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We have expressed muscle embryonic (alpha beta gamma delta) and adult (alpha beta delta epsilon) nicotinic acetylcholine receptors in Xenopus Laevis oocytes and measured their current reversal potentials in the presence of extracellular Na+, Ca2+, Sr2+ or Ba2+ ions. The ionic permeability ratio PCa2+/PNa+ was increased about 3 fold by the change in the subunit composition of the nicotinic acetylcholine receptor (replacement of the gamma by the epsilon subunit). A similar increase was also found when permeability to Ba2+ and Sr2+ ions was studies. Comparison of the nicotinic Ca2+ currents recorded from oocytes injected with embryonic or adult receptor subunit combinations also showed that the Ca2+ influx was significantly increased by expressing the epsilon subunit. This increase was estimated to change the contribution of the Ca2+ current to the total net inward current from 0.8% (in the case of the alpha beta gamma delta receptor) to 2.5% (in the case of the alpha beta delta epsilon receptor). Taken together, these results suggest that important modifications in the acetylcholine-mediated Ca2+ influx occurred during muscle innervation and underline the role of the nicotinic receptor in the developmental regulation of Ca2+ influx.

The class A Ca2+ channel alpha 1 subunit (alpha 1A) was expressed in Xenopus oocytes alone or in combination with the beta 1b, beta 2a, beta 3, or beta 4 subunit. Analysis of voltage-dependent activation and inactivation in the presence of 1.8 mM external Ca2+ showed an hyperpolarising shift of both relations when compared to similar recordings performed in the presence of 40 mM Ba2+. These shifts, which differed for activation and inactivation, were strongly modulated by the nature of the coexpressed beta subunit. On the other hand, for each combination, the kinetics of inactivation were similar in 1.8 mM Ca2+ and 40 mM Ba2+ (for example co-expression of the beta 2a subunit reduced inactivation using either 40 mM Ba2+ or 1.8 mM Ca2+). Thus, modulation of channel properties by the beta subunit is different in physiological Ca2+ or high Ba2+ concentrations. These results must be taken into consideration to extrapolate the role of the beta subunit in native cells.

Hypokalaemic periodic paralysis (HypoPP) is an autosomal dominant muscle disease which has been linked to point mutations in the skeletal muscle L-type calcium channel alpha 1 subunit (alpha 1s). Here, we have introduced one of the point mutations causing HypoPP (R528H) into cDNA of the rabbit alpha 1s. Expression of either the wild-type alpha 1s or the mutant R528H alpha 1s (alpha 1s-R528H) subunits was obtained in mouse Ltk- cells using a selectable expression vector. The alpha 1s-R528H subunit led to the expression of functional L-type Ca2+ channels. Corresponding whole-cell Ba2+ currents exhibit very slow activation and inactivation kinetics, typical for recombinant skeletal Ca2+ channel currents. Voltage-dependent activation and inactivation properties were similar for alpha 1s- and alpha 1s-R528H, as well as their sensitivity to the dihydropyridine agonist Bay K 8644. Differences in alpha 1s- and alpha 1s-R528H-directed channels reside in the Ba2+ current density, which was significantly reduced 3.2 fold in cells expressing alpha 1s-R528H. It was concluded that the R528H mutation af alpha 1s results in minor differences in the electrophysiological properties but significantly reduces the whole-cell Ca2+ channel current in its amplitude.

Voltage-dependent facilitation of L-type Ca2+ channels is an important regulatory mechanism by which excitable cells modulate Ca2+ entry during a train of action potentials. Expression of the alpha1 and beta subunits of the alpha1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation in Xenopus oocytes. Here we show that, by expressing the alpha1C together with different beta subunits in oocytes, the beta1, beta3 and beta4, but not the beta2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of the beta2 subunit mRNA, suggest that beta2 may be the beta subunit associated with functional cardiac L-type Ca2+ channels.

We have investigated whole-cell Ba2+ currents through Ca2+ channels (IBa) in single myocytes freshly isolated from the aortic media of neonatal (1-day-old) and adult (12-week-old) rats. In neonatal myocytes, (IBa) was undetectable even in presence of the dihydropyridine (DHP) agonist Bay K 8644. Binding of [3H]Nitrendipine on crude plasma membrane preparation of media confirmed the absence of DHP-receptors. By contrast, a robust DHP-sensitive 'L-type' IBa was recorded in adults which was consistent with the presence of specific [3H]Nitrendipine binding sites. In conclusion, neonatal aortic myocytes do not express any Ca2+ channels. The acquisition of L-type Ca2+ channels may be related to cell differentiation and acquisition of contractility during postnatal development.

