Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

Disruption of actin cytoskeleton attenuates sulfonylurea inhibition of cardiac ATP-sensitive K+ channels

 Two actin filament-depolymerizing agents, DNase I and cytochalasin D, were used to examine the involvement of the cytoskeleton in the functional interaction between the sulfonylurea receptor (SUR) and the ATP-sensitive K⁺ (K_ATP) channels. Isolated rat ventricular cardiomyocytes were studied using open cell-attached patches for single-channel recording. Bath application of DNase I (100 μg/ml) or cytochalasin D (10 μM) stimulated the K_ATP channel activities (in presence of 30 μM ATP), and these channels became resistant to inhibition by tolbutamide (0.5 mM). After exposure to tolbutamide, the relative NP_o value was 0.09 ± 0.02 in control patches in absence of actin disrupters, and 0.67 ± 0.22* or 0.65 ± 0.10*, respectively, in cells treated with DNase I or cytochalasine D (*P < 0.05 vs. control). The inhibitory action of glibenclamide (10 μM) on the K_ATP channels was also attenuated by DNase I. Thus, the disruption of the actin cytoskeleton attenuates the ability of SUR to inhibit the opening of K_ATP channels. Received: 3 February 1997 / Accepted: 25 March 1997     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (I _CCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. I _CCh was induced by carbachol (CCh, 50 μM) at a holding potential of –30 mV or –60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited I _CCh concentration dependently in a reversible manner (53 ± 8.6% at 1 μM, mean ± SE, n = 11). In addition, amplitudes of I _CCh were only 37 ± 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca²⁺ channel current. The peak value of Ba²⁺ current at 0 mV was reduced to 35 ± 7.4% (n = 9) by 3 μM of ML-7. As I _CCh is known to have an intracellular Ca²⁺ dependence, we tried to exclude the possibility that ML-7 inhibited I _CCh indirectly via suppression of Ca²⁺ current and the similar inhibitory effects of ML-7 on I _CChwere confirmed under the following conditions: (1) clamp of membrane potential at –60 mV; (2) clamp of intracellular [Ca²⁺] to 1 μM by 10 mM BAPTA; (3) pre-inhibition of Ca²⁺ channel by verapamil. Different from the effects on I _CCh, ML-7 barely inhibited the same cationic current induced by guanosine 5’-O-(3-thiotriphosphate) (GTP[γS], 0.2 mM) in the pipette solution. These results suggest that a Ca²⁺/calmodulin-MLCK-dependent pathway can modulate the activation of I _CCh in guinea-pig gastric antral myocytes. Received: 21 January 1997 / Received after revision and accepted: 2 April 1997     

Inhibitory effect of phorbol 12, 13 dibutyrate on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (I _CCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 μ M PDBu suppressed I _CCh by 96.5 ± 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 μM chelerythrine , a PKC inhibitor, inhibition of I _CChby PDBu was not seen. In whole-cell mode, the inhibition of I _CCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased I _CCh by 98.8 ± 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on I _CCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 ± 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[γS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[γS]-induced currents were 6.7 ± 2.4 pA and 236 ± 23 pA, respectively. These results suggest that PKC modulates I _CCh at postreceptor sites via protein phosphorylation. Received: 4 April 1997 / Received after revision: 27 June 1997 / Accepted: 3 June 1997     

Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations

 This study was undertaken to reassess the set of voltage-dependent Ca²⁺ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of ω-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba²⁺. Whole-cell Ba²⁺ currents (I_Ba) through Ca²⁺ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of –80 mV and depolarising pulses to 0 mV. Mean peak I _Ba was 425 pA in 2 mM Ba²⁺ (59 cells) and 787 pA in 10 mM Ba²⁺ (42 cells). In 2 mM Ba²⁺, ω-conotoxin MVIIC (3 μM) inhibited I _Ba by 79%; in 10 mM Ba²⁺, the blockade developed much more slowly and reached only 44%. A low concentration of ω-agatoxin IVA (20 nM) inhibited I _Ba by 9%; 2 μM inhibited I _Ba by 60%. This blockade was similar in low and high Ba²⁺ concentrations. After giving furnidipine (3 μM) and ω-conotoxin GVIA (1 μM), 2 μM ω-agatoxin IVA inhibited the remaining current (about 40–45%); this blockade was independent of the Ba²⁺ concentration. The current could be fully blocked by the cocktail furnidipine/ω-conotoxin GVIA/high ω-agatoxin IVA, both in low and high Ba²⁺ concentrations. The large Q-type channel component of I _Ba is blocked by micromolar concentrations of ω-agatoxin IVA and ω-conotoxin MVIIC. While solutions with a high Ba²⁺ concentration strongly delayed the development of blockade by ω-conotoxin MVIIC, the blockade by high concentrations of ω-agatoxin IVA was equally effective in solutions with a low or a high Ba²⁺ concentration. Hence, the use of appropriate Ba²⁺ and toxin concentrations in this study reveals that P-type Ca²⁺ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca²⁺ channels. An R-type residual current is not present in these cells. Received: 22 April 1996 / Received after revision: 11 June 1990 / Accepted: 11 June 1996     

Tyrosine kinase inhibitors suppress N-type and T-type Ca2+ channel currents in NG108–15 cells

 Modulation of Ca²⁺ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored the possible modulation of neuronal Ca²⁺ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels were analysed in differentiated NG108–15 neuroblastoma × glioma hybrid cells. Genistein suppressed I _Ba in a concentration-dependent fashion (IC₅₀ = 22 μM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed I _Ba, we estimated that specific PTK inhibition by genistein reduced I _Ba amplitude by 30%. In addition, lavendustin A (20 μM) and herbimycin A (20 μM), two other distinct PTK inhibitors, depressed I _Ba by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was independent of G protein activation. The results suggest that the activity of neuronal Ca²⁺ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated by Ca²⁺ channel modulation. Received: 21 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Modulation by mexiletine of action potentials, L-type Ca current and delayed rectifier K current recorded from isolated rabbit atrioventricular nodal myocytes

 Using whole-cell patch clamp recordings at 37°C, we have examined the effects of externally applied mexiletine (a class 1b antiarrhythmic agent) on action potentials, L-type Ca current (I_Ca,L) and delayed rectifier K current (I_K) in single isolated rabbit atrioventricular nodal (AVN) myocytes. In spontaneously active AVN cells, 30–100 μM mexiletine depolarised the maximum diastolic potential and slowed both action potential upstroke and repolarisation. Under selective recording conditions for I_Ca,L, mexiletine reduced peak I_Ca,L (at +10 mV) amplitude in a dose-dependent fashion (41.8 ± 3.0% inhibition by 100 μM and 16.4 ± 1.8% at 30 μM). The voltage dependence of I_Ca,L activation was unaffected by both concentrations of the drug. Under selective recording conditions, I_K amplitude was measured as the peak of the deactivating tail current following a depolarising voltage pulse to +20 mV. 30 μM mexiletine inhibited I_K by 34.3 ± 5.8%, whilst 100 μM mexiletine reduced the current by 52.7 ± 6.1%. The effects of mexiletine on I_Ca,L and I_K are likely to contribute significantly to the changes in action potentials observed in spontaneously active cells. These findings are also suggestive of key roles for I_Ca,L and I_K in determining the shape and rate of action potentials in this region of the heart. Received: 21 May 1997 / Received after revision: 25 July 1997 / Accepted: 19 August 1997     

Volume-sensitive chloride current activated by hyposmotic swelling in antral gastric myocytes of the guinea-pig

