Ca Channel Currents Research Articles

Different pharmacological properties of the optical isomers of MN9202, a novel 1,4‐dihydropyridine Ca2+ channel modulator, in rat ventricular myocytes

1. We have shown previously that 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid pentyl methyl ester (MN9202), a new 1,4-dihydropyridine Ca(2+) channel modulator, has significant hypotensive effects and favourable pharmacokinetic characteristics. As a chiral molecule, MN9202 has two optical isomers. The aim of the present study was to evaluate the pharmacological properties of the two enantiomers. 2. The two enantiomers, S-(-)- and R-(+)-MN9202, were obtained by HPLC. At 1 micromol/L, both racemic MN9202 and S-(-)-MN9202 decreased the contractility of rat ventricular myocytes by 54.0 and 64.4%, respectively, compared with control, whereas R-(+)-MN9202 enhanced cell shortening by 10.1%. At 1 micromol/L, racemic MN9202 markedly reduced calcium transient (CaT) and L-type Ca(2+) channel current (I(Ca,L)) by 60.0 and 50.7%, respectively, whereas the reductions in CaT and I(Ca,L) produced by 1 micromol/L S-(-)-MN9202 were greater still (62.2 and 65.7%, respectively). In contrast, 1 micromol/L R-(+)-MN9202 increased CaT and I(Ca,L) by 11.4 and 10.6%, respectively. Furthermore, findings from kinetics studies of I(Ca,L) revealed that the steady state inactivation curve of I(Ca,L) was shifted towards a hyperpolarizing potential by S-(-)-MN9202, but towards a depolarizing potential by R-(+)-MN9202. These results demonstrate different effects of R-(+)-MN9202 and S-(-)-MN9202. 3. In conclusion, the findings of the present study suggest that the chirality of MN9202 results in opposing pharmacological properties of its two enantiomers: S-(-)-MN9202 may be responsible for the therapeutic effects of racemic MN9202, whereas R-(+)-MN9202 contributes to it unwanted effects. The findings of the present study also indicate that MN9202 may be used as a new probe with which to investigate the structure-function relationships of Ca(2+) channels.

Modulation of High-Voltage Activated Ca 2+ Channels by Membrane Phosphatidylinositol 4,5-Bisphosphate

Modulation of voltage-gated Ca(2+) channels controls activities of excitable cells. We show that high-voltage activated Ca(2+) channels are regulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) with different sensitivities. Plasma membrane PIP(2) depletion by rapamycin-induced translocation of an inositol lipid 5-phosphatase or by a voltage-sensitive 5-phosphatase (VSP) suppresses Ca(V)1.2 and Ca(V)1.3 channel currents by approximately 35% and Ca(V)2.1 and Ca(V)2.2 currents by 29% and 55%, respectively. Other Ca(V) channels are less sensitive. Inhibition is not relieved by strong depolarizing prepulses. It changes the voltage dependence of channel gating little. Recovery of currents from inhibition needs intracellular hydrolysable ATP, presumably for PIP(2) resynthesis. When PIP(2) is increased by overexpressing PIP 5-kinase, activation and inactivation of Ca(V)2.2 current slow and voltage-dependent gating shifts to slightly higher voltages. Thus, endogenous membrane PIP(2) supports high-voltage activated L-, N-, and P/Q-type Ca(2+) channels, and stimuli that activate phospholipase C deplete PIP(2) and reduce those Ca(2+) channel currents.

The effect of tyrosine nitration of L‐type Ca2+ channels on excitation–transcription coupling in colonic inflammation

Excitation-transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L-type Ca(2+) channels induces phosphorylation of the transcription factor, cyclic-AMP response element binding protein (CREB) and downstream activation of the cyclic-AMP response element (CRE) promoter regions. Tyrosine nitration of Ca(2+) channels attenuates interactions with c-Src kinase, decreasing Ca(2+) channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca(2+) channel mediated phosphorylation of CREB and CRE activation. CREB and phospho-CREB were detected by Western blots and CRE activation measured by dual luciferase assay. Chinese hamster ovary (CHO) cells were transfected with hCa(v)1.2b and hCa(v)1.2b c-terminal mutants. Colonic inflammation was induced by intracolonic instillation of 2,4,6 trinitrobenzene sulphonic acid in mouse colon. In hCa(v)1.2b transfected CHO cells and in native colonic smooth muscle, depolarization with 80 mM KCl induced CREB phosphorylation (pCREB). Treatment with peroxynitrite inhibited KCl-induced pCREB. Following experimental colitis, KCl-induced CREB phosphorylation was abolished in smooth muscle, concomitant with tyrosine nitration of Ca(2+) channels. Depolarization increased CRE activation in hCa(v)1.2b CHO cells by 2.35 fold which was blocked by nifedipine and by protein nitration of Ca(2+) channels with peroxynitrite. The Src-kinase inhibitor, PP2, blocked depolarization-induced CRE activation. Mutation of the C-terminus tyrosine residue, Y2134F, but not Y1861F, blocked CRE activation. Post-translational modification of Ca(2+) channels due to tyrosine nitration modified excitation-transcriptional coupling in colonic inflammation.