Background: In mammalian heart cells, Ca2+ influx through voltage-gated L-type Ca2+ channels can be upregulated by high rates of stimulation. We have investigated this important adaptive regulation in human cardiomyocytes., Methods and Results: Using the whole-cell patch-clamp technique, we found a high frequency-induced upregulation (HFIUR) of the dihydropyridine-sensitive L-type Ca2+ current (ICa) in human cardiomyocytes. ICa was potentiated in a graded manner with increasing rates of stimulation between 0.3 and 5 Hz. Both moderate increase of ICa peak amplitude and marked slowing of current decay contributed to large increases of Ca2+ influx (up to 80%). The maximal potentiation of ICa was reached rapidly after the change in the rate of stimulation (no more than a few seconds). Beta-Adrenergic stimulation of the cells by isoproterenol (1 micromol/L), which is well known to induce a slow (approximately 1 minute) cAMP-mediated potentiation of ICa, could enhance (when present) or promote (when absent) the HFIUR of ICa. As a consequence, the increasing effect of isoproterenol on Ca2+ influx through Ca2+ channels was dependent on the rate of stimulation. HFIUR of ICa was altered in patients with ejection fraction lower than 40% and in patients pretreated with Ca2+ antagonists or beta-blockers., Conclusions: Upregulation of Ca2+ entry through voltage-gated Ca2+ channels by high rates of beating may be involved in the frequency-dependent regulation of contractility (Bowditch "staircase") of the human heart. This process, which is highly sensitive to beta-adrenergic stimulation, may be crucial in adaptation to exercise and stress.

Voltage-dependent calcium channels control various physiological functions such as the excitation-contraction coupling, the secretion of hormones or the release of neurotransmitters in the nervous system. Molecular genetics has allowed to provide a structural basis to the functional diversity of calcium channels and to initiate studies to understand the relations between the structure and the function of these excitable proteins. The aim of our research is to compare both the functional and structural properties of calcium channels from various tissues. The studies on dissociated or cultured cells allow to describe their properties, regulation, pharmacology and pathophysiology in native tissues. Structure-functions studies using reconstitution models such as Xenopus oocytes aim to understand the molecular basis underlying their diversity. Calcium channels are composed of several subunits (alpha 1, alpha 2-delta, beta, gamma). Six genes have been identified as coding for the pore subunit (alpha 1) which determines the general profile and in particular the pharmacology of a given calcium channel. However, the auxilliary subunits and mainly beta subunits for which 4 genes and several variants have been isolated, are able to modify the level of expression and the properties of a calcium current directed by an alpha 1 subunit in a reconstitution model. The structure-function studies are now mainly designed to investigate the functional consequences of the interaction alpha 1-beta on the electrophysiological and pharmacological properties. These studies should lead to a better understanding of the molecular basis underlying the diversity between cardiac and vascular calcium channels and also of their respective implication in pathophysiology. The co-expression of several families of calcium channels in a single neuron do not allow properly to investigate the properties and the regulation (by phosphorylation or G proteins) of the neuronal calcium channels which are involved in neurosecretion. The use of reconstitution models will provide a better characterization of neuronal calcium channels and should help to the development of new drugs of therapeutical interest.

We compared the effects of representative members of three major classes of cardiac L-type channel antagonists, i.e. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs) on high-voltage-activated (HVA) Ca2+ channel currents recorded from a holding potential of -100 mV in rat ventricular cells, mouse sensory neurons and rat motoneurons. Nimodipine (DHP), verapamil (PAA) and diltiazem (BTZ) block the cardiac L-type Ca2+ channel current (EC50: 1 microM, 4 microM and 40 microM, respectively). At these concentrations, the drugs could also inhibit HVA Ca2+ channel currents in both sensory and motor neurons. Large blocking effects (> 50%) could be observed at 2-10 times these concentrations. The omega -conotoxin-GVIA-sensitive (omega -CTx-GVIA, N-type), omega -agatoxin-IVA-sensitive (omega -Aga-IVA, P- and Q-types) and non-L-type omega -CTx-GVIA-, omega -Aga-IVA-insensitive (R-types) currents accounted for more than 90% of the global current. Furthermore, our data showed that omega -CTx-GVIA and omega -Aga-IVA spare L-type currents and have only additive blocking effects on neuronal HVA currents. We conclude that DHPs, PAAs and BTZs have substantial inhibitory effects on neuronal non-L-type Ca2+ channels. Inhibitions occur at concentrations that are not maximally active on cardiac L-type Ca2+ channels.