 The characteristics of volume-sensitive chloride current (I _Cl) induced by osmotic cell swelling were studied using the whole-cell patch-clamp technique and cell diameters of antral circular guinea-pig myocytes were simultaneously measured under isosmotic and hyposmotic conditions by using a video image analysis system. At –60 mV, osmotic cell swelling (200 mosmol/l) activated a sustained inward current. Instantaneous current/voltage (I-V) relations obtained by step voltage pulses showed an outward rectification. At potentials above +40 mV, the current exhibited time-dependent decay. The outward current amplitude was decreased and the reversal potential was shifted to more positive potentials by replacement of external Cl^– with gluconate^–, while the current amplitude and the I/V relation were not affected by replacing extracellular Na⁺ with N-methyl-D-glucamine. The anion permeability sequence of the swelling-induced current was I^– (1.80) > Br^– (1.31) > Cl^– (1) > F^– (0.85) > gluconate^– (0.18). The I _Cl was effectively inhibited by the Cl^– channel blockers, 4,4′-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS, 100 μM), and niflumic acid (10 μM). DIDS suppressed outward current more effectively than inward current. Also, the I _Cl was dose-dependently inhibited by arachidonic acid, an unsaturated fatty acid and also inhibited by other unsaturated fatty acids (linoleic acid and oleic acid) but not by stearic acid, a saturated fatty acid. The inhibitory effect of arachidonic acid on I _Cl was not prevented by indomethacin, a cyclo-oxygenase inhibitor and chelerythrine, a protein kinase C inhibitor. Under whole-cell patch-clamp conditions, the cell diameter was continuously measured using video image analysis, which reflects the change in cell volume. A hyposmotic-stimulation-induced increase of cell diameter was followed by I _Cl activation. In intact single gastric myocytes, relatively severe hyposmotic (176 mosmol/l) superfusing solution increased the cell diameter and the pretreatment with DIDS or with niflumic acid significantly potentiated the above effect of hyposmotic superfusion. These results suggest that volume-sensitive outwardly rectifying chloride current (I _Cl) is present in guinea-pig gastric myocyte and the I _Cl may play a role in smooth muscle cell volume regulation. Received: 6 June 1997 / Received after revision and accepted: 24 July 1997     

cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes

 The effect of guanosine 3′,5′-cyclic monophosphate (cGMP) on L-type Ca current (I _Ca) was investigated in a study of rabbit ventricular myocytes using the whole-cell patch-clamp technique. Intracellular application of cGMP (100 μM) increased I _Ca in the absence of isoprenaline or forskolin. 8-Bromo-cGMP (100 μM) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP, 400 μM), relatively specific stimulators of cGMP-dependent protein kinase (cGMP-PK), also increased I _Ca. The stimulatory effect of 8-pCPT-cGMP was suppressed by Rp-8-chlorophenylthio-cGMP (400 μM), a phosphodiesterase-resistant cGMP-PK inhibitor. When I _Ca was increased by bath application of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM), 8-pCPT-cGMP (400 μM) resulted in additional stimulation of I _Ca. In the presence of 8-pCPT-cGMP, additional applications of isoprenaline (1 μM) or forskolin (1 μM) induced a further increase in I _Ca. From these results, it could be concluded that the activation of cGMP-dependent protein kinase is involved in the facilitation of I _Ca by cGMP in rabbit ventricular myocytes. Received: 17 March 1997 / Received after revision: 28 August 1997 / Accepted: 16 September 1997     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells

 Using the whole-cell patch-clamp technique, a calcium-activated chloride conductance (CACC) could be elicited in HPAF cells by addition of 1 μM ionomycin to the bath solution (66 ± 22 pA/pF;V _m + 60 mV) or by addition of 1 μM calcium to the pipette solution (136 ± 17 pA/pF; V _m + 60 mV). Both conductances had similar biophysical characteristics, including time-dependent inactivation at hyperpolarising potentials and a linear/slightly outwardly rectifying current/voltage (I/V) curve with a reversal potential (E _rev) close to the calculated cloride equilibrium potential. The anion permeability sequence obtained from shifts in E _rev was I > Br ≥ Cl. 4,4′-Diisothiocyanatostilbene disulphonic acid (DIDS, 500 μM) caused a 13% inhibition of the current (V _m + 60 mV) while 100 μM glibenclamide, 30 nM TS-TM-calix[4]arene and 10 μM tamoxifen, all chloride channel blockers, had no marked effects (8%, –6% and –2% inhibition respectively). Niflumic acid (100 μM) caused a voltage-dependent inhibition of the current of 48% and 17% (V _m ± 60 mV, respectively). In freshly isolated human pancreatic duct cells (PDCs) a CACC was elicited with 1 μM calcium in the pipette solution (260 ± 62 pA/pF; V _m + 60 mV). The presence of this CACC in human PDCs could provide a possible therapeutic pathway for treatment of pancreatic insufficiency of the human pancreas in cystic fibrosis. Received: 2 October 1997 / Received after revision: 28 November 1997 / Accepted: 1 December 1997     

