Dihydropyridine Agonist Research Articles

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel.

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.

Dihydropyridine-sensitive Ca(2+) channels in human glomerular mesangial cells.

In mesangial cells (MC), the response of intracellular Ca(2+) concentration ([Ca(2+)](i)) to a contractile agonist is biphasic with a large, transient increase in [Ca(2+)](i) followed by a smaller but sustained elevation as Ca(2+) flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca(2+) channels in the plasmalemmal membrane. However, a role for voltage-gated Ca(2+) channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca(2+) entry pathway responsible for the sustained response of [Ca(2+)](i) in human MC. We found that ANG II at 10 nM, 100 nM, and 1 microM increased [Ca(2+)](i) to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 microM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca(2+). None of the peak responses (due to release of intracellular stores of Ca(2+)) were affected by removal of external Ca(2+) or addition of diltiazem. Upon elevating the extracellular [K(+)] from 5 mM to 75 mM, [Ca(2+)](i) reached a sustained level of 48% greater than baseline. This effect of high K(+) was attenuated by either Ca(2+) removal or addition of diltiazem. In the presence of 75 or 140 mM K(+), the dihydropyridine agonist BAY K 8644 (1 microM and 10 microM) initiated sustained [Ca(2+)](i) responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca(2+) in the external solution, BAY K 8644 did not significantly affect [Ca(2+)](i). In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl(2) and 10 microM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between -30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca(2+) channels responsible for the voltage-regulated entry of Ca(2+) into the cell during an agonist-induced contraction.

Omega-AgaIVA-sensitive (P/Q-type) and -resistant (R-type) high-voltage-activated Ba(2+) currents in embryonic cockroach brain neurons.

By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba(2+) currents (I(Ba)) were characterized in embryonic cockroach brain neurons in primary culture. I(Ba) was characterized by a threshold of approximately -30 mV, a maximum at approximately 0 mV, and a reversal potential near +40 mV. Varying the holding potential from -100 to -40 mV did not modify these properties. The steady-state, voltage-dependent activation and inactivation properties of the current were determined by fitting the corresponding curves with the Boltzmann equation and yielded V(0.5) of -10 +/- 2 (SE) mV and -30 +/- 1 mV, respectively. I(Ba) was insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 microM) and antagonist isradipine (10 microM) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner (IC(50) = 170 microM). The toxin omega-CgTxGVIA (1 microM) had no significant effect on I(Ba). Micromolar doses of omega-CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 microM, 38% of the peak current was blocked after 1 h. In contrast, I(Ba) was potently and irreversibly blocked by nanomolar concentrations of omega-AgaTxIVA in approximately 81% of the neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0. 4-1 microM). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC(50) of 17 nM and a Hill coefficient (n) of 0.6. A better fit was obtained with a combination of two Hill equations corresponding to specific (IC(50) = 9 nM; n = 1) and nonspecific (IC(50) = 900 nM; n = 1) omega-AgaTxIVA-sensitive components. In the remaining 19% of the neurons, concentrations >/=100 nM omega-AgaTxIVA had no visible effect on I(Ba). On the basis of these results, it is concluded that embryonic cockroach brain neurons in primary culture express at least two types of voltage-dependent, high-voltage-activated (HVA) calcium channels: a specific omega-AgaTxIVA-sensitive component and DHP-, omega-CgTxGVIA-, and omega-AgaTxIVA-resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.

Calcium channels involved in synaptic transmission from reticulospinal axons in lamprey.