Atrial Natriuretic Peptide Regulates Ca2+ Channel in Early Developmental Cardiomyocytes

BackgroundCardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca2+ currents has not been investigated in developmental cardiomyocytes.Methodology/Principal FindingsWe investigated the role of ANP in regulating L-type Ca2+ channel current (ICaL) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal ICaL in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of ICaL by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of ICaL induced by ANP was not affected by either Nω, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of ICaL by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested.Conclusion/SignificancesTaken together, these results indicate that ANP induced depression of action potentials and ICaL is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3′, 5′–cyclic monophophate (cAMP)–cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.

Characterization of L-Type Ca Channel Currents (ICa,L) in Zebrafish Cardiomyocytes

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations for functional interrogation. The similarity of the zebrafish cardiac action potential duration with that of humans further enhances the role of this model to study cardiac arrhythmias. Despite this, little is known about basic excitation-contraction coupling in the zebrafish heart. Single cardiomyocytes were isolated from the adult zebrafish heart by enzymatic perfusion of the cannulated ventricle.

Association of Voltage-Gated Calcium Channel Subunit α2δ-3 with Lipid Rafts: Structural and Functional Implications

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the main functional properties of the channel. The α1 subunit is associated with auxiliary subunits including intracellular β and α2δ, which modulate trafficking and functional properties of the channels.α2δ subunits consists of two peptides: α2 which is entirely extracellular is disulfide-bonded to a δ subunit that links the protein into the plasma membrane. There are four genes encoding α2δ subunits, which are believed to have similar structure. We have shown previously that α2δ-2 subunits associate with lipid rafts, that are sub-domains of the cell membrane enriched in cholesterol and glycosphingolipids.We have addressed the ability of α2δ-3 to associate with lipid rafts in both native tissues (it is highly expressed in brain) and in overexpression systems. We have generated mutations which reduced expression of the subunit in lipid rafts as well as the surface expression of the protein. These mutations reduced the enhancing effect of α2δ-3 on calcium channel currents.The α2 and δ peptides are product of a single gene, and they are encoded as an uninterrupted α2δ pre-protein, which is further processed post-translationally. In native tissues we observed exclusively the mature form of the protein, which was strongly associated with the lipid rafts. However, in several overexpression systems we could also detect unprocessed α2δ-3 pre-protein coexisting with the mature α2δ3. The unprocessed form was localized both in the rafts and non-raft protein fractions, suggesting that maturation of the protein might occur in localized membrane domains.These results further demonstrate the role of lipid rafts in the regulation of Ca channel currents by α2δ and their involvement in the maturation of the α2δ protein.

Lactobacillus reuteri ingestion and IKCa channel blockade have similar effects on rat colon motility and myenteric neurones

We have previously shown that ingestion of Lactobacillus reuteri may modulate colonic enteric neuron activity but with unknown effects on colon motility. The aim of the present report was to elucidate the neuronal mechanisms of action of the probiotic by comparing the effects on motility of L. reuteri ingestion with blockade of a specific ionic current in enteric neurons. We have used intraluminal pressure recordings from ex vivo rat colon segments and whole cell patch clamp recordings from neurons of rat longitudinal muscle myenteric plexus preparations to investigate the effects of L. reuteri and TRAM-34 on colon motility and neurophysiology. The effects of daily feeding of 10(9) L. reuteri bacteria or acute application of TRAM-34 on threshold fluid filling pressure or pulse pressure was measured. Lactobacillus reuteri increased intraluminal fluid filling pressure thresholds for evoking pressure pulses by 51% from 0.47 +/- 0.17 hPa; the probiotic also decreased the pulse pressure amplitudes, but not frequency, by 18% from 3.91 +/- 0.52 hPa. The intermediate conductance calcium-dependent potassium (IK(Ca)) channel blocker TRAM-34 (3 micromol L(-1)) increased filling threshold pressure by 43% from 0.52 +/- 0.22 hPa and reduced pulse pressure amplitude by 40% from 2.63 +/- 1.11 hPa; contraction frequency was unaltered. TRAM-34 (3 micromol L(-1)) reduced membrane polarization, leak conductance and the slow afterhyperpolarization current in 16/16 myenteric rat colon AH cells but 19/19 S cells were unaffected. The present results are consistent with L. reuteri enhancing tonic inhibition of colon contractile activity by acting via the IK(Ca) channel current in AH cells.