Action of the hyperpolarization-activated current (I h) blocker ZD 7288 in hippocampal CA1 neurons

 The effects of ZD 7288, a ”bradycardic” agent, in young rat hippocampal slices in vitro were studied. ZD 7288 (1–1000 μM) reduced the hyperpolarization-activated current (I _h) in CA1 pyramidal neurons by a voltage-independent blocking mechanism. Under current-clamp conditions, the bradycardic agent (10 μM) caused membrane hyperpolarization (by 5.9 ± 0.5 mV) and a reduction of membrane conductance (by 17.9 ± 4.1%). These data are consistent with the block of an inward current which is active at rest. The drug-induced hyperpolarization depressed the cell’s excitability by increasing the threshold current necessary to induce firing. When the drug-induced hyperpolarization was compensated for by injection of a tonic depolarizing current, ZD 7288 caused a reduction of the inhibitory post-synaptic potential (IPSP) in EPSP-IPSP sequences. Since Cs⁺, another known blocker of I _h, is able to reverse long-term depression (LTD) of the CA3-CA1 synapse in hippocampal slices, we tested the effect of ZD 7288 on synaptic transmission. We found that ZD 7288 did not significantly modify LTD, suggesting that Cs⁺-induced inhibition of LTD maintenance is not directly related to block of I _h. Received: 14 February 1997 / Received after revision: 4 July 1997 / Accepted: 21 July 1997     

Involvement of tyrosine kinase in the hyposmotic stimulation of I Ks in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the hyposmotic stimulation of cardiac I _Ks, a slowly activating delayed-rectifier K⁺ current that promotes repolarization of the action potential. The current was recorded from whole-cell-configured guinea-pig ventricular myocytes before, during, and after their exposure to solution whose osmolarity was 0.75 times normal. Exposure to hyposmotic solution caused a near-doubling of the amplitude of I _Ks, with little change in the voltage dependence of current activation. Stable, hyposmotically stimulated I _Ks (I _Ks,Hypo) was decreased by broadspectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀ ≈ 5 μM) and tyrphostin A25 (IC₅₀ 15.8 ± 1.6 μM) but not by TK-inactive tyrphostin analogs, suggesting that tyrosine phosphorylation is important for maintenance of the current. In agreement with that view, we found that the TK-inhibitor action on I _Ks,Hypo was strongly antagonized by vanadate compounds known to inhibit phosphotyrosyl phosphatase. When myocytes were pretreated with TK inhibitors, the stimulation of I _Ks was attenuated in a concentration-dependent manner. The attenuation was not due to concomitant attenuation of a stimulation of tyrosine phosphorylation because neither the stimulation of I _Ks nor its rate of decay following removal of hyposmotic solution was affected by pretreatment with vanadates. We suggest that the stimulation of I _Ks by hyposmotic solution is dependent on a basal tyrosine phosphorylation that modulates a swelling-induced I _Ks-stimulatory signal and/or the receptivity of Ks channels to that signal.     

The inhibitory effect of β-stimulation on the Na/K pump current in guinea pig ventricular myocytes is mediated by a cAMP-dependent PKA pathway

 The β-agonist isoproterenol (ISO) reduces the Na/K pump current (I _p) via β-adrenergic receptors when the intracellular calcium concentration ([Ca²⁺]_i) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 μM), or the adenylyl cyclase activator forskolin (50 μM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 μM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on I _p is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H₇ (100 μM) nor the protein phosphatase inhibitor calyculin A (0.5 μM) had any effect on I _p in the absence of ISO. However, H₇ could increase I _p and calyculin A could reduce it in the presence of ISO (1 μM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H₇ and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level. Received: 21 July1997 / Received after revision: 14 October 1997 / Accepted: 15 October 1997     