The pharmacology of calcium channels involved in glutamatergic synaptic transmission from reticulospinal axons in the lamprey spinal cord was analyzed with specific agonists and antagonists of different high-voltage activated calcium channels. The N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTx) induced a large decrease of the amplitude of reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The P/Q-type calcium channel blocker omega-agatoxin IVA (omega-Aga) also reduced the amplitude of the reticulospinal EPSPs, but to a lesser extent than omega-CgTx. The dihydropyridine agonist Bay K and antagonist nimodipine had no effect on the amplitude of the reticulospinal EPSP. Combined application of omega-CgTx and omega-Aga strongly decreased the amplitude the EPSPs but was never able to completely block them, indicating that calcium channels insensitive to these toxins (R-type) are also involved in synaptic transmission from reticulospinal axons. We have previously shown that the group III metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) mediates presynaptic inhibition at the reticulospinal synapse. To test if this presynaptic effect is mediated through inhibition of calcium influx, the effect of L-AP4 on reticulospinal transmission was tested before and after blockade of N-type channels, which contribute predominantly to transmitter release at this synapse. Blocking the N-type channels with omega-CgTx did not prevent inhibition of reticulospinal synaptic transmission by L-AP4. In addition, L-AP4 had no affect on the calcium current recorded in the somata of reticulospinal neurons or on the calcium component of action potentials in reticulospinal axons. These results show that synaptic transmission from reticulospinal axons in the lamprey is mediated by calcium influx through N-, P/Q- and R-type channels, with N-type channels playing the major role. Furthermore, presynaptic inhibition of reticulospinal transmission by L-AP4 appears not to be mediated through inhibition of presynaptic calcium channels.

Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons.

Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 microM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed approximately 60% of the LVA ICa, whereas approximately 10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega-conotoxin GVIA (omega-CgTx), an N-type ICa blocker, suppressed approximately 65% of the whole cell HVA ICa at the concentration of 1 microM. The omega-CgTx-resistant HVA ICa was sensitive to nifedipine (10 microM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by approximately 25% of the drug-free control ( approximately 70% of the omega-CgTx-resistant ICa). The combination of 10 microM nifedipine and 1 microM omega-CgTx left approximately 13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 microM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.

Crucial Role of N Terminus in Function of Cardiac L-type Ca2+ Channel and Its Modulation by Protein Kinase C

The role of the cytosolic N terminus of the main subunit (alpha1C) of cardiac L-type voltage-dependent Ca2+ channel was studied in Xenopus oocyte expression system. Deletion of the initial 46 or 139 amino acids (a.a.) of rabbit heart alpha1C caused a 5-10-fold increase in the whole cell Ca2+ channel current carried by Ba2+ (IBa), as reported previously (Wei, X., Neely, A., Olcese, R., Lang, W., Stefani, E., and Birnbaumer, L. (1996) Recept. Channels 4, 205-215). The plasma membrane content of alpha1C protein, measured immunochemically, was not altered by the 46-a.a. deletion. Patch clamp recordings in the presence of a dihydropyridine agonist showed that this deletion causes a approximately 10-fold increase in single channel open probability without changing channel density. Thus, the initial segment of the N terminus affects channel gating rather than expression. The increase in IBa caused by coexpression of the auxiliary beta2A subunit was substantially stronger in channels with full-length alpha1C than in 46- or 139-a.a. truncated mutants, suggesting an interaction between beta2A and N terminus. However, only the I-II domain linker of alpha1C, but not to N or C termini, bound beta2A in vitro. The well documented increase of IBa caused by activation of protein kinase C (PKC) was fully eliminated by the 46-a.a. deletion. Thus, the N terminus of alpha1C plays a crucial role in channel gating and PKC modulation. We propose that PKC and beta subunit enhance the activity of the channel in part by relieving an inhibitory control exerted by the N terminus. Since PKC up-regulation of L-type Ca2+ channels has been reported in many species, we predict that isoforms of alpha1C subunits containing the initial N-terminal 46 a.a. similar to those of the rabbit heart alpha1C are widespread in cardiac and smooth muscle cells.

Seasonal influences on the functional expression of the endogenous L-type Ca2+ channels in Pleurodeles oocytes: role of cAMP?

We have investigated the seasonal regulation of the Ba current (IBa) through the L-type Ca2+ channels in Pleurodeles oocytes and its relation with the intracellular cAMP level. IBa-density remained relatively constant from January to February, increased from March to April (with a maximum increase in March) and decreased from May to July. While the permeable cAMP analogue 8Br-cAMP reduced IBa during the breeding season, it was without effect during the resting season. However, IBa recorded during the two seasons was increased by a dihydropyridine agonist Bay K 8644 and blocked by cadmium. The main conclusion is that the seasonal reduction in the L-type Ca2+ current amplitude may be correlated with a high intracellular cAMP level and may account for the inability of oocytes to mature during the resting season.