A tetrodotoxin (TTX)-sensitive fast inward Ca2+ current (ICa,TTX) was recorded at physiological Ca2+ levels (2 mM) in human single atrial cells. The whole-cell patch-clamp method and Na(+)-free solutions (20-22 degrees C) were used. ICa,TTX depended upon extracellular Ca2+. It was prominent at rather negative test potentials (maximal peak amplitude at approximately -40 mV) and was observed only at holding potentials lower than -80 mV. It had the same size and kinetics when Ca2+ was exchanged for Ba2+ as the charge carrier. Its rapid activation and inactivation kinetics, voltage-dependent availability and fast recovery from inactivation resembled that of the Na+ currents (INa). ICa,TTX was insensitive to 250 microM Ni2+ and 10 microM La3+, both known to block totally T-type ICa (not evidenced here). ICa,TTX was suppressed by Na+ channel inhibitors such as TTX (10 microM) and Cd2+ (20 microM) and its decay was slowed by the specific Na+ channel activator veratrine (200 micrograms/ml). We found that both time to peak and time-constant of inactivation of ICa,TTX were slower than that of INa. There was no correlation between the presence and size of ICa,TTX and that of INa. In conclusion, ICa,TTX may reflect the presence and activation of either Ca(2+)-conducting channels related to Na+ channels or, alternatively, of a fraction of Na+ channels with an increased permeability for Ca2+ and Ba2+ ions.

Xenopus oocytes expressing neuronal alpha 1C, alpha 2 and beta 1b calcium channel subunit cDNAs were used in this study. During two-electric voltage clamp recording the oocyte was injected with 10-20 nl of a 100 mM BAPTA solution. Under these conditions, the endogenous Ca-activated Cl current was completely suppressed resulting in an alpha 1C Ba current free from Cl current contamination. BAPTA injection also allowed alpha 1C currents with different permeating ions, including Ca, to be examined. Compared to Ba and Sr, alpha 1C whole cell Ca currents were smaller in magnitude and showed kinetic and voltage-dependent properties more similar to those for L-type Ca currents recorded in native cells. That Ca-dependent inactivation occurs in BAPTA-buffered cells suggests that the Ca-binding site involved in this type of inactivation is very close to the pore of the channel.

A novel human-voltage-dependent-calcium-channel (VDCC) beta subunit was isolated from a 9-week-old human total-embryo cDNA library. Of the four genes encoding beta-subunit isoforms that have been identified in animal species, this isoform shares strong similarity with the rat and rabbit beta 3-related gene product and is referred to here as H beta 3 subunit. The H beta 3 isoform is the second beta subunit identified in human. Its open reading frame encodes a 482-amino-acid protein with a predicted molecular mass of 54.571 kDa. The H beta 3 mRNA is expressed mostly in brain, smooth muscle and ovary. The gene for the human H beta 3 was specifically localized on chromosome 12q13. The cloned H beta 3 subunit was further expressed in Xenopus oocytes to demonstrate its ability to modulate VDCC activity.

The rat brain class C calcium channel alpha 1 subunit cDNA, rbC-II, was subcloned into a vertebrate expression vector and transient expression was assayed following nuclear injection into Xenopus oocytes. Whole cell recordings showed that rbC-II currents (recorded with 40 mM Ba2+ as the charge carrier) had variable activation rates and minimal inactivation over an approximately 700 msec depolarizing step pulse. The pharmacological properties of the rbC-II current were consistent with those of an L-type calcium channel, being sensitive to dihydropyridines (10 microM nifedipine blocked approximately 85% of the current, 10 microM Bay K 8644 enhanced the current between 2- and 10-fold) and not affected by the N- and P-type calcium channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, respectively. Coexpression of rbC-II with cloned rat neuronal calcium channel alpha 2 and beta subunits resulted in several changes to the electrophysiological properties of the rbC-II current including, an increased whole cell peak current, an increased rate of activation and a hyperpolarizing shift in the voltage dependence of activation. Taken together with results showing that the neuronal class D alpha 1 subunit also encodes an L-type calcium channel [Williams M. E., Feldman D. H., McCue A. F., Brenner R., Velicelebi G., Ellis S. B. and Harpold M. M. (1992a) Neuron 8: 71-84], these results indicate that the mammalian nervous system expresses two distinct genes encoding L-type calcium channels.

