Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (K _ATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the K _ATP current started to rise and reached a maximum of 21.3 ± 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1–3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Ca_i) did not change significantly before hypoxic shortening. After hypoxic contracture, the K _ATP current decreased and Ca_i started to rise, reaching about 1 μmol/l. The presence of glibenclamide (10 μmol/l) in the bath reduced the anoxia-induced K _ATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Ca_i, or the magnitude of Ca_i. Metabolic inhibition with 1.5 mmol/l CN resulted in K _ATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 μmol/l glibenclamide had no significant effect on the magnitude of the Ca_i increase caused by exposure of the cells to 1.5 mmol/l CN⁻. After CN⁻ wash-out in non-patch-clamped cells, Ca_i declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced K _ATP currents only partially and has no significant effect on anoxia-induced rise in resting Ca_i. Received: 3 November 1995/Received after revision: 9 January 1996/Accepted: 16 January 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Run-down of the cardiac Ca2+ channel: characterization and restoration of channel activity by cytoplasmic factors

 Possible mechanisms for run-down in the Ca²⁺ channel, such as proteolysis or dephosphorylation of the channel, were examined in guinea-pig ventricular myocytes. The Ca²⁺ channel current, recorded in inside-out patches using a pipette solution containing 50 mM Ba²⁺ and 3 μM Bay K 8644, ran down with a mean survival time of 2.35 min. The survival time was not significantly affected by adenosine triphosphate (ATP) (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (2 mM), isoprenaline (l–5 μM), phosphate (l20 mM) and leupeptin (l0 μM). Stimulation of guanosine triphosphate (GTP)-binding proteins was also ineffective. The catalytic subunit of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA, 0.5–2 μM) slightly and transiently increased channel activity, but had minimal effects on the channel when applied after complete run-down. On the other hand, cytoplasm from the heart, skeletal muscle, brain and liver, but not kidney, induced channel activity. There was a positive correlation between NP _o (the product of the number of channels N and the open probability P _o) value before run-down and that after the application of cytoplasm, suggesting that the activity of once-active channels was restored ba the exogenous cytoplasm. The potency of cytoplasm in tissues in inducing channel activity was not related to PKA activity nor to the number of dihydropyridine binding sites. These results suggest that the run-down of the cardiac Ca²⁺ channel is not mediated by dephosphorylation or proteolysis of the channel, but involves other factor(s), possibly interaction of the channel protein with a cytoplasmic regulatory protein. Received: 7 June 1996 / Received after revision: 5 November 1996 / Accepted: 15 November 1996     

Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells

The effects of changing the intracellular concentrations of Ca²⁺ or Mg²⁺ ([Ca²⁺]_i, [Mg²⁺]_i) on Ca current (I _Ca) was studied in frog ventricular myocytes using the whole-cell and cell-attached patch clamp techniques. In the physiological range of [Mg²⁺]_i an increase in [Ca²⁺]_i enhancedI _Ca whereas at lower [Mg²⁺]_i I _Ca was suppressed. The increase inI _Ca caused by Ca²⁺ loading was not mediated by phosphorylation since the kinase inhibitors H-8 {N-[2-(methylamino)-ethyl]-5-isoquinolinesulphonamide dihydrochloride}, staurosporine and KN-62 {1-[N,O-bis(5-isoquinoline-sulphonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine} and a non-hydrolysable adenosine 5-triphosphate analogue ,-methyleneadenosine 5-triphosphate did not prevent the Ca²⁺-inducedI _Ca increase.I _Ca was dramatically increased from 10 ± 6 (n = 4) to 71 ± 7 nA/nF (n = 4) when [Mg²⁺]_i was lowered from 1.0 × 10^–3 to 1.0 × 10^–6 M at a [Ca²⁺]_i of 10^–8 M. The concentration response relation for inhibition of Ca channels by [Mg²⁺]_i is modulated by [Ca²⁺]_i. To account for the experimental results it is postulated that competitive binding of Ca²⁺ or Mg²⁺ to the Ca channel accelerates the transition of the channel from an active to a silent mode. Single-channel recordings support this hypothesis. The regulation may have clinical relevance in cytoprotection during cardiac ischaemia.     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

 Ca²⁺-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 μM free Ca²⁺ and 300 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca²⁺ (225 nM to 10 μM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca²⁺ buffering (0.1 mM EGTA), activation of Ca²⁺-release-activated Ca²⁺ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP ₃) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca²⁺ATPases were inhibited by thapsigargin, InsP ₃ and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca²⁺ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca²⁺ influx because it was effectively suppressed when external Ca²⁺ was removed. Our results demonstrate that I _CRAC represents an important source of Ca²⁺ for triggering a secretory response. Received: 1 May 1998 / Received after revision: 15 June 1998 / Accepted: 2 July 1998     

Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca²⁺ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K⁺ ions; the unitary conductance was 112 pS in symmetrical K⁺, and the K⁺ permeability (P _K) was 1.3 × 10⁻¹³ cm ⋅s⁻¹. An inward rectification was observed when intracellular K⁺ was reduced. Using a quasi-physiological K⁺ gradient, a non-linear K⁺ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (P _o) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K⁺ concentrations, the channel was activated from a threshold of about −60 mV, and the activation midpoint was −2 mV. P _o decreased noticeably at 50 μM internal adenosine 5′-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 μM. Internal application of 5′-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased P _o. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca²⁺ ions (0.1 to 10 μM) was required for the activation of this channel. The glucose--sensitive K⁺ channel of XO neurons could be considered as a subtype of ATP-sensitive K⁺ channel, contributing substantially to macroscopic outward current. Received: 13 November 1995/Received after revision and accepted: 13 December 1995     

Regulation of Cl–transport by IBMX in renal A6 epithelium

 We studied regulation of Cl^–transport by cAMP and Ca²⁺ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (I _sc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl^–channel blocker) markedly and dose-dependently inhibited the IBMX-induced I _sc. Pretreatment with nifedipine (100 μM, a Ca²⁺ channel blocker) or 1,2-bis (o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 μM, a Ca²⁺ chelator) partially but markedly inhibited the I _sc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 μM for 1 h), also reduced the IBMX-induced I _sc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced I _sc in cells treated with H89. Ionomycin (a Ca²⁺ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced I _sc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl^–release is regulated by an increased cytosolic Ca²⁺ through nifedipine-sensitive Ca²⁺ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca²⁺ concentration but not for activation of Cl^–channels, which are directly activated by cytosolic Ca²⁺; and (3) the IBMX-induced sustained Cl^–release requires cAMP elevation in addition to an increase in the cytosolic Ca²⁺ concentration. Received: 9 September 1996 / Received after revision: 14 January 1997 / Accepted: 28 February 1997     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Simultaneous presence of cAMP and cGMP exert a co-ordinated inhibitory effect on the agonist-evoked Ca2+ signal in pancreatic acinar cells

The stimulation of the pancreatic acinar cells by physiological secretagogues, such as acetycholine (ACh), activates a well-established intracellular signalling pathway, which involves the generation of Inositol 1,4,5-trisphosphate (InsP₃) and the release of Ca²⁺ from intracellular stores. Caffeine, which inhibits this agonist-evoked Ca²⁺ response reversibly and competitively also blocks the Ca²⁺ signal generated by the non-specific activation of the membrane guanine nucleotide-binding proteins (G-proteins). Removal of caffeine is associated with an increase of intracellular [Ca²⁺] ([Ca²⁺]_i) and the spatial and temporal characteristics of this Ca²⁺ signal are identical to those of the signal generated by the initial agonist stimulation. Caffeine is also a potent non-specific inhibitor of various cellular phosphodiesterases (PDE) and its inhibitory effect can be reproduced by other PDE inhibitors, chemically related (theophylline) or not (papaverine). Various protocols designed to increase the concentration of either of the major intracellular cyclic nucleotides [adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP)] failed to reproduce the full extent of the caffeine inhibition: at maximal agonist concentration (1 μM ACh) increases of either cAMP or cGMP did not affect the Ca²⁺ signal, whereas at submaximal doses of agonist (0.1–0.3 μM ACh) they induced partial inhibition. Here we show that only the simultaneous increase of the cellular concentrations of both cyclic nucleotides (either simultaneous or sequential) are effective in mimicking the blocking effect of caffeine and other non-specific PDE inhibitors. These data indicate, thus, that, in addition to other independent intracellular effects, cAMP and cGMP can exert a co-ordinated inhibitory effect of the agonist-evoked Ca²⁺ signal in pancreatic acinar cells. Received: 25 March 1996/Accepted: 23 May 1996     

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells

 The role of ATP in both the activation of store-operated Ca²⁺ current I _CRAC and in Ca²⁺-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, I _CRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4,5-trisphosphate (InsP ₃) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of I _CRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca²⁺-dependent vesicular fusion was triggered by dialysing cells with 10 μM Ca²⁺ and guanosine-5′-O-(3-thiotriphosphate (GTP[γ-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of I _CRAC or for the capacitance increases elicited by high concentrations of intracellular Ca²⁺. Received: 1 May 1998 / Received after revision: 16 July 1998 / Accepted: 16 July 1998     

