Ultraviolet photoalteration of late Na+ current in guinea-pig ventricular myocytes.

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca(2+) imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at -80 mV, dialyzed with K(+)-, Na(+)-free pipette solution, and bathed with K(+)-free Tyrode's solution at 22 degrees C. During experiments that lasted for approximately 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from -80 to -40 mV, but had little effect on background current or on L-type Ca(2+) current. Trials with depolarized holding potential, Ca(2+) channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na(+) current (I(Na)). The amplitude of the late inward current sensitive to 100 microM: TTX was increased by 3.5-fold after 20-30 min of irradiation. UVA modulation of late I(Na) may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I(Na).

Phosphorylation-dependent modulation of cardiac calcium current by intracellular free magnesium.

We compared the effects of cytosolic free magnesium (Mg(2+)(i)) on L-type Ca(2+) current (I(Ca,L)) in patch-clamped guinea pig ventricular cardiomyocytes under basal conditions, after inhibition of protein phosphorylation, and after stimulation of cAMP-mediated phosphorylation. Basal I(Ca,L) density displayed a bimodal dependence on the concentration of Mg(2+)(i) ([Mg(2+)](i); 10(-6)-10(-2) M), which changed significantly as cell dialysis progressed due to a pronounced and long-lasting rundown of I(Ca,L) in low-Mg(2+) dialysates. Ten minutes after patch breakthrough, I(Ca,L) density (at +10 mV) in Mg(2+)(i)-depleted cells ([Mg(2+)](i) approximately 1 microM) was elevated, increased to a maximum at approximately 20 microM [Mg(2+)](i), and declined steeply at higher [Mg(2+)](i). Treatment with the broad-spectrum protein kinase inhibitor K252a (10 microM) reduced I(Ca,L) density and abolished these effects of Mg(2+)(i) except for a negative shift of I(Ca,L)-voltage relations with increasing [Mg(2+)](i). Maximal stimulation of cAMP-mediated phosphorylation occluded the Mg(2+)(i)-induced stimulation of I(Ca,L) and prevented inhibitory effects of the ion at [Mg(2+)](i) <1 mM but not at higher concentrations. These results show that the modulation of I(Ca,L) by Mg(2+)(i) requires protein kinase activity and likely originates from interactions of the ion with proteins involved in the regulation of protein phosphorylation/dephosphorylation. Stimulatory effects of Mg(2+)(i) on I(Ca,L) seem to increase the cAMP-mediated phosphorylation of Ca(2+) channels, whereas inhibitory effects of Mg(2+)(i) appear to curtail and/or reverse cAMP-mediated phosphorylation.

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.

Beta-adrenoceptor-coupled Gs protein facilitates the activation of cAMP-dependent cardiac Cl- current.

Here a comparison is made between adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- current (ICl) density and activation time course in response to beta-adrenoceptor stimulation with isoproterenol and adenylyl cyclase activation with forskolin. Saturating concentrations of isoproterenol and forskolin failed to activate an ICl in guinea pig atrial as well as in rat and frog ventricular cardiomyocytes. In guinea pig ventricular cardiomyocytes, step application of 1 microM isoproterenol induced an ICl of -0.89 +/- 0.32 pA/pF (holding potential -40 mV, temperature 22 +/- 1 degrees C). ICl activation started after 3 +/- 1 s, was complete within 44 +/- 9 s, and was abolished after cell dialysis with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. Stimulation with increasing concentrations of forskolin (0.01-10 microM) increased ICl density and accelerated ICl activation. With 1 microM forskolin, ICl density was maximal (-0.57 +/- 0.30 pA/pF) but significantly smaller than that achieved with 1 microM isoproterenol. Although ICl density could not be further augmented by forskolin > 1 microM, current activation (latency 28 +/- 8 s, full activation after 112 +/- 8 s with 1 microM forskolin) was further accelerated by 3 and 10 microM forskolin. However, ICl activation with 10 microM forskolin was still slower than that with 1 microM isoproterenol. A low isoproterenol concentration (1 nM), which did not activate ICl by itself, accelerated the 1 microM forskolin-induced activation of ICl by 35%; this speeding up was abolished after cell dialysis with guanosine 5'-O-(2-thiodiphosphate). ICl deactivation after the washout of 1 microM forskolin or 1 microM isoproterenol followed a similar time course. After stimulation with 10 microM forskolin or 1 microM forskolin + 1 microM isoproterenol, but not with 1 microM forskolin + 1 nM isoproterenol, the decay of ICl was significantly delayed. These results indicate that both cAMP-dependent and cAMP-independent G protein pathways contribute to the regulation of guinea pig ventricular ICl.