In smooth muscle cells, essentially two distinct types of voltage-gated Ca2+ channels have been shown, on the basis of their distinct electrophysiological and pharmacological properties, to coexist. Here we report that, in addition to a dihydropyridine (DHP)-sensitive, low-voltage-activated Ba2+ current (IBa,LVA), two types of high-voltage-activated Ba2+ currents with distinct waveforms were recorded in whole-cell clamped aortic myocytes; these were referred to as IBa,HVA1 and IBa,HVA2. They were investigated in cells where no IBa,LVA was detectable. IBa,HVA1 had a slow, monoexponential decay. In contrast, the decay of IBa,HVA2 was much faster and biexponential. In addition, IBa,HVA2 had more negative ranges of activation and steady-state inactivation than IBa,HVA1 and was more sensitive to the DHP antagonist nicardipine (concentrations for half maximum inhibition 0.2 microM and 2 microM, respectively). When using the physiological ion Ca2+ as the charge carrier, the decay of HVA1 currents was not altered, whereas both time constants of HVA2 current decay were accelerated five-fold. Moreover, permeability ratios (ICa/IBa) were also significantly different (0.2 and 0.6 for HVA1 and HVA2 respectively). IBa,HVA1 and IBa,HVA2 are consistent either with the existence and activation of two functionally distinct subtypes of the so-called "DHP-sensitive L-type" Ca2+ channel or with different gating behaviours of a single type of channel. Potentially, they may serve distinct biological functions and constitute distinct targets for neurotransmitters and drugs.

We have studied a fast inward current expressed in oocytes from one Xenopus laevis. This current was characterized as a sodium current. It was activated by depolarizations to -50 mV or higher, peaked within 3-5 ms, and then decayed following a mono-exponential timecourse. When clamped at different holding potentials, the current displayed voltage-dependent inactivation with a V0.5 of -51 mV. The channel responsible for this Na+ entry was blocked by tetrodotoxin with a K0.5 of 8 nM, and was resistant to block by lidocaine at concentrations up to 100 microM. The pharmacological similarities between neuronal and oocyte sodium channels suggest that the two channels share a conserved structure.

Cyclic AMP-dependent modulation of cardiac L-type voltage-dependent Ca channel (VDCC) has been probed in Xenopus laevis oocytes injected with poly(A+) RNA from rat heart. A 2 to 3 fold increase of the Ba current amplitude was routinely obtained upon microinjection of cAMP (50-500 microM). Inhibition of protein kinase A (PKA) dramatically reduced the Ba current amplitude, indicating that cAMP-dependent modulation plays an important role in maintaining the basal activity of expressed Ca channels. Moreover, the effects of the DHP agonist Bay K 8644 on kinetic properties of expressed Ba current (IBa,C) were dependent on PKA activation. The results suggest that most expressed cardiac L-type VDCCs are phosphorylated and demonstrate that reconstitution in Xenopus oocytes is a suitable approach to address how phosphorylation regulates VDCC activity.

The beta-adrenergic cascade is important for the regulation of voltage-dependent Ca channels by phosphorylation. Here we report that isoproterenol (ISO) profoundly alters the voltage-dependent properties of L-type Ca channels studied in rat ventricular cells. ISO (1 microM) shifted both threshold and maximal activation of Ba current (IBa) towards more negative potentials (approx. 10 mV). An equivalent shift was observed in the steady-state voltage-dependent inactivation curve. As a consequence, the potentiation induced by ISO on IBa was greater for weak depolarizations and from negative holding potentials (Vh). We have excluded that the contribution of minor uncompensated series resistances, the activation of Cl currents or changes in junction potential during the experiments account for these effects. In addition, ISO had a dual effect on IBa decay depending on the voltage step (acceleration below, slowing above -10 mV). In conclusion, it is postulated that the voltage dependence of the potentiating effects of ISO on Ca channels activity may ensure a selective regulation among heart tissues with different membrane resting potentials.

The effects of dihydropyridines (DHPs) normally considered to be specific for L-type calcium channels were studied on the T-type Ca channel current of acutely isolated dorsal root ganglion (DRG) neurons taken from 13-day-old (E13) mouse embryos. Potent but reversible inhibitory effects of the DHP nicardipine were found in the micromolar range. For example, 5 microM nicardipine suppressed 93 +/- 5% of T-type currents. In comparison, other classical DHPs such as nifedipine, PN 200-110 and nitrendipine had only weak effects (less than 20% inhibition) at the same concentration. The inhibition by nicardipine was found slightly to be voltage dependent and the drug induced a leftward shift in the steady-state inactivation. The DHP agonist (-)-Bay K 8644, which dramatically increased the L-type current, weakly decreased T-type Ca currents (17 +/- 8% at 5 microM). In conclusion, neuronal T-type Ca channels may be potential targets for some dihydropyridines. This property is not only a feature of the central nervous system (J. Physiol., 412 (1989) 181-195) and can be extended to peripheral neurons.

