 1998 / Accepted: 10 November 1998     

The cellular mechanisms of Cl– secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias

 We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [I _sc=transepithelial voltage/transepithelial resistance (R _te)] as well as basolateral membrane voltage (V _bl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a ”stimulation cocktail” (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in I _sc (–31.6±7.7 to –316±82.2 μA/cm², n=15). CNP significantly depolarized the luminal membrane from –87.4±1.0 to –82.3±2.6 mV (n=12). V _bl was not changed (n=12) but the fractional conductance for K⁺ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on I _sc were not additive (n=5). The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02±0.05 to 1.32±0.05 (n=8) with a time constant in the 1–2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in I _sc in the rectal gland. The concomitant fall in R _te corresponds to increases in the luminal membrane Cl^– conductance and in the basolateral membrane K⁺ conductance. The latter effect is probably due to an increase in [Ca²⁺]_i. Received: 21 December 1998 / Received after revision: 5 February 1999 / Accepted: 9 February 1999     

Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002

 Angiotensin II (AngII) is coupled to several important intracellular signaling pathways, and increases intracellular Ca²⁺. In vascular smooth muscle (VSM) cells, AngII is known to activate enzymes such as tyrosine protein kinase (Tyr-PK), phospholipase C (PLC), protein kinase C (PKC), and phophatidylinositol-3-kinase (PI-3-K). A non-receptor Tyr-PK, pp60^c-src, and PKC have been reported to stimulate the Ca²⁺ channels in VSM cells. However, less is known about AngII action on the voltage-gated Ca²⁺ channels. The Ca²⁺-channel currents of a cultured rat aortic smooth muscle cell line, A7r5, were recorded using whole-cell voltage clamp. Application of 50 nM AngII significantly increased the amplitude of Ba²⁺ currents through the voltage-gated Ca²⁺ channels (I _Ba) by 34.5±9.1% (n=10) within 1 min. In the presence of lavendustin-A (5 μM), a selective inhibitor of Tyr-PK, AngII failed to stimulate I _Ba (n=5). AngII stimulation of I _Ba was also prevented by (5 μM) LY-294002, an inhibitor of PI-3-K (n=5). In contrast, H-7 (30 μM), an inhibitor of PKC, did not prevent the effect of AngII on I _Ba (n=6). These results suggest that AngII may stimulate the Ca²⁺ channels of VSM cells through Tyr-PK and PI-3-K under conditions that probably exclude participation of PK-C. Received: 17 July 1998 / Received after revision: 10 September 1998 / Accepted: 29 September 1998     

Protein kinase C stimulates swelling-induced chloride current in canine atrial cells

 The whole-cell patch-clamp technique was used to study the effect of protein kinase C (PKC) stimulation and α-adrenergic agonists on the swelling-induced chloride current (I _Cl,swell) in canine atrial cells. I _Cl,swell was activated by positive-pressure inflation. 4β-Phorbol 12, 13-dibutyrate (PDBu) concentration-dependently stimulated I _Cl,swell. PDBu (500 nM) increased the current density of I _Cl,swell from 9.1±1.3 to 24.2±4.8 pA/pF at +20 mV (n=4). This effect developed slowly, reaching a steady-state after more than 5 min of exposure. 4α-Phorbol 12, 13-dibutyrate (4α-PDBu, 500 nM), an inactive analogue of PDBu, did not affect I _Cl,swell. The effect of PDBu was inhibited by bisindolylmaleimide I. After down regulation of PKC by phorbol 12-myristate 13-acetate (PMA, 1.6 μM, 24 h), I _Cl,swell no longer responded to PDBu (n=4). Neither the basal whole-cell current (prior to cell inflation) nor inflation-induced I _Cl,swell were affected by PKC down regulation. Phenylephrine did not affect I _Cl,swell. We conclude that PKC activity stimulates and does not prevent the activation of dog atrial I _Cl,swell. These results contrast with reports of PKC-dependent inhibition of rabbit atrial I _Cl,swell and currents conducted by ClC-3, a putative clone for I _Cl,swell. The data suggest species-dependent variations in the modulation of cardiac I _Cl,swell by PKC. Received: 19 June 1998 / Received after revision: 15 September 1998 / Accepted: 25 September 1998     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