Exercise training changes the gating properties of large-conductance Ca 2+-activated K + channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca 2+-activated K + (BK Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability ( Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca 2+ and voltage without altering the unitary conductance and the K + selectivity. Our results suggest a novel mechanism by which exercise training increases the K + currents by changing the BK Ca channel activities and kinetics.

Plasma membrane‐associated proline‐rich extensin‐like receptor kinase 4, a novel regulator of Ca2+ signalling, is required for abscisic acid responses in Arabidopsis thaliana

Plant roots respond to environmental stresses or the exogenous plant hormone abscisic acid (ABA) by undergoing marked physiological and morphological changes. We show here that PERK4, a gene that encodes a member of the Arabidopsis thaliana proline-rich extensin-like receptor kinase family, plays an important role in ABA responses. Mutation of PERK4 by T-DNA insertion decreased sensitivity to ABA with respect to seed germination, seedling growth and primary root tip growth. The effect on root growth was due to enhanced cell elongation rather than cell division. The cytosolic free calcium concentration and Ca(2+) channel currents were lower in perk4 root cells than in wild-type cells in the presence of ABA. Root growth was similar in wild-type and perk4 plants after the application of a Ca(2+) channel blocker. PERK4 localised to the plasma membrane, and was shown to be an ABA- and Ca(2+)-activated protein kinase. Our data suggest that the receptor-like kinase encoded by PERK4 functions at an early stage of ABA signalling to inhibit root cell elongation by perturbing Ca(2+) homeostasis.

The negative inotropic action of canrenone is mediated by L‐type calcium current blockade and reduced intracellular calcium transients

Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis. Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques. Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone. Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.

Enhancement of BK Ca channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H 2O 2) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN wt, PTEN C124S, PTEN G129E in HEK 293 cells. Typical BK Ca channel currents could be recorded in cell-attached configurations. We found that PTEN wt reduced the H 2O 2-induced BK Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN C124S/PTEN G129E mutants that lack lipid phosphatase activity produced no regulation on the H 2O 2-induced BK Ca channel activation. These results demonstrated that PTEN regulated the H 2O 2-induced BK Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H 2O 2-induced BK Ca channel activation was attenuated when cells were treated with H 2O 2 at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca 2+] i) induced by H 2O 2 was also inhibited. These findings may elucidate a new mechanism for H 2O 2-induced BK Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H 2O 2.

Egg Coat Proteins Activate Calcium Entry into Mouse Sperm via CATSPER Channels1

During mammalian fertilization, the contact between sperm and egg triggers increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in sperm. Voltage-gated Ca(2+) channels (Ca(V)s) are believed to mediate the initial phase of [Ca(2+)](i) increases in sperm induced by egg coat (zona pellucida [ZP]) glycoproteins, while store depletion-activated Ca(2+) entry is thought to mediate the sustained phase. Using patch-clamp recording and Ca(2+) imaging, we show herein that Ca(V) channel currents, while found in spermatogenic cells, are not detectable in epididymal sperm and are not essential for the ZP-induced [Ca(2+)](i) changes. Instead, CATSPER channels localized in the distal portion of sperm (the principal piece) are required for the ZP-induced [Ca(2+)](i) increases. Furthermore, the ZP-induced [Ca(2+)](i) increase starts from the sperm tail and propagates toward the head.

Mutations Leu427, Asn428, and Leu431 Residues within Transmembrane Domain-I-Segment 6 Attenuate Ginsenoside-Mediated L-Type Ca2+ Channel Current Inhibitions

