Depression Of ICa Research Articles

Involvement of Src in L-type Ca2+ channel depression induced by macrophage migration inhibitory factor in atrial myocytes

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca2+ currents (ICa,L) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of ICa,L in human atrial myocytes and in HL-1 cells. Basal ICa,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α1C subunit of L-type Ca2+ channel (LCC α1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y416) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress ICa,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak ICa,L in HL-1 cells by ∼69% and ∼83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of ICa,L and down-regulated LCC α1C subunit levels. The depression of ICa,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing ICa,L amplitudes through impairment of channel function, down-regulation of LCC α1C subunit levels, and the activation of c-Src kinases in atrial myocytes.

Reply from N. Gamper and M. S. Shapiro

Ours is one of a number of groups that have shown N- and P/Q-type Ca2+ channels to be sensitive to the abundance of PIP2 in the plasma membrane (Wu et al. 2002; Gamper et al. 2004; Lechner et al. 2005; Robbins et al. 2006; Suh et al. 2006). The evidence comes from cloned channels expressed in Xenopus oocytes studied in inside-out patches and from endogenous channels of sympathetic neurons in which [PIP2] was manipulated with phosphoinositide (PI) phosphatases, blockers of PI kinases, PIP2 scavengers and by PIP2 dialysis from whole-cell pipettes. All these experiments strongly suggest that depletion of PIP2 from plasma membrane does induce suppression of ICa of N- and P/Q-types. Of particular note is the N-type ICa depression observed upon PIP2 depletion produced by rapamycin-induced plasma membrane recruitment of a PIP2 5-phosphatase in mammalian cells (Suh et al. 2006). This is a receptor- and PLC-free experiment in which no further second messengers are produced. On the other hand, much evidence also indicates that [PIP2] can be strongly depleted in the neurons by stimulation of Gq/11-coupled M1R stimulation (Suh & Hille, 2002; Zhang et al. 2003; Horowitz et al. 2005; Winks et al. 2005; Hughes et al. 2007). Taken together, these two lines of evidence make M1R-mediated suppression of ICa by depletion of [PIP2] a mechanism logical to most investigators. In spite of all this, however, we accept a role for arachadonic acid (AA) in tuning the sensitivity of the Ca2+ channels to small or local depletions of PIP2, as is plausible given that AA is produced downstream of PIP2 hydrolysis and consequential diacylglycerol production via the actions of diacylglycerol lipase. Indeed, we explicitly consider such a mechanism for ensuring fidelity in spatiotemporally limited receptor signalling (Gamper et al. 2004; Delmas et al. 2005). Finally, our review article in the special issue of The Journal of Physiology (Gamper & Shapiro, 2007), dedicated to the question of the regulation of ion channels by phosphoinositides, is not focused on voltage-gated Ca2+ channels (particularly, we do not discuss L-type channels at all) but rather on a few difficult issues in phosphoinositide signalling, such as receptor specificity, the affinity of PIP2-binding sites and technical problems in phosphoinositide detection and manipulation, which are often left behind in such discussions. In this sense, we whole-heartedly agree with the Rittenhouse lab: the biochemistry of phosphoinositide interactions with membrane proteins is not ‘simple’ and we do not hold a ‘PIP2-centric’ view of G protein-mediated signalling. Nevertheless, the widespread and powerful role of phosphoinositide signals in regulation of ion channels and transporters cannot be ignored, as the recent special issue of The Journal of Physiology (Robertson, 2007) clearly shows.

Reversible Heart Failure in Gαq Transgenic Mice

For many patients with cardiac insufficiency, the disease progresses inexorably to organ dilatation, pump failure, and death. Although there are examples of reversible heart failure in man, our understanding of how the myocardium repairs itself is limited. A well defined animal model of reversible heart failure would allow us to better investigate these restorative processes. Receptors that activate Galpha(q), a signal transduction molecule in the heterotrimeric G protein superfamily, are thought to play a key role in the development of heart failure. We demonstrated previously that mice expressing a recombinant Galpha(q) protein, the activity of which can be turned on or off at will in cardiac myocytes, develop a dilated cardiomyopathy with generalized edema and heart failure following activation of the protein (Fan, G., Jiang, Y.-P., Lu, Z., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (2005) J. Biol. Chem. 280, 40337-40346). Here we report that the contractile dysfunction and pathological structural changes in the myocardium improved significantly after termination of the Galpha(q) signal, even in animals with overt heart failure. Abnormalities in two proteins that regulate Ca(2+) handling in myocytes, phospholamban and the voltage-dependent L-type Ca(2+) channel, were also reversed, as was the increased expression of genes that are associated with heart failure. These results indicate that the heart has a substantial reparative capacity if the molecular signals responsible for the myocardial dysfunction can be identified and blocked.