Ca-channel currents have been investigated in single cells isolated from adult human atrium using the whole-cell patch clamp technique. Ca-channel currents are activated at voltage positive to -40 mV, peak between -10 and 0 mV and inactivate with a slow decay when Ba2+ ions (5 mM) are used as charges carrier. These properties correspond to those of the high voltage activated, DHP-sensitive, (L-type) Ca channel. No low voltage activated (T-type) currents have been evidenced. The present work also provides the first report about the modulation of Ca channels in adult human atrial cells by beta-adrenergic agonists and dihydropyridines (agonists and antagonists). Electrophysiological and pharmacological properties of these Ca channels are qualitatively similar to those of the L-type Ca currents recorded from cardiac animal cells. However, at a physiological calcium concentration (2 mM), basal Ca currents are often very small or even absent but are revealed following the addition of the dihydropyridine (DHP) agonist Bay K 8644. Whether the decrease of the basal Ca current amplitude may be related to the chronic pretreatment of the patients by Ca channel blockers or to the pathology is discussed.

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.

alpha-Thrombin (TH) initiates a program of intracellular events that lead to DNA replication in quiescent CCL39 Chinese hamster lung fibroblasts via membrane receptors that have yet to be characterized at a molecular level. Functional TH receptors were expressed in Xenopus laevis oocytes following injection of poly(A)+ RNA from TH-responsive CCL39 cells; their presence was demonstrated by TH-stimulated 45Ca2+ efflux or Ca2(+)-dependent Cl- channel activation. In voltage clamp experiments on microinjected oocytes a Ca2(+)-activated Cl- current was detected in response to TH (0.2-10 U/ml). The TH response was blocked by a specific TH inhibitor, and potentiated by addition of FGF or intracellular injection of GTP-gamma-S.

Current clamp studies showed that after 10 minutes under DNP 10(-4) M the membrane potential does not change significantly while an important shortening of the action potential duration and a diminished amplitude are observed. Voltage clamp studies have been performed on the slow inward and delayed outward currents. DNP 10(-4) M induced a marked decrease of the slow inward current related to the reduction in both conductance and driving force, and a decrease in the amplitude of the delayed current. The decrease of the slow inward current seems to be mainly responsible for the suppression of the plateau of the action potential during metabolic inhibition.

Voltage clamp experiments have been conducted on frog atrial fibers to study the mechanism of the negative inotropic effect induced by muscarinic agonists. The results have shown that muscarinic agonists exert a dual effect: 1.) A reduction of the slow inward current (Is) amplitude; 2.) An increase in the potassium conductance. Dose-effect curves for both Is and K+ extra-current show that the inhibition of Is appears at lower agonist concentrations and can mainly account for the negative inotropic effect. It is suggested that the action of muscarinic agonists may involve one or two types of muscarinic receptors. The slow channel conductance could be controlled by the intracellular cyclic nucleotides concentration (cAMP and cGMP) via a muscarinic receptor (R1) with a high affinity for agonists. The increase in the potassium conductance could be mediated either by an increase in the intracellular free Ca2+ concentration or by a second type of muscarinic receptor (R2) with a lower affinity for muscarinic agonists and controlling a specific potassium channel.

This paper concerns the specificity of nicardipine, a calcium antagonist from the dihydropyridine class which is used in the treatment of cardiovascular disorders. It is well established that in cardiac cells dihydropyridines inhibit the calcium current (Isi) and the correlated phasic tension. In this study we demonstrate that nicardipine, in the concentration range which blocks Isi (KD = 1 microM) also decreases the amplitude of the potassium-delayed current (KD = 3 microM) in frog atrial fibres. Moreover, tonic tension, which has been reported to be directly dependent on the Na+-Ca2+ exchange, was also reduced by nicardipine and the time course of the onset of both contraction and relaxation was significantly slowed. These results indicate that on depolarized membranes, dihydropyridines probably affect voltage-dependent mechanisms with a high threshold of activation that are unrelated to Ca2+ channels, such as potassium-delayed current and Na+-Ca2+ exchange.

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.

A double sucrose gap voltage clamp technic has been used to study the extra-current induced by acetylcholine (Iach) on the myocardial membrane on frog atrial trabeculae. I) No desensitization of the Iach current is noted for repeated perfusions of Ach. II) The Iach current is suppressed by atropine. III) The reversal potential Each is more negative than the resting potential --20 mV less than or equal to Each less than or equal to OmV.IV) The relationship Iach/Holding potential for various [K+]o shows a) That Each behaved as a potassium electrode, b) an inward going rectification. These results indicate that the cholinergic receptor might be related with the gk1 channel.

