Effects of purinergic stimulation on the Ca current in single frog cardiac cells

Ca current (I _Ca) was measured by whole-cell voltage clamp in single cells isolated from frog ventricle, in which the Na current was inhibited by tetrodotoxin (0.3 M) and K currents were blocked by substituting K with 120 mM intracellular and 20 mM extracellular Cs. The influence of stimulation by ATP (0.1–100 M) was assessed in the presence of propranolol (1 M) or pindolol (0.1 M), prazozin (0.1 M) and atropine (10 M). ATP, in the micromolar range, had two types of effect. Like other P₁-purinoagonists, it antagonized the increase in I _Ca elicited by -adrenostimulation. When added alone, 1 M ATP could increase I _Ca up to twofold. An increase in I _Ca was also observed even after it had been maximally enhanced by intracellularly applied cAMP (50 M). Voltage dependence and kinetics of I _Ca were not affected. These effects were considered to be related to P₂-purinoceptor activation. At higher ATP concentrations the increase in I _Ca was less; at 100 M, ATP reduced I _Ca. The ATP-induced increase in I _Ca was prevented by internal perfusion of the cells with GDP [-S] or neomycin, respectively, to block signal transduction to phospholipase C or its phosphodiesterase activity on the polyphosphoinositides. We conclude that P₂purinoceptor stimulation increases the Ca current in frog ventricular cells by a pathway that might involve phosphoinositide turnover.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes

The effect of intracellular perfusion with cyclic GMP (cGMP) on Ca current (I_Ca) was investigated in Cs-loaded isolated cells from guinea pig ventricle using the whole-cell patch-clamp technique and a perfused patch pipette. cGMP (5 M) strongly reduced I_Ca which had been elevated by intracellular perfusion with 50 M of either cyclic AMP (cAMP) or its hydrolysis-resistant analog 8-Bromo-cAMP. In addition, cGMP prevented the stimulation of I_Ca by IBMX, a phosphodiesterase inhibitor. The membrane permeant cGMP analog 8-Bromo-cGMP (100 M), when applied outside the cell, also antagonized the stimulatory effect of IBMX on I_Ca. It is concluded that cGMP inhibits I_Ca in guinea pig ventricular cells by a mechanism different from the activation of a cGMP-stimulated phosphodiesterase recently found in frog ventricular cells.This work was partly supported by an exchange program INSERM/CNR.     

Inhibition of the M current in NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1–5 M bradykinin inhibited I _M by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 M and 1 M PDBu diminished I _M to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35–43 min in a concentration of 0.3 M significantly reduced the effect of 1 M PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51–64 M), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185–207] that the bradykinin effect on I _M is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of I _M by 1 M PDBu was fully developed, 0.1 M bradykinin produced a further inhibition of I _M. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 M bradykinin significantly but did not abolish it. Staurosporine (0.3 M, applied for 31–46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutylmethylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.     

Role of the GTP-binding protein Gs in the β-adrenergic modulation of cardiac Ca channels

In the heart, the guanosine 5-triphosphate (GTP)-binding protein G_s is activated by hormone binding to -adrenergic receptors and stimulates the intracellular cyclic adenosine 3,5-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A [28]. Additionally, G_s can modulate cardiac Ca channels directly in cell-free systems [57]. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (I _Ca) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained.First, typically, the I _Ca density increased from 12 to 40 A/cm² following application of 1 M isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl₂. However, 1 M ISP enhanced I _Ca only from 9 to 17 A/cm² after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPS (the Rp-isomer of adenosine 3,5-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on I _Ca. Thus, Rpc-AMPS unmasks a GTP-dependent component of the -adrenergic stimulation of I _Ca, which probably reflects the direct stimulation of Ca channels by G_s under block of cAMP-dependent phosphorylation.Second, in cells under dialysis with 100 or 200 M cAMP, bath application of 20–40 M 3-isobutyl-1-methylxanthine (IBMX) enhanced the I _Ca density to about 41 A/cm² indicating saturation of the cAMP pathway. Under this condition, 1 M ISP was without significant effect on I _Ca. This result may suggests that direct G_s stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the G_s pathway in intact myocytes.Third, simultaneous application of 1 M ISP and 40 M IBMX enhanced I _Ca up to densities of around 75 A/cm² during cell dialysis with 100 M cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that G_s is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of G_s may act to prime L-type Ca channels for cAMP-dependent phosphorylation during -adrenergic stimulation of cardiac myocytes.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

Isoproterenol modulates the calcium channels through two different mechanisms in smooth-muscle cells from rabbit portal vein