Gαq Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase

Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Galpha q on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Galpha q-estrogen receptor hormone-binding domain fusion proteins (Galpha qQ209L-hbER or Galpha qQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but Galpha qQ209L-AA-hbER cannot activate the canonical Galpha q effector phospholipase Cbeta (PLCbeta). L-type Ca2+ current (I(Ca,L)) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Galpha q animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Galpha q does not require PLCbeta. To investigate the role of PI3K in this inhibitory effect, I(Ca,L) was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect I(Ca,L), but it fully restored I(Ca,L) density in both Galpha q transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110beta/p85alpha or p110gamma PI3K in the presence of PI(4,5)P2 also restored I(Ca,L) density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which sequesters PI(3,4,5)P3, reduced the peak I(Ca,L) density in wild-type myocytes by approximately 30%. Taken together, these results strongly suggest that the inhibitory effect of Galpha q on the cardiac LTCC is mediated by inhibition of PI3K.

Gabapentin decreases membrane calcium currents in injured as well as in control mammalian primary afferent neurons

Background and Objectives: Neuropathic pain following injury to peripheral sensory neurons is a common clinical problem and frequently difficult to treat. Gabapentin (GBP), a novel anticonvulsant, has significant analgesic effects in clinical neuropathic states and in relevant preclinical models, but its mechanism of action remains unclear. Because calcium currents play a significant role in neuronal function, this study was designed to assess the effect of GBP on the membrane voltage-activated inward calcium currents (ICa) in dorsal root ganglia (DRG) primary afferent neurons of neuropathic versus control rats. Methods: Male rats were prepared according to the chronic constriction injury (CCI) model. The L4 and L5 dorsal root ganglia of those selected as CCI or control after appropriate behavioral testing were removed, and neurons were enzymatically dissociated. Fluorescent dye (DiI) placed at the injury site allowed identification of neurons projecting to that site. These were acutely studied using whole-cell, perforated (with [beta ]-escin) patch-clamp recordings. Additionally, neurons from sham or nonoperated rats were also studied. Results: Although there was marked variability among cells, concentrations of GBP ranging from 0.1 to 300 [mu ]mol/L decreased neuronal peak ICa in midsized neurons (30 to 40 [mu ]m) of both sham and neuropathic rats, in a fast, reversible, and concentration-dependent manner. Intergroup differences were not significant, however the concentration-response EC50s were 2.7 [mu ]mol/L for the sham and 16.5 [mu ]mol/L for the CCI neurons. The drug suppressed ICa in nonoperated rats to a lesser degree, but changes did not differ significantly from the operated groups. Calcium currents in either small or large diameter neurons were also variably decreased by 10 [mu ]mol/L of GBP in sham and CCI neurons. Current inhibition by GBP was partly voltage dependent. Conclusions: GBP, at clinically relevant concentrations, results in significant reduction of ICa in both sham and neuropathic neurons, while in nonoperated rats reduced ICa to a smaller degree. Sensitivity to drug was not affected by neuropathy. This current inhibition is partly voltage dependent. Depression of ICa may be partly related to the binding of the drug to the [alpha ]2[delta ] modulatory subunit of the voltage activated calcium channels (VACC). Analgesia may be due to diminished release of neurotransmitter by sensory neurons, a Ca2+-dependent process. Reg Anesth Pain Med 2002;27:47-57.