Group III metabotropic glutamate receptor agonists modulate high voltage-activated Ca2+ currents in pyramidal neurons of the adult rat.

In pyramidal neurons of the rat sensorimotor cortex, we have investigated the modulation of high voltage-activated calcium currents by agonists at group III metabotropic glutamate receptors (mGluRs). l-2-Amino-4-phosphonobutyrate (l-AP4) and l-serine-O-phosphate (l-SOP) reduced calcium currents in the vast majority of cells isolated from the adult animal. Interestingly, this modulation was negligible in the young animals (2-14 postnatal days), becoming prominent only after full development (more than 21 days). The efficacy of l-SOP mimicked l-AP4 in reducing calcium currents. Yet, l-SOP produced saturating responses at about 3 microM and significant modulation at nanomolar concentrations (EC50=923 nM). The voltage-dependence of the group III mGluR-mediated responses was evaluated by comparing the inhibition of "standard" and "facilitated" conductances. On the calcium currents facilitated by depolarizing prepulse, 3 microM l-SOP produced a mean 13.4% inhibition compared with 19.6% in control condition, supporting the proposition that part of the inodulation was voltage-dependent. The calcium current inhibition caused by the activation of group III metabotropic glutamate receptors was only partially sensitive to omega-conotoxin GVIA, but largely inhibited by omega-agatoxin IVA, at concentrations (100 nM) known to block P- and Q-type channels. Conversely, the dihydropyridine antagonists nifedipine and nimodipine (50-500 nM) failed to prevent the group III mGluR-mediated response in the majority of tested cells (more than 65%). Furthermore, the long-lasting tail promoted by the inclusion of the dihydropyridine agonist Bay K 8644 was not consistently affected by l-SOP and l-AP4. These findings imply that the observed modulation involves different channel subtypes, namely N- and P- or Q-type channels, and suggests that group III mGluRs play an important role in the intrinsic and synaptic functions of adult cortical pyramidal neurons.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-L-type Ca2+ channel.

The activity of L-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming alpha1 subunits of L-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive L-type alpha1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type alpha1A subunit. The resulting chimeric alpha1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the L-type alpha1C subunit and 92% of the binding energy of the L-type alpha1S subunit for the high-affinity DHP antagonist PN200-110. The activity of chimeric Ca2+ channels containing alpha1A/DHPS was increased 3.5 +/- 0.7-fold by the DHP agonist (-)Bay K8644. The effect of this agonist was stereoselective as in L-type Ca2+ channels since (+) Bay K8644 inhibited the activity of alpha1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Molecular Studies on the Voltage Dependence of Dihydropyridine Action on L-type Ca2+ Channels

The interaction site(s) of dihydropyridine (DHP) antagonists and agonists have been identified by site-directed mutagenesis and localized on motifs IIIS5, IIIS6, and IVS6 of L-type voltage-gated calcium channels. In this study, we investigated the voltage-dependent action of DHPs with mutants of the IIIS6 and IVS6 segments of a cardiac calcium channel. Tyrosine residues in both motifs (Tyr1178 and Tyr1489) strongly contributed to the action of DHP agonists and antagonists. When these two sites were mutated, the communication between the voltage sensor and the DHP interaction site(s) was substantially impaired. In contrast, mutants of a nearby Ile (Ile1182) had much less influence on DHP agonist and antagonist interaction, and the voltage dependence of DHP antagonists was very similar to that of the wild type. The effect of a mutating of Ile1182, on agonist or antagonist action, however, depended strongly on the type of amino acid change. When Ile1182 was substituted with alanine, small changes were noted for DHP agonist and antagonist action. Changing this site into phenylalanine, however, significantly decreased the action of the DHP antagonist. These data show that Ile1182 can preferentially interact with DHP antagonists, but has a lesser contribution in agonist interaction. Thus, even though the agonist and antagonist interaction sites for DHPs with L-type calcium channels may overlap, some amino acids in this site may exhibit a preference for either DHP enantiomers.