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells

 The properties of the calcium stores coupled to a depletion-operated cation current (I _DOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 μM) activated an initial, large (≈ 200 pA), transient, calcium-dependent chloride current (I _ClCa) followed by a smaller (≈ 10 pA) sustained, non-selective cation current (I _DOC). Intracellular application of heparin (5 mg ml^–1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 μM) activated I _DOC but not I _ClCa; ryanodine (30 μM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of I _ClCa by carbachol. In the presence of 30 μM, but not 3 μM, ryanodine, carbachol was able to activate I _DOC. Cyclopiazonic acid (CPA; 10 μM) abolished the response to carbachol; however, caffeine was still able to activate I _ClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 μM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4,5-trisphosphate-(IP₃-) sensitive or ryanodine-sensitive calcium stores is able to activate I _DOC, and, by extension, capacitative calcium entry in this tissue. Received: 21 July 1997 / Received after revision: 29 August 1997 / Accepted: 1 September 1997     

Role of the cytoskeleton in stimulation of Na+ channels in A6 cells by changes in osmolality

 Permeable supports with A6 cell monolayers were mounted in an Ussing chamber and bilaterally bathed with Ringer solution at room temperature. Short-circuit current (I _sc) was recorded continuously, and noise analysis revealed microscopic channel current characteristics. Our investigation focuses on the stimulation of apical Na⁺ entry caused by exposing the serosal surface of the A6 cell monolayers to hyposmotic Ringer solution. To evaluate the possible role of the cytoskeleton in the regulation of Na⁺ channels in response to a change in osmolality we used four different experimental approaches. In the control group, which were not exposed to any cytoskeleton-influencing drugs, there was a 1.5-fold increase in I _sc and in the number of open Na⁺ channels after osmotic stimulation. For the second group cytochalasin D (0.1 μg/ml) was present on the serosal side during the experiments. Neither I _sc nor the number of open Na⁺ channels increased after osmotic stimulation. In the third group colchicine (0.2 mM) or nocodazole (20 μM) was present on the serosal side, which resulted in 1.8-fold and 1.5-fold increases in I _sc as well as 3-fold and 2-fold increases in the number of Na⁺ channels, respectively. In the fourth experimental group erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 0.5 mM), a dynein inhibitor, was present on the serosal side. In this group I _sc decreased to about 0.4 μA/cm², and subsequent application of amiloride abolished I _sc completely. Under hyposmolar conditions EHNA abolished entirely the sensitivity of I _sc to the osmotic challenge. Because of the EHNA-induced down-regulation of I _sc, the density of apical Na⁺ channels in this experimental group could not be determined. These results show that the cytoskeleton is dominantly involved in osmotic channel regulation at the apical membrane, and that actin filaments, microtubules and molecular motors are involved in the recruitment of additional Na⁺ channels. Received: 21 July 1997 / Received after revision: 4 December 1997 / Accepted: 16 December 1997     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons

 Using the patch-clamp technique, we studied the effect of intracellular Ca²⁺ on Cl^– current gated by type A γ-aminobutyric acid receptors (GABA_A) in mouse cortical neurons. When the rapid Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl^– current amplitude decreased over time to 49 ± 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 ± 10% increase in GABA current. An increased intracellular Ca²⁺ concentration caused a transient augmentation of the GABA current. This effect of Ca²⁺ was concentration dependent (10 nM to 34 μM). Ca²⁺ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC₅₀ = 5 ± 0.4 μM vs. 7 ± 0.3 μM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 μM) blocked the potentiating effect of Ca²⁺ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABA_A receptors by intracellular Ca²⁺ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca²⁺ on responses to GABA was mimicked by activating excitatory receptors with 100 μM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABA_A receptor activity by glutamate may be brought about via changes in intracellular Ca²⁺. Received: 20 May 1997 / Received after revision: 12 August 1997 / Accepted: 1 September 1997     

Separation of calcium channel current components in mouse chromaffin cells superfused with low- and high-barium solutions