Cellular Basis for the Acute Inhibitory Effects of IL-6 and TNF- α on Excitation-contraction Coupling

There is controversy over whether nitric oxide (NO) mediates acute negative inotropic actions of cytokines including tumor necrosis factor-alpha (TNF- α). The reports from established laboratories have appeared inconsistent, which could be due to species differences. Thus, we tried to elucidate the mechanisms underlying negative inotropic actions of interleukin-6 (IL-6) and TNF- α in the same model. We studied the effects of cytokines on [Ca2+]itransients (using indo-1), cell shortening (CS) (using a video motion detector) and thel -type Ca2+channel current (ICa) (using the whole cell perforated patch clamp technique) in isolated guinea-pig ventricular myocytes. IL-6 (1000 U/ml) or TNF- α (500 U/ml) decreased both peak systolic [Ca2+]i(IL-6: 0.43±0.01 to 0.40±0.01, n=5, P<0.05; TNF- α: 0.42±0.02 to 0.39±0.02, n=5, P<0.05) and the amplitude of CS (IL-6: 7.5±0.9 to 6.2±0.5 μ m, n=5, P<0.05; TNF- α: 6.7±0.7 to 5.8±0.7 μ m, n=5, P<0.05) without detectable reductions in ICa(IL-6: 0.9±0.1 to 0.9±0.1 nA, n=4, N.S.; TNF- α: 1.1±0.3 to 1.1±0.2 nA,n =4, N.S.) within 5 min. The nitric oxide synthase (NOS) inhibitor, NG-monomethyl- l arginine (300 μ mol/l), blocked the effects of IL-6 but not of TNF- α. When pretreated with 20 nmol/l isoproterenol, exposure to IL-6 decreased both ICa(2.8±0.5 to 2.0±0.3 nA) and the amplitude of CS (10.4±2.4 to 7.5±1.9 μ m) within 5 min. TNF- α also clearly depressed ICa(2.9±0.9 to 2.3±0.7 nA) and the amplitude of CS (7.0±1.4 to 5.5±1.3 μ m) inβ -adrenergic stimulated cells. TNF- α significantly increased the content of sphingosine (product of sphingomyelin pathway) in isolated heart. The effects of low dose sphingosine (5 μ mol/l) mimicked those of TNF-α on cardiac myocytes. IL-6 produced an acute negative inotropic effect through a NO-dependent pathway while TNF-α did so via a sphingomyelin-dependent pathway in isolated guinea-pig ventricular myocytes.

Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition.

In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.

Volatile Anesthetic Sensitivity of T-Type Calcium Currents in Various Cell Types

We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells-adrenal glomerulosa cells (AG) and thyroid C-cells-ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47% +/- 4% (n = 6) by isoflurane (0.7 mM) and 56% +/- 2% (n = 5) by enflurane (1.2 mM), but by only 24% +/- 1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability. Implications: Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics. (Anesth Analg 1999;88:168-73)

Volatile anesthetic sensitivity of T-type calcium currents in various cell types.

We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells--adrenal glomerulosa cells (AG) and thyroid C-cells--ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47%+/-4% (n = 6) by isoflurane (0.7 mM) and 56%+/-2% (n = 5) by enflurane (1.2 mM), but by only 24%+/-1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability. Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics.

The mode of action of several opioids on cardiac muscle.

The objective of the experiments was to investigate the cellular basis of the inotropic effect on myocardium of several opioids which have been implicated in producing cardiotoxic effects in human poisoning. Opioids exerted negative inotropic effects, with half-maximal concentrations between 10 microM (dextropropoxyphene) and 118 microM (pethidine); all agents reduced the magnitude of the intracellular Ca2+ transient and the L-type Ca2+ current, ICa, over a similar concentration range to that which reduced twitch tension. The depression of ICa correlated positively with the value of the opioid oil-water partition coefficient. Effects were not antagonized by the opioid receptor antagonist naloxone. Action potential upstroke rate was also reduced but at significantly higher concentrations. Resting potential and action potential duration were not consistently affected; none of the opioids tested altered intracellular pH. These data suggest that opioids exert a negative inotropic effect by their action on ICa; blockade of the Na+ current is not great enough to exert a significant action. The lack of effect of naloxone implies the actions are independent of the opioid receptor. The correlation of effects with the oil water partition coefficient implies a non-specific effect dependent on the hydrophobicity of the agent.