Mibefradil (Ro 40m5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones

The actions of the novel calcium (Ca 2+) channel antagonist mibefradil (Ro 40–5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca 2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca 2+ current flowed through N-type Ca 2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca 2+ channel currents. Inhibition was nearly complete at 10 μM suggesting mibefradil blockade of all subclasses of Ca 2+ channels. The IC 50 was approximately 1.4 μM on currents measured at 0 mV, from a holding potential of −90 mV. Inhibition of LVA Ca 2+ current occurred over the same concentration range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC 50 = 3.4 μM). These broad inhibitory effects of mibefradil on Ca 2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca 2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca 2+ channels other than type T.

Calcium channel subtypes in lamprey sensory and motor neurons.

Pharmacologically distinct calcium channels have been characterized in dissociated cutaneous sensory neurons and motoneurons of the larval lamprey spinal cord. To enable cell identification, sensory dorsal cells and motoneurons were selectively labeled with fluorescein-coupled dextran amine in the intact spinal cord in vitro before dissociation. Calcium channels present in sensory dorsal cells, motoneurons, and other spinal cord neurons were characterized with the use of whole cell voltage-clamp recordings and specific calcium channel agonist and antagonists. The results show that a transient low-voltage-activated (LVA) calcium current was present in a proportion of sensory dorsal cells but not in motoneurons, whereas high-voltage-activated (HVA) calcium currents were seen in all neurons recorded. The different components of HVA current were dissected pharmacologically and similar results were obtained for both dorsal cells and motoneurons. The N-type calcium channel antagonist omega-conotoxin-GVIA (omega-CgTx) blocked >70% of the HVA current. A large part of the omega-CgTx block was reversed after washout of the toxin. The L-type calcium channel antagonist nimodipine blocked approximately 15% of the total HVA current. The dihydropyridine agonist (+/-)-BayK 8644 markedly increased the amplitude of the calcium channel current. The BayK-potentiated current was not affected by omega-CgTx, indicating that the reversibility of the omega-CgTx effect is not due to a blockade of L-type channels. Simultaneous application of omega-CgTx and nimodipine left approximately 15% of the HVA calcium channel current, a small part of which was blocked by the P/Q-type channel antagonist omega-agatoxin-IVA. In the presence of the three antagonists, the persistent residual current (approximately 10%) was completely blocked by cadmium. Our results provide evidence for the existence of HVA calcium channels of the N, L, and P/Q types and other HVA calcium channels in lamprey sensory neurons and motoneurons. In addition, certain types of neurons express LVA calcium channels.

Depolarization and activation of dihydropyridine-sensitive Ca2+ channels stimulate inositol phosphate accumulation in photoreceptor-enriched chick retinal cell cultures.

Elevated concentrations of extracellular K+ increased inositol phosphate accumulation in primary cultures of chick retinal photoreceptors and multipolar neurons. K+-evoked stimulation of inositol phosphate accumulation was greater in photoreceptor-enriched cell cultures than in cultures where multipolar neurons were the predominant cell type. Destroying multipolar neurons, but not photoreceptors, with kainic acid and N-methyl-D-aspartate did not reduce the K+-evoked stimulation of inositol phosphate accumulation. Both of these observations indicate that the observed effects occur in photoreceptor cells. The K+-evoked stimulation of inositol phosphate accumulation was blocked by omitting Ca2+ from the incubation medium or by adding the dihydropyridine-sensitive Ca2+-channel antagonists, nitrendipine and nifedipine. Bay K 8644, a dihydropyridine agonist, stimulated inositol phosphate accumulation and enhanced the effect of K+. omega-Conotoxin GVIA, an inhibitor of N-type Ca2+ channels, had no significant effect on K+-stimulated inositol phosphate accumulation. Pretreatment with pertussis toxin neither blocked K+-evoked inositol phosphate accumulation nor altered the inhibitory effect of nifedipine. K+-evoked inositol phosphate accumulation appears to reflect activation of phosphatidylinositol-specific phospholipase C, as it is inhibited by U-73122. These results indicate that Ca2+ influx through voltage-gated, dihydropyridine-sensitive channels activates phospholipase C in photoreceptor inner segments and/or synaptic terminals.