Many lines of evidences have shown that Panax ginseng exhibits beneficial effects on cardiovascular systems. We previously demonstrated that ginsenoside Rg(3) (Rg(3)), one of active ingredients of Panax ginseng, inhibits Ca(2+) channel currents in a stereospecific manner and affects the steady-state activation but not inactivation. This points a possibility that Rg(3) regulates Ca(2+) channels through specific interaction site(s) for Ca(2+) influx inhibition through Ca(2+) channels. However, it was not known how Rg(3) interacts with Ca(2+) channel proteins. In the current study, we sought to identify these site(s) in Xenopus oocytes expressing cardiac wild-type and mutant L(alpha(1C))-type Ca(2+) channels using the two-microelectrode voltage-clamp technique. To this end, we assessed how various point mutations of the L-type Ca(2+) channel affected the Rg(3) action. Mutations of L427R, N428R and L431K in transmembrane domain-I-segment 6 (IS6) of the channel significantly attenuated the Rg(3) action and caused rightward shifts in dose-response curves. Rg(3) treatment produced a negative shift in the inactivation voltage but did not alter the steady-state activation voltage, and none of the mutant channels affected the Rg(3)-induced negative shift of inactivation voltage. Rg(3) had no effects on inactivation time constant in wild-type and mutant channels. These results indicate that Rg(3) inhibition of L-type Ca(2+) channel currents is attenuated by mutations of Leu427, Asn428 and Leu431 in transmembrane IS6 residues. Leu427, Asn428 and Leu431 residues of the L-type Ca(2+) channel play important roles in the Rg(3) effect on channel properties.

Calcium-Sensing Receptor Activation Depresses Synaptic Transmission

At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca](o)) and thus may impair neurotransmission. Increases in [Ca](o) activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits nonselective cation channels at the majority of cortical nerve terminals. This pathway may modulate synaptic transmission by attenuating the impact of decreases in [Ca](o) on synaptic transmission. Using patch-clamp recording from isolated cortical terminals, cortical neuronal pairs and isolated neuronal soma we examined the modulation of synaptic transmission by CaSR. EPSCs were increased on average by 88% in reduced affinity CaSR-mutant (CaSR(-/-)) neurons compared with wild-type. Variance-mean analysis indicates that the enhanced synaptic transmission was due largely to an increase in average probability of release (0.27 vs 0.46 for wild-type vs CaSR(-/-) pairs) with little change in quantal size (23 +/- 4 pA vs 22 +/- 4 pA) or number of release sites (11 vs 13). In addition, the CaSR agonist spermidine reduced synaptic transmission and increased paired-pulse depression at physiological [Ca](o). Spermidine did not affect quantal size, consistent with a presynaptic mechanism of action, nor did it affect voltage-activated Ca channel currents. In summary, reduced CaSR function enhanced synaptic transmission and CaSR stimulation had the opposite effect. Thus CaSR provides a mechanism that may compensate for the fall in release probability that accompanies decreases in [Ca](o).

The Janus‐faced atracotoxins are specific blockers of invertebrate KCa channels

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.

Cardiomyocyte Ca2+ overload in atrial tachycardia: is blockade of L-type Ca2+ channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca(2+) overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca(2+) overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca(2+) channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca(2+) channel current (I(Ca,L)) that may reinforce Ca(2+) overload thereby promoting AF in the atrium. Ca(2+) channel blockers which target T-type Ca(2+) channels in addition to I(Ca,L) (for instance, efonidipine) may be more efficient at preventing Ca(2+) overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca(2+) channel blockers that additionally target T-type Ca(2+) channels.

Protease Treatment of Cerebellar Purkinje Cells Renders ω-Agatoxin IVA-Sensitive Ca2+ Channels Insensitive to Inhibition by ω-Conotoxin GVIA

The identification of currents carried by N- and P-type Ca(2+) channels in the nervous system relies on the use of omega-conotoxin (CTx) GVIA and omega-agatoxin (Aga) IVA. The peptide omega-Aga-IVA inhibits P-type currents at nanomolar concentrations and N-type currents at micromolar concentrations. omega-CTx-GVIA blocks N-type currents, but there have been no reports that it can also inhibit P-type currents. To assess the effects of omega-CTx-GVIA on P-type channels, we made patch-clamp recordings from the soma of Purkinje cells in cerebellar slices of mature [postnatal days (P) 40-50, P40-50] and immature (P13-20) rats, in which P-type channels carry most of the Ca(2+) channel current (>/=85%). These showed that micromolar concentrations of omega-CTx-GVIA inhibited the current in P40-50 cells (66%, 3 microM; 78%, 10 microM) and in P13-20 Purkinje cells (86%, 3 muM; 89%, 10 microM). The inhibition appeared to be reversible, in contrast to the known irreversible inhibition of N-type current. Exposure of slices from young animals to the enzyme commonly used to dissociate Purkinje cells, protease XXIII, abolished the inhibition by omega-CTx-GVIA but not by omega-Aga-IVA (84%, 30 nM). Our finding that micromolar concentrations of omega-CTx-GVIA inhibit P-type currents suggests that specific block of N-type current requires the use of submicromolar concentrations. The protease-induced removal of block by omega-CTx-GVIA but not by omega-Aga-IVA indicates a selective proteolytic action at site(s) on P-type channels with which omega-CTx-GVIA interacts. It also suggests that Ca(2+) channel pharmacology in neurons dissociated using protease may not predict that in neurons not exposed to the enzyme.