 This study was carried out to characterize the set of voltage-dependent Ca²⁺ channel subtypes expressed by mouse adrenal chromaffin cells superfused with solutions containing low (2 mM) or high (10 mM) Ba²⁺ concentrations. Using 50-ms test pulses at 0 mV from a holding potential of –80 mV, averaged peak current in 10 mM Ba²⁺ was around 1 nA, and in 2 mM Ba²⁺ 0.36 nA. When using 2 mM Ba²⁺ as the charge carrier, nifedipine (3 μM) blocked I _Ba by 40–45%. ω-Conotoxin GVIA (1 μM) caused 26% inhibition, while ω-conotoxin MVIIC (3 μM) produced a 48% blockade. At low concentrations (20 nM), ω-agatoxin IVA caused 5–15% of current inhibition, while 2 μM gave rise to a 35–40% blockade. In 10 mM Ba²⁺, the blocking effects of nifedipine (40%) and ω-conotoxin GVIA (25%) were similar to those seen in 2 mM Ba²⁺. In contrast, blockade by ω-conotoxin MVIIC was markedly reduced in 10 mM Ba²⁺ (20–25%) as compared to 10 mM Ba²⁺ (48%). The blocking actions of ω-agatoxin IVA (2 μM) were also slowed down in 10 mM Ba²⁺, though the final blockade was unaffected. In 2 mM Ba²⁺, I _Ba was quickly inhibited by over 94% with combined nifedipine + ω-conotoxin MVIIC + ω-conotoxin GVIA; in 10 mM Ba²⁺, I _Ba was blocked by 70% with this combination. The data suggest that mouse chromaffin cells express L-type (40%) as well as non-L-type (60%) high-threshold voltage-dependent Ca²⁺ channels. The current carried by non-L-type Ca²⁺ channels consists of about 25% N-type and 35% P/Q-type; P-type channels, if anything, are poorly expressed. The data also indicate that the fraction of current blocked by ω-conotoxin MVIIC and ω-agatoxin IVA might considerably change as a function of the Ba²⁺ concentration of the extracellular solution; taking this fact into consideration, it seems that a residual R-type current is not expressed in mouse chromaffin cells. Received: 21 January 1998 / Received after revision and accepted: 20 February 1998     

Effects of temperature on human L-type cardiac Ca2+ channels expressed in Xenopus oocytes

 Temperature normally affects peak L-type Ca²⁺ channel (CaCh) current with a temperature coefficient (Q ₁₀) of between 1.8 and 3.5; in cardiomyocytes attenuating protein kinase A activity increases Q ₁₀ whilst activating it lowers Q ₁₀. We examine temperature effects using cloned human cardiac CaChs expressed in Xenopus oocytes. Peak inward currents (I _Ba) through expressed CaChs (i.e. α_1Cα₂/δ_aβ_1b) exhibited a Q ₁₀ of 5.8±0.4 when examined between 15 and 25°C. The nifedipine-sensitive I _Ba exhibited a higher Q ₁₀ of 8.7±0.5, whilst the nifedipine-insensitive I _Ba exhibited Q ₁₀ of 3.7±0.3. Current/voltage (I/V) relationships shifted to negative potentials on warming. Using instead a different CaCh β subunit isoform, β_2c, gave rise to an I _Ba similar to those expressed using β_1b. We utilized a carboxyl deletion mutant, α_1C-Δ1633, to determine the temperature sensitivity of the pore moiety in the absence of auxiliary subunits; I _Ba through this channel exhibited a Q ₁₀ of 9.3±0.3. However, the Q ₁₀ for macroscopic conductance was reduced compared to that of heteromeric channels; decreasing from 5.0 (i.e. α_1Cα₂/δ_aβ_1b) and 3.9 (i.e. α_1Cα₂/δ_aβ_2c) to 2.4 (α_1C-Δ1633). These observations differ markedly from those made in studies of cardiomyocytes, and suggest that enhanced sensitivity may depend on the membrane environment, channel assembly or other regulatory factors. Received: 16 December 1997 / Accepted: 23 February 1998