Sympathetic Modulation of the Effect of Nifedipine on Myocardial Contraction and Ca Current in the Rat

The regulation of cardiacl-type Ca2+current (ICa) and contraction by dihydropyridine antagonists andβ-adrenergic receptor agonists has been the subject of numerous studies over the last decade. However, little is known on the crosstalk between these two regulatory pathways. For instance, a fundamental question that remains unanswered is: does activation of theβ-adrenergic receptors modify the sensitivity of the myocardium to dihydropyridine agonists? To answer this question, we examined in the present study how activation of theβ-adrenergic receptors modifies the effects of nifedipine on the mechanical and energetic parameters of the isolated perfused rat heart. Activation of theβ-adrenergic receptors was achieved by perfusing the hearts with isoprenaline, a non-selectiveβ-adrenergic receptor agonist, and could be reduced by atenolol, aβ1-adrenergic receptor antagonist. To examine possible alterations during hypertension in the sensitivity of the hearts to the drugs tested, the study was performed in both normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive animals (SHR). While 0.1μMnifedipine reduced left ventricular pressure (LVP) by 36% and 34% in WKY and SHR rats, respectively, under basal conditions, its effects became negligible in both groups of rats after stimulation of the hearts with 0.1μMisoprenaline. Addition of 1μMatenolol in the presence of isoprenaline restored the inhibitory effect of nifedipine to control values in both WKY and SHR rats. Additional experiments were performed in isolated ventricular myocytes from WKY rats using the whole-cell patch-clamp technique. The inhibitory effects of 0.1 to 1μMnifedipine were significantly larger on basal ICathan after the current had been previously elevated by 0.1μMisoprenaline. Addition of 1μMatenolol in the presence of isoprenaline partially restored the inhibitory effect of nifedipine on ICa. Our results demonstrate a reduced sensitivity of the heart muscle to nifedipine during activation ofβ1-adrenergic receptors. This effect is partly explained by a reduced inhibitory effect of nifedipine on ICaduring activation of cAMP-dependent phosphorylation

Dihydropyridine- and neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala.

The central amygdala (CeA) is an area involved in emotional learning and stress, and identification of Ca2+ currents is essential to understanding interneuronal communication through this nucleus. The purpose of this study was to separate and characterize dihydropyridine (DHP)- and neurotoxin-sensitive and -resistant components of the whole cell Ca2+ current (ICa) in acutely dissociated rat CeA neurons with the use of whole cell patch-clamp recording. Saturating concentrations of nimodipine (NIM, 5 microM), a DHP antagonist, blocked 22% of ICa: this NIM-sensitive (L-type) current was recorded in 68% of CeA neurons. The DHP agonist Bay K 8644 (5 microM) produced a 36% increase in ICa in a similar proportion of CeA neurons (70%). omega-Conotoxin GVIA (CgTx GVIA, 1 microM) in saturating concentrations inhibited 30% of ICa, whereas omega-agatoxin IVA (Aga IVA, 100 nM), in concentrations known to block P-type currents, did not affect ICa. Higher concentrations of Aga IVA (1 microM) alone reduced ICa by 34%, but in the presence of NIM (5 microM) and CgTx GVIA (1 microM) blocked only 18% of ICa. omega-Conotoxin MVIIC (CgTx MVIIC, 250 nM) reduced ICa by 13% in the presence of CgTx GVIA (1 microM). Application of NIM (5 mM), CgTx GVIA (1 microM); and Aga IVA (1 microM) blocked approximately 67% of ICa. A similar portion (63%) of Ca2+ current was blocked with CgTx MVIIC (250 nM) in the presence of NIM (5 microM) and CgTx GVIA (1 microM). The current resistant to NIM and the neurotoxins represented 37% of ICa, whereas in neurons not having L-type currents the resistant current made up approximately 53% of ICa (49 +/- 2%, mean +/- SE). The resistant current activated at around -40 mV and peaked at approximately 0 mV with half-activation and -inactivation potentials of -17 and -58 mV and slopes for activation and inactivation of -5 and 13 mV, respectively. The resistant current was sensitive to Cd2+ (IC50 = 2.5 microM) and Ni2+ (IC50 = 86 microM), was larger in Ca2+ than in Ba2+ (ratio = 1.31:1), and showed a moderate rate of decay. In summary, our results show that the high-voltage-activated calcium current in rat CeA neurons is composed of at least four pharmacologically distinct components: L-type current (NIM sensitive, 22%), N-type current (CgTx GVIA sensitive, 30%), Q-type current [Aga IVA (1 microM) and CgTx MVIIC sensitive, approximately 13-18%], and a resistant current (Non-L, -N, and -Q current, 33 approximately 37%), amounting to 37-53% of the total current. The resistant current has some electrophysiological and pharmacological characteristics in common with doe-1, alpha 1E, and R-type calcium currents, but remains unclassified.