We have shown in the first part of this work, how pharmacological technics can be associated to electrophysiological measurements, to study the action of neurotransmitters on the excitation-contraction coupling in cardiac cells. However, to better understand their mechanism of action, it is necessary to have informations about the link between the activation of the receptor and the conductance changes. Kinetics measurements have been very useful to investigate this problem at the nicotinic cholinergic receptor. This study was performed with technics such as iontophoresis, voltage relaxations, noise analysis and recently patch clamp measurements. Nicotinic responses are very fast (in the order of the msec) and are however the best known. The binding of the agonist to the receptor would lead to the opening of a channel only by molecular conformation change. By comparison, beta-adrenergic and muscarinic responses in heart or smooth muscle can be considered as "slow responses" because they are thought to be mediated by intracellular biochemical steps. We have developed a new technique using photosensitive compounds, which allows to jump the extra- or intracellular concentration of some active molecules. The experiments have been performed on frog atrial fibers, in current or voltage clamp conditions using a double sucrose gap technique. Various photochemical reactions have been exploited. The photoisomerization of an azobenzene derivative (Bis-Q) allows to study the muscarinic-induced potassium conductance. Bis-Q binds to muscarinic receptors and blocks the effect of agonists. These drugs have the additional property that their cis and trans configuration have different potencies. The photoisomerization cis----trans or trans----cis is obtained a few msec after a 1 msec duration light flash (respectively visible or UV), and then induces a concentration jump of antagonist near the receptor. The results show that the potassium conductance change has a slow relaxation time constant (tau = 500 msec.) and this suggest rather successive intracellular steps than the opening of a channel directly linked to the receptor. Cyclic nucleotides are considered as the intracellular messengers for various neurotransmitters or hormones. Our photochemical technique allows to jump the intracellular concentration of cAMP and cGMP in a few msec. cAMP (or cGMP) orthonitrobenzyl derivatives are able to penetrate cell membrane without injection and have no effect up to 100 microM. A UV flash (1 msec duration) induces a photolysis of the derivative and generates instantaneously cAMP (or cGMP) inside the cell.(ABSTRACT TRUNCATED AT 400 WORDS)

These experiments employ the photoisomerizable compound, 3,3'-bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans-Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.

Electrophysiological recording was used to study non-N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors after injection of rat brain ribonucleic acid (RNA) in Xenopus laevis oocytes. Quisqualate (QA) induced two types of responses, a smooth one and an oscillatory one. These responses are probably mediated by the ionotropic (QAi, a cationic channel) and the metabotropic (QAp, a newly discovered receptor coupled to phospholipase C) QA receptors respectively. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) only induced a smooth inward current suggesting that it acts only on QAi. Kainate (KA) also induced a smooth inward current, the maximal KA response being 10-fold higher than the maximal AMPA. AMPA inhibited the KA response in a dose-dependent and competitive manner. Amongst various complex hypotheses the simplest to explain these results would be that KA and AMPA both activate the same receptor-channel complex, AMPA inducing a smaller response than KA.

The organic Ca2+ antagonists are potent inhibitors of Ca2+ influx in cardiac and smooth muscle and are widely used clinically in the treatment of various cardiovascular disorders. It appears that Ca2+ antagonist binding prevents the normal movement of ions through Ca2+ channels, perhaps via an open-channel blockade mechanism. Although this concept is generally accepted, questions do remain about the detailed relationship between binding and blockade in the case of the structurally diverse organic Ca2+ channel blockers; e.g., (1) do they bind preferentially to open, closed and/or inactivated channels; (2) are there multiple binding sites; (3) do they act at extracellular and/or intracellular sites; and (4) does blocking or unblocking depend on membrane potential or its history? The dihydropyridine Ca2+ antagonist, nifedipine, contains an o-nitrobenzyl moiety and is photolabile; irradiation yields a molecule devoid of channel blocking activity and the photoconversion reactions are complete within 100 microseconds. Taking advantage of these properties to study the mechanistic details of nifedipine blockade of Ca2+ channels, we examined the waveform of the slow inward Ca2+ current (Isi) in atrial fibers before and following flash-induced removal of nifedipine. After flashes, we find that nifedipine blockade is reversed within at most a few milliseconds and that the rate of Isi reactivation parallels the normal, voltage-dependent activation rate. Our results imply that nifedipine binds to and stabilizes resting, closed Ca2+ channels and are not in agreement with the recent conclusions of Morad and coworkers that photoconversion of nifedipine must be followed by membrane repolarization in order to effect recovery of Isi and tension.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of acetylcholine (ACh) on membrane potential and currents in frog atrial muscle has been studied with a double sucrose gap technique. The results show the following. 1. ACh induces the development of an extra current, outward at the resting potential, which is dependent on the ACh concentration. 2. The preparation does not show any sign of desensitization. 3. The reversal potential of the current induced by ACh is between 0 and 20 mV more negative than the resting potential and behaves as a K electrode. 4. The mechanism of ACh-induced K conductance presents inward going rectification properties. 5. The delayed outward current is not affected by ACh. However the evolution of its tail current seems to indicate a process of K accumulation related to the ACh-induced current.