 This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca²⁺]_i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca²⁺]_i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114±18 μmol l^–1 and K _d of 0.59±0.17 μM (n=8). During the systolic rise of [Ca²⁺]_i, the measured changes of [Ca²⁺]_i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca. Received: 14 October 1998 / Accepted: 6 November 1998     

A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate

 The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (P_i) and H⁺ ([MgADP], [Pi] and [H⁺], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k₁ [5.6±0.1 s^–1 for barnacle, n=30; 26.3±0.7 s^–1 for frog, n=14 (mean±SEM)] and k₂ [0.6±0.1 s^–1 in barnacle, n=30; 10.4±1.0 s^–1 in frog, n=14 (mean±SEM)], at 10°C. Decreasing the pH by 0.5 pH units did not significantly affect k₁ for barnacle relaxation [5.6±0.1 s^–1 (mean±SEM), n=15] compared to the decrease in k₁ of 40% seen in frog. Use of the Ca²⁺-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa₅₀ value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in k_off from the labelled protein [322±32 s^–1 at pH 7.0 and 381±24 s^–1 (mean±SEM) at pH 6.6]. Increasing [MgADP] by 20 μM (50 μM added ADP) from control values of 50 μM in frog decreased k₁ to 12.3±0.4 s^–1 (mean±SEM, n=8), and at 400 μM MgADP, k₁=9.6±0.1 s^–1 (mean±SEM, n=12). In barnacle, 500 μM MgADP had a much smaller effect on k₁ (4.0±0.9 s^–1, mean±SEM, n=8). Increasing the free [P_i] from the contaminant level of 0.36 mM to 1.9 mM slowed k₁ by ≈15% in barnacle [4.8±0.8 s^–1, mean±SEM, n=7], compared to a ≈30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation. Received: 18 May 1998 / Received after revision and accepted: / 1 September 1998     

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium

 In the present study we investigated the possible existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na⁺ transport. Cytosolic calcium ([Ca²⁺]_i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na⁺ transport was measured as the amiloride-sensitive short-circuit current (I _sc) using a conventional voltage clamp. Basal [Ca²⁺]_i was 65±6 nM (n=15). Removal of Na⁺ from the mucosal solution had no effect on [Ca²⁺]_i. When Na⁺ was removed from the serosal solution, [Ca²⁺]_i increased biphasically to a peak of 220±38 nM (n=8, P=0.006). Readdition of Na⁺ to the serosal solution returned [Ca²⁺]_i to control level. The serosal Na⁺ gradient and changes in [Ca²⁺]_i were closely correlated; stepwise changes in serosal Na⁺ were followed by stepwise changes in [Ca²⁺]_i. These observations indicate the existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na⁺ transport decreased from 13.2±1.8 to 9.2±1.5 μA cm^–2 (n=8, P=0.049) when Na⁺ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca²⁺, Na⁺ transport decreased similarly from 16.7±1.7 to 11.6 ±1.8 μA cm^–2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in I _sc after removal of serosal Na⁺ is due to an increase in [Ca²⁺]_i per se. Received: 10 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Adrenoceptor-mediated regulation of myofibrillar Ca2+ sensitivity through the GTP-binding protein-related mechanisms: tension recording in β-escin-skinned single rat cardiac cells with preserved receptor functions