Okadaic acid enhances prepulse facilitation of cardiac α1-subunit but not endogenous calcium channel currents in Xenopus laevis oocytes

Xenopus laevis oocytes can be selected to express relatively high levels of endogenous Ca currents. These currents are facilitated by prepulses. Facilitated endogenous Ca currents are unaffected by okadaic acid, RpcAMPS or the dihydropyridine (DHP) antagonist (+) PN 200-110. The endogenous currents and facilitation of endogenous currents by depolarizing prepulses are fully blocked by 1 mM Cd2+. In contrast, oocytes injected with mRNA encoding for the rabbit cardiac alpha 1-subunit express prepulse-facilitated Ca channel currents that are highly enhanced by the phosphoprotein phosphatase inhibitor okadaic acid (3-fold) and blocked by RpcAMPS and the DHP antagonist (+) PN 200-110. While okadaic acid selectively stimulates prepulse facilitation of cardiac alpha 1-subunit Ca currents, the DHP agonist (+) SDZ 202-791 largely increases (5-fold) both the control (before prepulse) and facilitated currents (after prepulse). (+) SDZ 202-791 did not prevent the effect of RpcAMPS or okadaic acid on facilitation of cardiac alpha 1-subunit, suggesting that DHP stimulation is independent of phosphorylation leading to channel facilitation. The enhancement of prepulse facilitation of cardiac alpha 1L-subunit Ca channel current by okadaic acid can be accounted for by a speeding up in the rates of onset during the prepulse. Inhibition of phosphoprotein phosphatases by okadaic acid has only modest effects on the rates of recovery of cardiac alpha 1-subunit Ca channel current from facilitation in the time immediately following the prepulse.

Bay K 8644 reveals two components of L-type Ca2+ channel current in clonal rat pituitary cells.

Whole-cell L-type Ca2+ channel current was recorded in GH3 clonal rat pituitary cells using Ba2+ as a charge carrier. In the presence of the dihydropyridine agonist Bay K 8644, deactivation was best described by two exponential components with time constants of approximately 2 and approximately 8 ms when recorded at -40 mV. The slow component activated at more negative potentials than the fast component: Half-maximal activation for the slow and fast components occurred at approximately -15 and approximately 1 mV, respectively. The fast component was more sensitive to enhancement by racemic Bay K 8644 than the slow component: ED50fast = approximately 21 nM, ED50slow = approximately 74 nM. Thyrotropin-releasing hormone (TRH; 1 microM) inhibited the slow component by approximately 46%, whereas the fast component was inhibited by approximately 22%. TRH inhibition of total L-current showed some voltage dependence, but each Bay K 8644-revealed component of L-current was inhibited in a voltage-independent manner. Therefore, the apparent voltage dependence of TRH action is derived from complexities in channel gating rather than from relief of inhibition at high voltages. In summary, Bay K 8644-enhanced L-currents in GH3 cells consist of two components with different sensitivities to voltage, racemic Bay K 8644, and the neuropeptide TRH.

Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes.