The effects of nifedipine and nicardipine, 2 dihydropyridines (DHP) used in the treatment of cardiovascular disorders, were compared in frog atrial fibers. Rapid photolysis of nifedipine with a single UV flash (1-ms duration) reversed the block, allowing comparison of effects of both drugs on the same preparation, and manipulation of the calcium channel on a millisecond timescale. The results show that inhibition of the action potential (AP) and slow inward current (Isi) is more pronounced with nifedipine than with nicardipine. Concentration-effect relationships confirm that nicardipine (IC50 = 1 microM) is less potent than nifedipine (IC50 = 0.2 microM) in blocking cardiac calcium channels. Both DHP block calcium channels in the closed state at the resting potential, inducing a large tonic block (in the absence of stimulation). An additional phasic block can be observed with nifedipine and nicardipine. A slight voltage dependence to the block is observed for both DHP, their effects being enhanced depolarization holding potentials. Rapid unblocking of calcium channels by a single light flash, presented during the decay phase of Isi, reveals a higher affinity of DHP for inactivated channels. This effect is most pronounced when inactivation is slowed by using Ba++, Sr++, or Na+ ions as the current carriers. Open channel block is also suggested. The mechanism of DHP action on calcium channels can be described according to the "modulated receptor hypothesis". These DHP exhibit an additional nonspecific effect on potassium channels. It is concluded that nicardipine is a less potent Ca++ antagonist than nifedipine in atrial fibers and that the reduction of delayed potassium current, which occurs in a similar range of concentrations to the blockade of Isi, could also be involved in its therapeutic effects.

An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD2, PGI2, inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5-dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP ("cAMP jumps") following light flashes. A single flash, delivered after 2 min incubation in 100-200 microM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation.

The cyclic nucleotides cyclic AMP and cyclic GMP are important intracellular messengers mediating the responses to neurotransmitters and neurohormones and regulating cellular function over a wide range of time scales. Despite the widespread acceptance of this second messenger mechanism in many systems, much remains unknown about their mechanism of action, except that such events are associated with increases or decreases in intracellular cyclic nucleotides. Quantitative descriptions of cyclic nucleotide-dependent processes are hampered by the absence of a means by which intracellular cyclic nucleotide concentrations can be accurately controlled. We have now designed, synthesized and characterized new, substituted photolabile cyclic nucleotide analogues, the 4,5-dimethoxy-2-nitrobenzyl esters of cyclic AMP and cyclic GMP (Fig. 1), which are physiologically inert before irradiation and which liberate free cyclic AMP or cyclic GMP on absorption of a photon. The thermal properties and photolysis rates and efficiencies of light-induced release of cyclic nucleotides from these analogues are more favourable than for the simple o-nitrobenzyl derivatives used previously. These molecules should permit intracellular "concentration jumps' of cyclic AMP or cyclic GMP to be produced in cells under physiological investigation with spatial and temporal resolution unmatched by conventional techniques.

Functional endothelin and angiotensin receptors have been expressed in Xenopus oocyte following the microinjection of rat heart mRNA. Under voltage clamp conditions, application of these peptides clearly induced oscillatory Ca2+-activated chloride currents in a dose-dependent manner. In addition, no direct modulation of expressed or native cardiac Ca channels was observed.

The effect of alkali-earth cations (Ca2+, Sr2+, Ba2+) on the excitation-contraction coupling events of the frog atrial fibres were studied using a double mannitol gap voltage clamp technique coupled with a mechano-electric transducer. Photoremoval of the suppressive effect of nifedipine on the calcium channels allowed to obtain rapid transient Ca2+, Sr2+ or Ba2+ ions current jumps. The effect on the amplitude of the associated contraction was proportional to the current jumps. These results together with the correlation established between the estimated increase in the internal concentration of divalent cations and the amplitude of the phasic tension suggest that the essential source of divalent cations for activation of contraction is the extracellular space. Also Ba2+ ions reduced the tonic tension and strongly slowed the relaxation of the phasic component whereas Sr2+ exhibited smaller effects. Sr2+ ions could be more efficient than Ba2+ ions in substituting for Ca2+ ions in the Na+-Ca2+ exchange mechanism known to regulate these two mechanical events. The conclusions are that the order of effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is the same with regard to transarcolemmal exchange for Na+ ions, presumed uptake by a "second relaxing system", activation of contraction, and inactivation of the slow inward current.