 To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using β-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca²⁺ sensitivity and Ca²⁺ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 μM isoproterenol plus 100 μM guanosine 5′-triphosphate (GTP) decreased the myofibrillar Ca²⁺ sensitivity (pCa₅₀, the [Ca²⁺] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 μM metoprolol or 1 mM guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Phenylephrine (10 μM) plus 100 μM GTP increased the Ca²⁺ sensitivity (pCa₅₀; from 6.12 to 6.28), and this effect was blocked by 1 μM phentolamine or 1 mM GDPβS. After Ca²⁺ loading into the SR, 10 μM phenylephrine plus 100 μM GTP in a low-ethylene- glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 0.1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 μM phentolamine or pre-treatment with 10 μM inositol 1,4,5-trisphosphate. Our results demonstrate that β₁-adrenergic stimulation decreases myofibrillar Ca²⁺ sensitivity and that α₁-adrenergic stimulation both increases the Ca²⁺ sensitivity and activates Ca²⁺ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms. Received: 17 August 1998 / Received after revision: 23 October 1998 / Accepted: 15 December 1998     

Accentuated antagonism by angiotensin II on guinea-pig cardiac L-type Ca-currents enhanced by β-adrenergic stimulation

 To examine mechanism(s) underlying the accentuated antagonism by angiotensin II (A-II) on twitch tension, we recorded L-type Ca²⁺ currents (I _Ca,L) using conventional patch-clamp techniques in single, guinea-pig, ventricular myocytes. I _Ca,L was recorded by a step-pulse protocol after eliminating K⁺ conductances (internal Cs⁺ plus tetraethylammonium chloride and K⁺-free extracellular solution). A-II (100 nM) did not affect basal I _Ca,L, but inhibited I _Ca,L that had been enhanced (approximately 200% of control) by (ISO, isoproterenol 100 nM). The inhibitory action of A-II was concentration dependent (concentration eliciting 50% inhibition 88±9 pM, n=41) and the ISO-enhanced component of I _Ca,L was completely blocked by A-II at concentrations above 10 nM. CV-11974 (500 nM), an A-II type-1 receptor (AT₁) antagonist, prevented the inhibitory action of A-II. Pre-incubation with pertussis toxin (PTX) abolished the inhibitory effect of A-II. A-II also inhibited the I _Ca,L enhanced by histamine (500 nM) and forskolin (1 μM), but failed to affect I _Ca,L enhanced by intracellular cyclic adenosine monophosphate (1 mM). The inhibitory action of A-II may therefore involve AT₁ receptors/PTX-sensitive, guanine nucleotide-binding (G) proteins (Gi)/adenylate cyclase and partially explains the A-II-dependent accentuated antagonism of inotropy.     

Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes

 In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, ”Myristyl- (Myr-) FRCRCFa”. Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35–37°C. The Na-Ca exchange current (I _Na-Ca), L-type calcium current (I _Ca,L), inward rectifier potassium current (I _K1) and delayed rectifier potassium current (I _K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, I _Na-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of –40 mV, between +80 and –120 mV (ramp velocity 0.1 V s^–1). In untreated cells, I _Na-Ca at +60 mV was 7.1±0.6 pA/pF and at –100 mV was –2.7±0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 μM Myr-FRCRCFa, I _Na-Ca was reduced to 4.2±0.3 pA/pF at +60 mV and –1.5±0.2 pA/pF at –100 mV (P<0.02; n=7). After incubation with 20 μM Myr-FRCRCFa for 1 h, I _Na-Ca at both potentials was further reduced (2.3±0.8 pA/pF at +60 mV; –0.9±0.3 pA/pF at –100 mV; P<0.008 compared with control; n=4). Under selective recording conditions for I _Ca,L, there was little difference in I _Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I _Ca,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I _K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I _K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of I _Na-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an I _Na-Ca blocker. I _Ca,L, I _K1 and I _K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger. Received: 6 February 1997 / Received after revision: 8 April 1998 / Accepted: 9 April 1998     

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes

 We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca²⁺ regulation in Fura-2-loaded ferret myocytes. H-89 (10 μmol/l) decreased the amplitude of the Fura-2 transient to 28.2±4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca²⁺ to diastolic levels: t _1/2 increased from 311±35 ms to 547±43 ms (P<0.001, n=7). Reduced Ca²⁺ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca²⁺ content, as assessed with caffeine. The apparent slowing of the SR Ca²⁺-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca²⁺ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca²⁺-ATPase. In electrophysiological experiments, 5.0 μmol/l H-89 decreased the L-type Ca²⁺ current (I _Ca) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca²⁺ ]_i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of β-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca²⁺-ATPase. Received: 4 September 1998 / Received after revision: 19 October 1998 / Accepted: 20 October 1998     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Use of replication-deficient adenoviruses to study signal transduction pathways. Muscarinic responses in HSG and HT29 epithelial cell lines are mediated by G protein βγ-subunits

 HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca²⁺ ([Ca²⁺]_i) by activating phospholipase Cβ. In the present study, we have used the measurement of [Ca²⁺]_i with Fura-2 to show that these receptors are of the M₃ sub-type and that the increase in [Ca²⁺]_i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses expressing wild-type and dominant-negative mutants of the α-subunits of the heterotrimeric G proteins, G_q and G_i2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca²⁺ response to 100 μmol/l carbachol is not affected by increased expression of the wild-type α-subunit of G_q, but is blocked by the dominant-negative mutant of G_q and by both the wild-type and the dominant-negative mutant α-subunits of G_i2. Expression of α-subunits of G_i2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca²⁺ response to M₃ receptor activation is mediated by the βγ- rather than the α-subunits of G_q. Received: 23 September 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999     

The effect of phosphate on the relaxation of frog skeletal muscle

 The effect of phosphate on the relaxation of isometrically contracting single skinned fibres from the semitendinosus muscle of the frog Rana temporaria has been investigated using laser pulse photolysis of the photolabile caged calcium-chelator diazo-2 to rapidly reduce the Ca²⁺ (<2 ms) within the fibre and produce >90% relaxation of force. Relaxation occurred in two phases – an initial linear shoulder which lasted approximately 20 ms followed by a double-exponential phase which gave two rate constants, k ₁ (43.4±1.8 s^–1, mean ±SEM, n=14) and k ₂ (15.6±0.3 s^–1, mean ±SEM, n=14) at 12°C. Increased phosphate concentrations did not affect the linear phase, but slowed the double-exponential phase following photolysis of diazo-2 in a dose-dependent fashion (k ₅₀= 0.9 mM for k ₁, 1.15 mM for k ₂). Reducing the concentration of contaminating phosphate (from 640 μM to 100 μM) led to an increase in the rate of the double-exponential phase (k ₁=67.1±4.4 s^–1, k ₂=19.7±0.6 s^–1, mean ±SEM, n=12). Time-resolved measurements of sarcomere length during relaxation, both in control fibres and in the presence of a raised phosphate concentration, reveal a <2% change throughout the whole relaxation transient, and less than 0.1% at the end of the linear phase. This finding implies that gross changes in sarcomere length do not contribute to the decay of the relaxation transient seen upon diazo-2 photolysis. Our results suggest that cross-bridges in states prior to phosphate release are already committed to force generation and must relax by releasing phosphate, rather than by a reversal of the force-generating step to a weakly bound, low-force phosphate-bound state. These findings also indicate that an increase in the phosphate concentration within muscle fibres plays an important part in the slowing of relaxation observed in skeletal muscle fatigue and that the relaxation transients observed upon diazo-2 photolysis represent a disengagement of the cross-bridges. Received: 14 September 1998 / Received after revision and accepted: 20 October 1998     

Losartan antagonism of angiotensin-II-induced potassium secretion across rat colon