Cyclic AMP Does Not Induce the Down-Regulation of Calcium Current in Molluscan Neurons Through Kinase A Activation or Cytoplasmic Ca2+ Elevation

In isolated neurons of the land snail, a high threshold voltage-activated calcium current (ICa) was recorded using the two microelectrode voltage clamp method. Iontophoretic intracellular injection of cAMP or extracellular application of dbcAMP evoked a rapid and reversible decrease in peak amplitude of ICa in 30 out of 54 cells tested. The effects of cAMP on ICa and IBa were similar. cGMP injected into the same cell from another barrel of a multi-barreled micropipette did not mimic the effect of cAMP. The cyclic nucleotide phosphodiesterase activator, imidazole, caused an augmentation of ICa in 8 out of 13 cells tested. Intracellular injection of the Ca2+-chelator, EGTA, had no influence on the extent of cAMP-evoked depression of ICa. The inhibitors of kinase A, tolbutamide and H-8, evoked an effect resembling the effect of cAMP, i.e. depression of ICa. The results suggest that the down-regulation of ICa by cAMP is achieved through neither kinase A activation nor through cytoplasmic Ca2+ elevation.

Comparative actions of propofol and thiopentone on cell membranes of isolated guineapig ventricular myocytes

We have examined the effects of propofol and thiopentone on membrane potentials and currents of isolated guineapig ventricular myocytes using the whole-cell patch-clamp technique. After current clamping, propofol at concentrations greater than 0.5 mumol litre-1 shortened the plateau and action potential duration (APD) (P < 0.05). Thiopentone 10 mumol litre-1 prolonged APD (P < 0.05), whereas concentrations of 50 mumol litre-1 or higher decreased plateau height (P < 0.05) and resting membrane potential (RMP) (P < 0.05) with abbreviation of the prolonged APD. With voltage clamping, propofol 1 mumol litre-1 decreased the L-type Ca2+ current (ICa,L) to 88.4% of control (P < 0.01) without affecting the delayed rectifier K+ current (IK) and propofol 10 mumol litre-1 decreased ICa,L and IK to 75.0% (P < 0.01) and 78.4% (P < 0.01), respectively, with no effect on the inward rectifier K+ current (IK1). Thiopentone 10 mumol litre-1 decreased ICa,L to 88.5% (P < 0.01) and IK to 78.3% (P < 0.05), while thiopentone 100 mumol litre-1 depressed ICa,L to 82.8% (P < 0.01), IK to 27.0% (P < 0.01) and IK1 to 67.3% (P < 0.05). These results indicated that propofol, at concentrations greater than those that are clinically relevant, shortened APD mainly by suppression of ICa,L, and the biphasic effects on APD by thiopentone were caused by depression of IK, and concomitant suppression of ICa,L and IK1 at higher concentrations. The distinct cardiodepressant effects of propofol and thiopentone may be, at least in part, attributed to different actions on membrane Ca2+ and K+ currents.

Isoflurane and sevoflurane modulate inactivation kinetics of Ca2+ currents in single bullfrog atrial myocytes.

To clarify the mechanism(s) of anesthetic depression of myocardial Ca2+ currents, the effects of isoflurane and sevoflurane on the inactivation kinetics of Ca2+ current in single bullfrog atrial myocytes were studied. Freshly isolated bullfrog atrial myocytes were obtained with an enzymatic dispersion procedure. Ca2+ currents were recorded with a whole-cell voltage-clamp technique. Both isoflurane (1.25, 2.5, 5.0 vol%) and sevoflurane (2.5, 5.0 vol%) decreased the peak amplitude of Ca2+ current ICa with a minimal change in the time to peak and the reversal potential. The inactivation kinetics studies revealed that (1) isoflurane (2.5 vol%) and sevoflurane (5.0 vol%) markedly reduced the time constant of inactivation in ICa to 55% and 75% of control, respectively; (2) isoflurane (2.5 vol%) shifted the midpoint (V1/2) of steady-state inactivation curve of ICa toward negative by 2.3 mV; and (3) isoflurane (2.5 vol%) delayed the reactivation time constant of ICa to 119% of control. The further computer-simulation study demonstrated that the observed decrease of time constant by isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) can explain the reduction in amplitude of ICa. The depression of ICa by lower concentration of isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) mainly is due to the decrease of time constant and, at higher concentration, isoflurane and sevoflurane may affect the other membrane components.