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     

Store depletion triggers the calcium release-activated calcium current (I CRAC) in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell lines

 In endothelial cells, different types of Ca²⁺ conductances have been described, but none of them has been clearly identified as I _CRAC, the Ca²⁺ release-activated Ca²⁺ current originally described in mast and lymphoma cells. Here we show that in bovine pulmonary artery endothelial cells (CPAE) depletion of intracellular Ca²⁺ stores by inositol 1,4,5-trisphosphate (InsP ₃), Ca²⁺ ionophores and Ca²⁺ pump inhibitors activates a Ca²⁺-selective conductance in the presence of the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The current shows inward rectification, a highly positive reversal potential and is blocked by micromolar concentrations of La³⁺. The conditions used in studies of endothelial cells were also employed in those of HEK-293, an embryonic kidney cell line commonly used to express putative store-operated channels, and Jurkat cells, the reference cell model. Similar to CPAE, HEK cells also have an I _CRAC-like current. At 0 mV holding potential the estimated current density is –0.1 and –0.2 pA/pF in CPAE and HEK cells respectively, i.e. 15 and 30% of that measured in Jurkat cells. As shown in studies of Jurkat cells, larger Na⁺ currents are detectable in CPAE and HEK cells following store depletion in Ca²⁺- and Mg²⁺-free medium. The current carried by Na⁺ ions is similarly blocked by micromolar La³⁺, is inwardly rectifying and has a positive reversal potential. Received: 12 November 1997 / Received after revision: 11 February 1998 / Accepted: 12 February 1998     

Role of calcium-dependent K+ channels in the regulation of arterial and venous tone by nitric oxide in pigs

Effects of inhibition of calcium-dependent potassium channels (K⁺ _Ca channels) on the regulation of arterial and venous tone by nitric oxide (NO) were studied in anaesthetized pigs following vagotomy and blockade of autonomic ganglia. Selective inhibition of K⁺ _Ca channels by charybdotoxin (CTX, 2 μg/kg iv) or iberiotoxin (IbTX, 1 μg/kg) significantly augmented mean total peripheral resistance (TPR) to levels 30–60% above control. Venous and pulmonary vascular tone were assessed by changes in effective compliances of the venous (EVC) and pulmonary (EPC) vascular beds as calculated from changes in central venous and diastolic pulmonary arterial blood pressure during haemorrhagia (−5 ml/kg) and hypervolaemia (+5 ml/kg). Blockade of K⁺ _Ca channels significantly decreased both EVC (−20 to −30%) and EPC (−30 to −50%). Both CTX and IbTX significantly diminished the vasodilation caused by the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) both during control conditions and following experimental vasoconstriction induced by systemic inhibition of NO-synthesis by NG-nitro-L-arginine methyl ester (L-NAME) or infusion of vasoconstrictor agonists. Dilator effects of the adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent agonist adenosine were only slightly reduced. However, blockade of K⁺ _Ca channels did not increase vasoconstriction induced by L-NAME significantly. These results suggest that activation of vascular K⁺ _Ca channels is an important mechanism by which NO attenuates the constrictor tone of resistance and capacitance vessels in vivo. + _Ca channel blockade during vasoconstriction by agonists The effects of CTX on the haemodynamic responses to infusions of AII, AVP, NA and ET-1, at doses producing similar increases in MAP of about 50 mmHg, are listed in Table 1. The increase in TPR caused by NA and ET-1 was significantly smaller after CTX, whereas the responses to AII and AVP were similar both before and after CTX. To characterize this effect of K⁺ _Ca channel blockade further, we constructed dose-response curves for AII and NA with and without pretreatment with IbTX. The results for TPR are shown in Fig. 5. The constrictor responses to the two lower doses of NA were significantly reduced by IbTX. Received: 12 February 1996 / Accepted: 31 March 1996     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 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     

Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells

Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (K _d = 15.4 ± 4.6 μM; B _max = 1.7 ± 0.5 nmol mg⁻¹ protein); and a low affinity site (K _d = 170 ± 94.5 μM; B _max = 14.2 ± 3.9 nmol mg⁻¹ protein). In myotube membranes only a low affinity binding site for GTP (K _d = 169 ± 39 μM; B _max = 12.3 ± 1.4 nmol mg⁻¹ protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca²⁺]_i. The patterns of Ca²⁺ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca²⁺ responses: (1) a fast increase in [Ca²⁺]_i, followed by a sustained [Ca²⁺]_i elevation, and (2) a slow raising and steady prolonged increase in [Ca²⁺]_i. In myotubes, however only fast Ca²⁺ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca²⁺]_i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca²⁺ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle. This revised version was published online in July 2006 with corrections to the Cover Date.