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2-5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. IBa in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 +/- 4 nA, n = 21). In contrast, injected oocytes showed a peak IBa of 339 +/- 42 nA (n = 33). The threshold for activation of IBa was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (tau = 650 +/- 50 msec at +20 mV). The maintained current was 67 +/- 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward IBa could be observed in BaCl2 solutions at potentials positive to ECl and also in Na-free salines, indicating that neither Cl- nor Na+ was carrying the inward current. Although IBa displayed voltage-independent blockade by Cd (50% inhibition at 6 microM), the peptide Ca channel antagonist, omega-CgTX (1 microM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 microM, or when cells were held at -40 mV) or agonist Bay K-8644. However, IBa was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT2). In contrast, use of forskolin to activate protein kinase A did not alter IBa.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-clamped atrial trabeculae from bullfrog hearts were exposed to membrane-permeant photolyzable o-nitrobenzyl esters of cAMP and cGMP. UV flashes produced intracellular concentration jumps of cAMP or cGMP. With the cAMP derivative, flashes resulted in an increased slow inward current (Isi), producing a broadened action potential. The Isi reached a maximum 10-30 sec after the flash and decreased over the next 60-300 sec. The first increases were observable within 150 msec; this value is an upper limit imposed by the instrumentation. Responses to flashes lasted longer at higher drug concentrations and in the presence of the phosphodiesterase inhibitor papaverine; effects of flashes developed and decreased faster at higher temperature. Although the amplitude of the Isi was increased, its waveform and voltage sensitivity were not affected. Intracellular concentration jumps of cAMP failed to affect the muscarinic K+ conductance. There were no observable effects of cGMP concentration jumps. The data confirm (i) that cAMP regulates the Isi and (ii) that the 5- to 10-sec delay between application of beta-agonists and the onset of positive inotropic effects, observed in previous studies, has been correctly ascribed to events prior to the interaction between cAMP and protein kinase.

Ba2+ and Sr2+ ions are widely used to replace Ca2+ ions for the study of Ca2+ channel currents in electrophysiological experiments. Using the double sucrose gap technique, we investigated the effects of Sr2+ and Ba2+ ions on the Na+ Ca2+ exchange activity in frog atrial fibres where it is the major relaxation mechanism. With either Sr2+ or Ba2+ ions instead of Ca2+ in the extracellular bath, Na-free contractures reversibly developed but with different kinetics. Voltage clamp experiments showed that the tonic tension recorded in the presence of Sr2+ or Ba2+ was markedly increased following the addition of monensin, a Na+ ionophore known to increase the intracellular Na+ activity. In Na-free solutions (Li-substituted), it was possible to induce contractures by substituting Sr2+ or Ba2+ ions for extracellular Ca2+. These contractures could be relaxed by reintroducing Na+ or Ca2+ ions in the extracellular medium. Taken together, these results suggest that Sr2+ and Ba2+ ions can interact with the Na+-Ca2+ exchange mechanism and potentially participate not only in Na+-cation but also in Ca2+-cation exchanges on either side of the sarcolemmal membrane.

We have previously reported that intraplatelet "cyclic AMP jumps" produced with newly synthesized photoactivatable cyclic AMP analogue, inhibited washed rat platelet aggregation and serotonin release as induced by thrombin. Using the same approach on human platelets, thrombin-induced platelet aggregation was dose-dependently inhibited only when a flash was delivered. The mechanism of action of intraplatelet cyclic AMP as resulting from photolysis could be by controlling the level of cytosolic Ca2+. In order to test this hypothesis, the same protocol was used on human platelets preloaded with the internal Ca2+ fluorescent indicator, Quin 2, we found that the extent and the rate of the rise of the cytosolic Ca2+ induced by thrombin were dramatically decreased, in the presence of the photoactivatable cyclic AMP, only following photoirradiation. In addition, the flashes were produced, in the presence of photoactivatable cyclic AMP, after the thrombin-induced rise of internal Ca2+ had reached its peak. In these conditions, photoirradiation caused a rapid fall in fluorescence. These experiments provide the first direct evidence that intracellular cyclic AMP is involved in the control of platelet cytosolic Ca2+ by inhibition of its mobilization and by stimulation of its sequestration.