Inhibition of L-type Ca2+ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes

We have previously shown an increase in arachidonic acid (AA) release in response to proinflammatory cytokines in adult rat ventricular myocytes (ARVM). AA is known to alter channel activities; however, its effects on cardiac L-type Ca(2+) channel current (I(Ca,L)) and excitation-contraction coupling remain unclear. The present study examined effects of AA on I(Ca,L), using the whole cell patch-clamp technique, and on cell shortening (CS) and the Ca(2+) transient of ARVM. I(Ca,L) was monitored in myocytes held at -70 mV and internally equilibrated and externally perfused with Na(+)- and K(+)-free solutions. Exposure to AA caused a voltage-dependent block of I(Ca,L) concentration dependently (IC(50) 8.5 microM). The AA-induced inhibition of I(Ca,L) is consistent with its hyperpolarizing shift in the voltage-dependent properties and reduction in maximum slope conductance. In the presence of AA, BSA completely blocked the AA-induced suppression of I(Ca,L) and CS. Intracellular load with AA had no effect on the current density but caused a small depolarizing shift in the I(Ca,L) activation curve, suggesting a site-specific action of AA. Moreover, intracellular AA had no effect on the extracellular AA-induced decrease in I(Ca,L). Pretreatment with indomethacin, an inhibitor of cyclooxygenase, or addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase, had no effect on AA-induced changes in I(Ca,L). Furthermore, AA suppressed CS and Ca(2+) transients of intact ARVM with no significant effect on SR function and myofilament Ca(2+) sensitivity. Therefore, these results suggest that AA inhibits contractile function of ARVM, primarily due to its direct inhibition of I(Ca,L) at an extracellular site.

Increased Ca2+ channel currents in cerebellar Purkinje cells of the ataxic groggy rat

The ataxic groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats. Recent genetic analysis has identified the missense (M251K) mutation in the alpha(1) subunit of the Ca(V)2.1 (P/Q-type) voltage-dependent Ca(2+) channel gene (Cacna1a) of GRY rat. In this study, we found that high-voltage-activated (HVA) Ca(2+) channel currents in acutely dissociated Purkinje cells of GRY rats showed increased (not decreased) current density and depolarizing shift of the activation and inactivation curves compared with those of normal Wistar rats. In contrast low-voltage-activated (LVA) Ca(2+) channel currents of GRY rats showed no significant changes. These results suggest that functional alteration of Ca(2+) channel currents in cerebellar Purkinje cells of GRY rats is attributed to the change of HVA Ca(2+) channel currents, and that increased HVA Ca(2+) channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.

Effects of local anesthetics on opioid inhibition of calcium current in rat dorsal root ganglion neurons

Neuraxial analgesia is often provided using a mixture of local anesthetics and opioids. This combination of agents provides better pain relief and is generally associated with fewer side effects than when either drug is given alone. Local anesthetics have been shown to alter signaling of other G protein-coupled receptors, but little is known about their effect on opioid receptor signaling. Because opioids produce analgesia at least in part by inhibiting presynaptic Ca channels, we have evaluated the effects of tetracaine and bupivacaine on opioid-mediated inhibition of Ca channels in dorsal root ganglion neurons. The μ-opioid specific agonist DAMGO (1 μM) inhibited Ca channels in both the absence and presence of tetracaine (50 or 100 μM). However, the extent of DAMGO inhibition in the presence of both concentrations of tetracaine was less than that observed in the absence of tetracaine. DAMGO inhibition decreased from 39.2 ± 24.4% in control to 34.2 ± 24.4% with 50 μM tetracaine ( n = 16; p < 0.05), and from 40.5 ± 19.6% in control to 34.6 ± 20.5% with 100 μM tetracaine ( n = 10; p < 0.05). Similar results were seen with bupivacaine. Tetracaine also decreased the voltage-dependent facilitation of Ca channel currents when G proteins were activated by either DAMGO or the non-hydrolyzable GTP analogue (GTPγS), suggesting that tetracaine weakens the interaction between G protein βγ subunits and the Ca channel. Overall, these results suggest that local anesthetics decrease opioid inhibition of Ca channel activity by interfering with the GTP-mediated signal transduction between opioid receptors and Ca channels.