Previous data from our laboratory indicated that the slow Ca²⁺ channel of vascular smooth muscle cells was regulated by cyclic nucleotides. In the present study, the effects of isoproterenol (ISO) on L-type calcium current (I _Ca(L)) were investigated in freshly-isolated single smooth-muscle cells from the rabbit portal vein using the whole-cell voltage-clamp technique. With high-Cs⁺ solution in the pipette and physiolocial salt solution (containing 2.0 mM Ca²⁺) in the bath, (I _Ca(L)) was recorded. At a holding potential of –80 mV, low concentrations of ISO ( 100 nM) increased I _Ca, whereas higher concentrations (1–100 M) transiently increased I _Ca but then inhibited it persistently. At 10 M ISO, I _Ca was initially increased by 44±9%, and was subsequently decreased by 24±3%. Pretreatment of cells with 30 M H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride] caused the first phase to persist and the second inhibitory phase to disappear. Intracellular application of 1 mM GDP[S] (guanosine 5-O-2-thiodiphosphate) abolished both phases of ISO action. In contrast, intracellular application of 100 M GTP caused the initial stimulatory phase of ISO action to be significantly potentiated; the later inhibitory phase was slightly diminished. In addition, the activated G protein subunit (G_s ) mimicked the stimulatory effect of ISO. Pertussis toxin had no effect on either phase of the ISO action. These results suggest that ISO modulates the Ca²⁺ channel through mechanisms that involve the pertussis-toxin-insensitive G protein(s). That H-7, a nonspecific inhibitor of protein kinases, blocked the second phase but not the first phase indicates that the actions of ISO are mediated via two different pathways. One pathway (for inhibition) is more indirect, and may be mediated by the adenylate cyclase/cAMP/protein-kinase-A cascade. The other pathway (for stimulation) is more direct, and may reflect a type of G protein gating of the Ca²⁺ channel.     

On the mechanism of β-adrenergic regulation of the Ca channel in the guinea-pig heart

Dose-response relations for the increase in the amplitude of Ca current (I _Ca) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10^–9 M, for cAMP 3 M (pipette concentration to which 10^–5 M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 M (pipette concentration). The concentrations for the half-maximal effect were 3.7×10^–8 M (ISP), 5.0 M (cAMP) and 0.95 M (C subunit) and for the maximum effect 10^–6 M (ISP), 15–20 M (cAMP) and 3–4 M (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3–4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit onI _Ca were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects onI _Ca could be estimated and were compared with those obtained in broken cell preparations.A competitive inhibitor of phosphorylation, 5-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit onI _Ca. Cell dialysis with 3 mM adenosine-5-(-thio)-triphosphate, which produces a dephosphorylationresistant phosphorylation, markedly potentiated the effects of ISP and cAMP onI _Ca.The results support the hypothesis that phosphorylation of a protein within, or close to, the Ca channel by cAMP-dependent protein kinase is the mechanism of -adrenergic stimulation.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38 (membranforschung), Projekt G, and H0579/6-2     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

α 1- and β-adrenergic interactions on L-type calcium current in cardiac myocytes

We investigated the mechanism by which ₁-adrenergic activation regulates basal and stimulated whole cell L-type Ca current (I_Ca) in rat ventricular myocytes using the physiological neurotransmitter, norepinephrine (NE, 10M). Stimulation of ₁-adrenoceptors, achieved by NE+10M esmolol (a -receptor antagonist), had no significant effect on basal I_Ca. ₁-adrenergic activation had a marked inhibitory effect on I_Ca elevated by activation (NE+1M) prazosin, an ₁-receptor antagonist) or activation of adenylyl cyclase by forskolin (25M); the inhibitory effect was reversible upon washout. However, ₁-adrenergic stimulation had no significant effect on I_Ca previously increased by intracellular application of cAMP (25M). The inhibitory effect seen on I_Ca elevated by NE showed no significant shift of either I–V or inactivation curves. It is unlikely that the inhibitory effect of ₁-adrenergic stimulation on NE or forskolin-elevated I_Ca is mediated through activation of Ca-dependent protein kinase C or changes in intracellular free Ca (pCa=8.5, EGTA 5 mM) or cAMP-dependent phosphodiesterase. We conclude that ₁-adrenergic inhibition of -adrenergic stimulated-I_Ca is probably mediated through an as yet unknown G-protein. This inhibitory effect could serve as a regulatory feedback mechanism in physiological and pathophysiological settings.     

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.     

Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons

Modulation by protein phosphorylation of the relation between acetylcholine (ACh)-activated current (I _ACh) and adenosine triphosphate-(ATP)-activated current (I _ATP) was investigated with the whole-cell voltage-clamp technique in rat sympathetic neurons. During simultaneous activation by 100 M ATP of an inward current, the current evoked by 100 M ACh was reduced to 60–70% of that in the absence of ATP. Effects of compounds that are known to modulate protein phosphorylation were tested by including them in the intracellular solution. The reduction ofI _ACh by ATP was not observed when K252a (1 M), a non-selective protein kinase inhibitor, adenosine 5-O-(3-thiotriphosphate) (ATP[S], 1 mM) or,-methylene ATP (1 mM) were included in the intracellular solution. Activators of protein kinases, adenosine 3,5-cyclic monophosphate (cAMP, 100 M), guanosine 3,5-cyclic monophosphate (cGMP, 100 M), phorbol 12-myristate 13-acetate (PMA, 1 M), also abolished the reduction by ATP ofI _ACh. The effects of okadaic acid, a protein phosphatase inhibitor, were paradoxical: okadaic acid (2 M) itself abolished the reduction by ATP ofI _ACh but it antagonized the abolishment by cAMP or cGMP of the reduction ofI _ACh. Okadaic acid did not affect the disappearance of the reduction ofI _ACh by ATP in the presence of intracellular PMA. The results suggest that the interaction betweenI _ACh andI _ATP is regulated by protein phosphorylation/dephosphorylation. Possible mechanisms underlying the effects of these modulators of protein phosphorylation are discussed.     

Effects of a protein phosphatase inhibitor,okadaic acid,on membrane currents of isolated guinea-pig cardiac myocytes

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca²⁺ current (I _Ca) was recorded as peak inward current in response to test pulse to O mV. Extracellular application of OA (5–100M) produced an increase ofI _Ca. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.I _Ca was increased from 11.3±0.8A cm^–2 to 19.0±1.1A cm^–2 (n=4) by 5M-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the -adrenergic increase ofI _Ca was markedly prolonged by OA. The -adrenergic stimulation of cardiac Ca²⁺ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA onI _Ca is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.     

Effect of a series of 1-alkyl ether lipids on inhibition of phospholipase A2 activity and PAF responses

Several 1-alkyl ether lipids were studied for their ability to inhibit PLA₂ and antagonize PAF responses. Studies with synthetic micellar substrate (1-stearyl-2-arachidonyl phosphocholine), at concentrations ranging from 0.02 to 1000M, demonstrate that CL 118326 inhibits porcine pancreatic PLA₂ in vitro. As the substrate concentration increases, there is a dose-dependent increase in the IC₅₀ value (IC₅₀ ranges: 1.6–84.6g/ml or 2.6–137M). CL 118326 inhibits mammalian pancreatic PLA₂, but not snake or bee venom PLA₂. CL 118326 inhibits thrombin (IC₅₀ =7.9M), but not Na arachidonate- (IC₅₀ > 100M) induced platelet aggregation, indicative of inhibition of cellular PLA₂. CL 118326 inhibits other PLA₂-dependent processes such as antigen-induced leukotriene (LTC₄) release (IC₅₀=2.3g/ml or 3.8M) and histamine release (IC₅₀=1.4g/ml or 2.2M) in basophil-enriched WBCs. Intradermal coinjection of CL 118326 (10g) with PLA₂ into guinea pig skin inhibits pancreatic PLA₂-induced increase in vascular permeability and leakage, but not snake or bee venom PLA₂-induced leakage. CL 118326 shows no PAF-like agonist activity in stimulating rabbit platelet-rich plasma. It inhibits PAF-induced aggregation (IC₅₀=5.8M), but not ADP-induced aggregation. CL 118326 has greater efficacy as a PLA₂ inhibitor than as a PAF antagonist since the IC₅₀-substrate concentration ratio for PLA₂ inhibition is <- 1.0 at substrate concentrations of 10–1000M while the IC₅₀-agonist ratio for PAF antagonism is > 100. Results for four other compounds related to CL 118326 are also presented.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

Guanylate-cyclase-mediated inhibition of cardiac I Ca by carbachol and sodium nitroprusside

We studied the role of cyclic guanosine monophosphate (cGMP) as a mediator of the reduction of L-type calcium current (I _Ca) induced by muscarinic receptor stimulation and by nitric oxide in isolated guinea-pig ventricular cells using the whole-cell patchclamp technique. Our results show that when the level of cyclic adenosine monophosphate was increased by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), stimulation of a pertussis-toxin (PTX)-sensitive muscarinic receptor by carbachol (1 M) reduced the calcium current increase from 80.6±23.5% to 19.8±9.6% over the control and this effect was prevented by methylene blue (10 M), an inhibitor of the soluble guanylate cyclase. Pipette solution containing 10 M cGMP reduced the enhancement of I _Ca by IBMX from 121.9±11.6% to 14.2±5.4% above the control. Sodium nitroprusside (10 M), a spontaneous donor of nitric oxide, and consequently a stimulator of soluble guanylate cyclase, also reduced IBMX-stimulated I _Ca from 115.2±13.2% to 32.2±6.9% above control and the sodium nitroprusside effect was also suppressed by methylene blue. The latter two reagents were ineffective on basal I _Ca.