 The effect of angiotensin II (ANG II) on potassium transport across the short-circuited rat distal colon was investigated using ⁸⁶Rb⁺ as a tracer for unidirectional K⁺ fluxes. The addition of high concentrations of ANG II (≥10^–6 M) to the serosal bathing solution had no effect on the mucosal to serosal flux of Rb⁺, but significantly increased the serosal to mucosal flux and abolished the basal net absorptive Rb⁺ flux. These ANG-II-induced alterations in Rb⁺ transport were blocked by the AT₁ receptor antagonist losartan and its metabolite EXP3174, which is also known to have AT₁ receptor antagonistic activity. In contrast, an AT₂ receptor antagonist, PD123319, did not prevent the alterations in colonic Rb⁺ transport induced by ANG II in vitro. At lower bath concentrations (10^–7 M to 10^–10 M ), ANG II had no measurable effects on Rb⁺ transport across this tissue but ANG II did have a bimodal effect on short-circuit current (I _sc), indicating additional effects on the electrogenic transport of other ions. Dose-dependent reductions in I _sc were observed between 10^–7 M (↓ΔI _sc=1.96±0.49 μEq·cm^–2·h^–1, n=6) and 10^–10 M (↓ΔI _sc=0.16±0.19 μEq·cm^–2·h^–1, n=7) ANG II, whereas I _sc was increased at the higher concentrations (↑ΔI _sc= 3.36±0.52 μEq·cm^–2·h^–1, n=7, at 10^–4 M). The ANG-II-induced increases and decreases in I _sc were both blocked by losartan but not by PD123319. These studies are the first to demonstrate an effect of ANG II on K⁺ transport across rat colon that is independent of aldosterone and mediated by AT₁ receptors. Received: 13 March 1998 Received after revision and accepted: 26 May 1998     

Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes

 The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (I _CCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, I _CCh was induced by bath application of 50 μM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]_i), I _CCh decayed spontaneously (desensitization of I _CCh) to around 20% within 10 min. Desensitization of I _CCh was significantly attenuated with 2 mM [EGTA]_i. At a concentration of 20 μM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I _CCh at 0.5 mM [EGTA]_i but far less at 2 mM [EGTA]_i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5′-O-(3-thiotriphosphate) (GTP[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA]_i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 μM chelerythrine or 1 μM peptide inhibitor, attenuated the desensitization of I _CCh. [Ca²⁺]_i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]_i, [Ca²⁺]_i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]_i. These results suggest that the desensitization of I _CCh is partly due to the Ca²⁺-dependent PKC pathway in guinea-pig gastric myocytes. Received: 27 August 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

α1-Adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium

 The effects of adrenoceptor agonists on the transepithelial Cl^– conductance (G ^Cl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively and reversibly inhibited the voltage-activated G ^Cl in toad skin and the short-circuit G ^Cl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands, since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations of Epi were variable among individual tissues. G ^Cl was reduced to 34±17% (n=46) with 1 μmol/l Epi, but in some tissues 0.1 μmol/l inhibited more than 80% of G ^Cl, whereas some preparations were little influenced at >3 μmol/l Epi. The affected receptor type was identified by the use of the α₁-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 μmol/l, whereas the α₂-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific α₁-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The α₂-antagonist yohimbine, at concentrations ≤0.3 μmol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations of 1–10 μmol/l. This might indicate affinity to α₁-adrenoceptors in amphibian skin. Activation of β-adrenoceptors by isoproterenol (0.1–5 μmol/l) led to a transient increase of the baseline inactivated component of G ^Cl with a slight reduction of the voltage-activated G ^Cl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented nor reversed the induction of a voltage-insensitive G ^Cl in toad skin caused by application of cAMP at supramaximal concentrations (>100 μmol/l CPT-cAMP). Preincubation of the serosal medium with Ca²⁺-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It is concluded that α₁-adrenoceptors participate in the physiological control of voltage-activated Cl^– conductance in amphibian skin epithelium via modulation of intracellular Ca²⁺, presumably by efflux from intracellular stores. Received: 11 March 1998 / Received after revision: 2 June 1998 / Accepted: 8 June 1998     

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

 The effect of adenosine triphosphate (ATP) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca²⁺]_i increase, which was dose dependent in the 2.5–50 μM range (EC₅₀=4.3 μM). The ATP-evoked [Ca²⁺]_i rise was not modified during the first minute following the removal of external Ca²⁺. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca²⁺]_i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca²⁺]_i rise. The pituicytes responded to various P₂ purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P₂ purinoceptor antagonists blocked the ATP-evoked [Ca²⁺]_i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca²⁺]_i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca²⁺]_i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca²⁺]_i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P_2Y receptor. Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998