SD‐3212, a new class I and IV antiarrhythmic drug: a potent inhibitor of the muscarinic acetylcholine‐receptor‐operated potassium current in guinea‐pig atrial cells

1. By use of patch-clamp techniques, the effects of SD-3212, a novel antiarrhythmic drug, on the calcium current (Ica), the sodium current (INa) and the muscarinic acetylcholine-receptor-operated potassium current (IK.ACh) were examined and compared with those of bepridil in guinea-pig single atrial cells. 2. SD-3212 inhibited ICa and INa in a concentration-dependent manner. The IC50 values of SD-3212 for inhibition of ICa and INa were 1.29 microM and 3.92 microM, respectively. The steady state inactivation curves of ICa and INa were shifted in the hyperpolarizing direction in the presence of 1 microM SD-3212. Similar inhibition of ICa and INa was also observed with bepridil. The IC50 values of bepridil for depression of ICa and INa were 1.55 microM and 4.43 microM, respectively. 3. The muscarinic acetylcholine-receptor-operated potassium current (IK.ACh) was activated by the extracellular application of 1 microM carbachol in the GTP-loaded cells or by the intracellular loading of GTP gamma S, a nonhydrolysable GTP analogue. SD-3212 potently inhibited the carbachol- and GTP gamma S-induced IK.ACh and the IC50 values were 0.38 microM and 0.20 microM, respectively. These IC50 values were very close and about 10 times lower than those for inhibiting ICa and INa. Bepridil also suppressed the carbachol- and GTP gamma S-induced IK.ACh with the IC50 values of 0.69 microM and 0.84 microM, respectively. 4. In guinea-pig atrial cells stimulated at 0.2 Hz, carbachol at a concentration of 1 microM markedly shortened action potential duration. Both SD-3212 (0.1-1 microM) and bepridil (1-10 microM) reversed the action potential shortening in a concentration-dependent manner. The antagonizing effect of SD-3212 on the carbachol-induced action potential shortening was more potent than that of bepridil. 5. These results suggest that SD-3212 inhibits IK.ACh by depressing the function of the potassium channel itself and/or associated GTP-binding proteins. SD-3212 is a unique antiarrhythmic drug, which potently inhibits IK.Ach in addition to its class I and IV effects. SD-3212 and bepridil may be useful for the termination and prevention of vagally-induced atrial flutter and fibrillation.

Activity-induced facilitation of L-type Ca2+ current in cardiomyocytes isolated from guinea-pig ventricles.

The facilitation of L-type Ca2+ current (ICa), which sometimes occurs with an increase in stimulation frequency, was investigated in single guinea-pig ventricular cardiomyocytes using whole-cell recording and K(+)-free solutions. With a holding potential of -80 mV, an increase in frequency from 0.5 to 1, 2, 3 or 4 Hz caused either a small or large initial reduction, or a transient enhancement (facilitation) of peak ICa, which developed rapidly and was followed by a reduction of ICa. Reducing the frequency to 0.1 or 0.2 Hz caused a depression of ICa on the first pulse that was followed by a slower increment. Transient facilitation and depression were entirely absent when either Ba2+ or Na+ was used as the charge carrier in Ca(2+)-free solutions. High concentrations of isoprenaline (1-3 microM), forskolin (1-3 microM), or 8-(4-chlorophenylthio)-cAMP (150 microM) suppressed but did not abolish the incidence and size of facilitation; employing a holding potential of -40 mV also suppressed the incidence of ICa facilitation. Lower isoprenaline concentrations (0.1 and 0.3 microM) greatly enhanced the incidence and magnitude of the transient facilitation occurring with an increase in stimulation rate, but did not diminish the magnitude of the ensuing reduction. When facilitation occurred with 0.1 mM EGTA in the dialysate, or the usual 5 mM EGTA with 0.01 microM extracellular isoprenaline, it developed more slowly after an increase in frequency. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, an increase in stimulation rate from 0.5 to 1, 2 or 3 Hz sometimes caused a large and sustained facilitation of ICa, which developed over tens of seconds, declined slowly with continued stimulation and was maintained after returning to 0.5 Hz. It is concluded that the levels of intracellular cAMP and Ca2+ mediate the initial sensitivity of ICa to changes in stimulation rate, to the extent that they determine whether or not transient facilitation will occur. Because the reduction of ICa was relatively constant, facilitation dictates the level of steady-state ICa that will be reached at the higher rate. Taken together with the fact that facilitation can be modulated, the results argue for separate mechanisms of facilitation and reduction for ICa. It is suggested that the mechanism of facilitation is partly enzymatic, insofar as sustained facilitation could be a manifestation of a stimulatory Ca(2+)-dependent process, which is normally counteracted by the action of phosphodiesterases.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of halothane and diltiazem on L-type calcium currents in single smooth muscle cells from rabbit portal veins