Skeletal and cardiac dihydropyridine receptors function both as voltage-dependent L-type calcium channels (L-channels) and as critical proteins that trigger calcium release from the sarcoplasmic reticulum in muscle. In spite of these similarities, skeletal L-channels exhibit a markedly slower activation rate than cardiac L-channels. We investigated the mechanisms underlying this difference by comparing the unitary behavior of L-channels in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, or chimeric dihydropyridine receptors. Our results demonstrate that ensemble averages activate rapidly for the purely cardiac dihydropyridine receptor and approximately five times more slowly for L-channels attributable to the purely skeletal dihydropyridine receptor or a chimeric dihydropyridine receptor in which only the first internal repeat and all of the putative intracellular loops are of skeletal origin. All of the constructs studied similarly exhibit a brief (2-ms) and a long (> or = 15-ms) open time in the presence of Bay K 8644, neither of which depend significantly on voltage. In the absence of Bay K 8644, the fraction of total open events is markedly shifted to the briefer open time without altering the rate of ensemble activation. Closed time analysis of L-channels with cardiac-like, rapid activation (recorded in the presence of dihydropyridine agonist) reveals both a brief (approximately 1-ms) closed time and a second, voltage-dependent, long-lasting closed time. The time until first opening after depolarization is three to six times faster for rapidly activating L-channels than for slowly activating L-channels and depends strongly on voltage for both types of channels. The results suggest that a voltage-dependent, closed-closed transition that is fast in cardiac L-channels and slow in skeletal L-channels can account for the difference in activation rate between these two channels.

Electrophysiological properties of the hypokalaemic periodic paralysis mutation (R528H) of the skeletal muscle α1S subunit as expressed in mouse L 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 α 1 subunit ( α 1 S ). Here, we have introduced one of the point mutations causing HypoPP (R528H) into cDNA of the rabbit α 1 S . Expression of either the wild-type α 1S or the mutant R528H α 1 S ( α 1 S− R528 H) subunit was obtained in mouse Ltk − cell using a selectable expression vector. The α 1 S− R528 H subunit led to the expression of functional L-type Ca 2+ channels. Corresponding whole-cell Ba 2+ currents exhibit very slow activation and inactivation kinetics, typical for recombinant skeletal Ca 2+ channel currents. Voltage-dependent activation and inactivation properties were similar for α 1 S -and α 1 S − R528 H , as well as their sensitivity to the dihydropyridine agonist Bay K 8644. Differences in α 1 S -and α 1 S− R528 H -directed channels reside in the Ba 2+ current density, which was significantly reduced 3.2 fold in cells expressing α 1 S − R528 H . It was concluded that the R528H mutation of α 1 S results in minor differences in the electrophysiological properties but significantly reduces the whole-cell Ca 2+ channel current in its amplitude.

Serotonin modulates N- and P-type calcium currents in neocortical pyramidal neurons via a membrane-delimited pathway.

1. The effects of serotonin (5HT) on neocortical pyramidal neurons were studied using whole cell and ON-cell patch-clamp recordings from acutely dissociated neurons. 2. 5HT decreased high voltage-activated calcium channel currents in a dose-dependent and reversible manner in acutely dissociated neocortical pyramidal neurons. The maximum block was 30% of the peak whole cell current (at -10 mV). 3. The 5HT modulation was mimicked by 5HT1A agonists and was reduced by 5HT1A antagonists. 5HT2 antagonists had no effect on the modulation. These data suggest that the 5HT effects were mediated by 5HT1A receptors. 4. The 5HT1A modulation was reduced in the presence of the specific N-type blocker omega-conotoxin GVIA (CgTx) and by the P-type channel blocker omega-agatoxin IVA (AgTx), but not by the L-type blocker nifedipine. 5HT did not modulate the slowed tail currents in the presence of the dihydropyridine agonist Bay K 8644. These data suggest that N- and P-type channels (but not L-type channels) were targeted by 5HT. 5. The modulation involved G proteins and utilized a membrane-delimited pathway. The modulation was rapid in onset (tau approximately 600 ms) and offset. About 50% of the reduction in current by 5HT1A agonists was overcome by prepulses to 120 mV. 6. Slowing of current onset kinetics in response to 5HT1A agonists was seen rarely in neocortical pyramidal neurons (11% of cases). The presence of slowing depended on agonist concentration, being evident only with high micromolar doses.