PKC-independent inhibition of cardiac L-type Ca2+ channel current by phorbol esters.

Active and inactive phorbol esters were applied to guinea pig ventricular myocytes to study the responses of L-type Ca2+ (ICa,L) and L-type Na+ (INa,L) currents. Phorbol 12-myristate 13-acetate (PMA) (10-100 rM) never stimulated ICa,L or INa,L and frequently depressed them by 5-30% in a voltage-independent manner. However, the phorbol ester consistently activated delayed-rectifying K+ (IK) and Cl- currents. The inhibition of ICa,L occurred approximately 3 times faster than comonitored stimulation of IK, and ICa,L and INa,L were unaffected by two interventions that suppressed IK stimulation [pretreatment with 50 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and dialysis with pCa 11 versus standard pCa 9 solution]. Inactive phorbol esters 4 alpha-phorbol 12,13-didecanoate (alpha-PDD) and 4 alpha-phorbol had little effect on IK, but alpha-PDD had a PMA-like inhibitory effect on Ca2+ channel currents. We conclude that, unlike the stimulation of IK by PMA, inhibition of Ca2+ channel current by phorbol esters is a protein kinase C-independent action.

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells.

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.

Modulation by stimulation rate of basal and cAMP-elevated Ca2+ channel current in guinea pig ventricular cardiomyocytes.

The modulation of L-type Ca2+ current (ICa) by changes in stimulation frequency was investigated in single ventricular cardiomyocytes isolated from guinea pig hearts. Electrical recordings were carried out at 21-25 degrees C and at 33-37 degrees C with the whole-cell patch clamp method, under K(+)-free conditions. A comparison is made between the response to frequency changes for ICa in the basal state and after the application of drugs which elevate the level of adenosine-3',5'-cyclic monophosphate (cAMP) within the cells. Peak basal ICa was reduced with an increase in stimulation rate from 0.5 Hz to 1, 2, 3, 4, or 5 Hz. This frequency-induced reduction of ICa was enhanced by reduced temperature, was unchanged when Na+ or Ba2+ carried the basal Ca2+ channel current, and was greatly enhanced after elevating cAMP levels with forskolin, isoprenaline, or 8-(4-chlorophenylthio)-cyclic AMP. We examined the mechanism of the enhancement of the frequency-induced reduction of ICa by cAMP, and found two conditions which abolished it: (a) application of isoprenaline when Na+ carried the Ca2+ channel current in Ca(2+)-free solution, or (b) application of 3-isobutyl-1-methylxanthine, a broad-spectrum phosphodiesterase inhibitor. It was further shown that an elevation of both ICa and cAMP (induced by isoprenaline), and not an increase of ICa alone (induced by Bay K 8644), is required to produce the extra component of reduction by frequency. It is concluded that Ca2+ entry results in feedback regulation of ICa, through the activation of Ca(2+)-dependent phosphodiesterase(s). This is important in the context of sympathetic stimulation, which produces the companion conditions of an elevated heart rate and increases in cAMP levels and Ca2+ entry.

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)

Modulation of calcium current density by intracellular calcium in isolated guinea pig ventricular cardiomyocytes.

This study describes quantitatively the relation between L-type calcium current (ICa) density and resting cytosolic free calcium concentration ([Ca2+]i) in isolated guinea pig ventricular cardiomyocytes. ICa was measured simultaneously with [Ca2+]i at 22 and 35 degrees C using the whole-cell patch-clamp method in combination with the Fura-2 ratio-fluorescence technique. At 22 degrees C, the increase in [Ca2+]i upon replacing external Na+ with tetraethylammonium resulted in a monotonic decrease in ICa density. Half-maximal inhibition of ICa density occurred at a [Ca2+]i value (K0.5) of 60 +/- 17 nM (mean +/- S.D., n = 59). At 35 degrees C, with a holding potential of -80 mV, a similar relation between [Ca2+]i and ICa was observed (K0.5 of 62 +/- 12 nM, n = 34). When the cells were depolarized from -40 mV, however, the initial decrease in ICa density with increasing [Ca2+]i was usually followed by transient stimulation (< or = 2-fold), which was again overcome by inhibition. Stimulation was not observed in the presence of broad spectrum protein kinase inhibitors. These data suggest that Ca2+i-dependent facilitation of cardiac ICa is mediated by a temperature-sensitive enzymatic pathway that ends in voltage-dependent phosphorylation of Ca2+ channels.