We have studied the effects of halothane and diltiazem on L-type voltage-dependent calcium currents (ICa) in single smooth muscle cells from rabbit portal veins using a whole cell voltage clamp technique. The threshold of ICa was -30mV and the peak current was reached at 0mV. Halothane (0.25, 0.5, 1.0, 1.5 and 2.0%) decreased ICa in a concentration-dependent manner and shifted the ICa activation threshold to the depolarizing side. Halothane 2.0% abolished ICa. Diltiazem 10(-8)-10(-6) mol litre-1, a calcium channel antagonist, also depressed ICa in a concentration-dependent manner. Administration of both 0.5% halothane and diltiazem 10(-7) mol litre-1 (concentrations lower than the clinical therapeutic range) abolished ICa; however, halothane did not exhibit use-dependent inhibition of ICa whereas diltiazem showed partial use-dependency. We conclude that the decrease in ICa produced by halothane is associated with a direct vasodilator effect of this anaesthetic, but is not explained by block of Ca2+ channels similar to the action of diltiazem. Furthermore, administration of low concentrations of both halothane and diltiazem decreased ICa and may reduce the contractility of the vascular smooth muscle cells.

Potentiating and depressant effects of metabotropic glutamate receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion neurons from postnatal mice

This study was aimed at clarifying the role of metabotropic glutamate receptors (mGluRs) in the regulation of intracellular Ca2+ concentration ([Ca2+]i in postnatal mouse retinal ganglion neurons (RGNs). RGNs were maintained for 1-2 weeks in vitro by adding brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) to the culture medium. In order to select these cells for electrophysiological measurements, RGNs were vitally labelled with an antibody against Thy-1.2. Voltage-activated Ca2+ currents [ICa(V)] were recorded with patch electrodes in the whole-cell configuration. It was found that racemic +/--1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) or its active enantiomer 1S,3R-ACPD rapidly and reversibly either enhanced or depressed ICa(V). Quisqualate (QA), L-2-amino-4-phosphonobutyrate (L-AP4) and the endogenous transmitter glutamate induced similar effects when ionotropic glutamate receptors were blocked with D-2-amino-5-phosphonovalerate (D-APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). omega-Conotoxin GVIA (omega-CgTx GVIA), but not nifedipine prevented modulation of ICa(V) by mGluR agonists. The depression of ICa(V) by t-ACPD was irreversible when cells were dialysed with guanosine-5'-O-(3-thiotriphosphate) (GTP[gamma-S]). Ratio measurements of fura-2 fluorescence in Thy-1+ cells showed that neither t-ACPD, QA nor L-AP4 affected [Ca2+]i by liberation of Ca2+ from intracellular stores. Our results suggest that cultured RGNs express mGluRs. These receptors cannot induce Ca2+ release from intracellular stores but regulate [Ca2+]i by a fast and reversible, G-protein-mediated action on a subpopulation of voltage-activated Ca2+ channels.

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels, and it is suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). Figure 11 summarizes the possible mechanisms by which uterine contractility can be modulated. In contrast to vascular smooth muscle, neither ISO nor adenosine, which produce elevation of cyclic AMP, affected ICa and INa. Therefore, no arrow can be drawn between cA-PK/cG-PK and the Ca2+ slow channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).