Halothane attenuates the cerebroprotective action of several Na+ and Ca2+ channel blockers via reversal of their ion channel blockade

We have previously shown the involvement of Na(+) channel as well as N-type and P/Q-type Ca(2+) channels in the oxygen and glucose deprivation-induced injury in rat cerebrocortical slices. In the present study, we investigated the influence of halothane on the cerebroprotective effects of a variety of Na(+) and Ca(2+) channel blockers in rat cerebrocortical slices. The hypoxic injury was attenuated by Na(+) channel blockers including tetrodotoxin, lidocaine and dibucaine, and Ca(2+) channel blockers, such as verapamil, omega-agatoxin IVA and omega-conotoxin GVIA. Halothane abolished the protective effects of lidocaine, dibucaine and verapamil, all of which block the respective cation channels in a voltage-dependent manner, without affecting the actions of tetrodotoxin, omega-agatoxin IVA and omega-conotoxin GVIA, which reveal voltage-independent blockade. On the other hand, the nitric oxide synthesis estimated from the extracellular cyclic GMP formation was elevated during exposure to hypoxia. All channel blockers tested here attenuated hypoxia-evoked nitric oxide synthesis. Halothane blocked almost completely these actions of lidocaine and verapamil. Moreover, the Na(+) and Ca(2+) channel blockade by these compounds, as determined by veratridine- and KCl-stimulated nitric oxide synthesis, respectively, was also reversed by halothane. These findings suggest that an anesthetic agent halothane reversed the Na(+) and Ca(2+) channel blockade of several voltage-dependent ion channel blockers, leading to the attenuation of their cerebroprotective actions. Therefore, the influence of halothane anesthesia should be taken into consideration for the evaluation of neuroprotective action of Na(+) and Ca(2+) channel blockers.     

Gabapentin inhibits high-threshold calcium channel currents in cultured rat dorsal root ganglion neurones.

1. This study examined the action of gabapentin (gabapentin,1-(aminomethyl) cyclohexane acetic acid (Neurontin) on voltage-gated calcium (Ca(2+)) channel influx recorded in cultured rat dorsal root ganglion (DRG) neurones. 2. Voltage-gated Ca(2+) influx was monitored using both fura-2 based fluorescence Ca(2+) imaging and the whole-cell patch clamp technique. 3. Imaging of intracellular Ca(2+) transients revealed that gabapentin inhibited KCl (30 mM)-evoked voltage-dependent Ca(2+) influx. Both the duration for 50% of the maximum response (W50) and total Ca(2+) influx were significantly reduced by approximately 25-30% in the presence of gabapentin (25 microM). 4. Gabapentin potently inhibited the peak whole-cell Ca(2+) channel current (I(Ba)) in a dose-dependent manner with an estimated IC(50) value of 167 nM. Block was incomplete and saturated at a maximal concentration of 25 microM. 5. Inhibition was significantly decreased in the presence of the neutral amino acid L-isoleucine (25 microM) but unaffected by application of the GABA(B) antagonist, saclofen (200 microM), suggesting a direct action on the alpha(2)delta subunit of the Ca(2+) channel. 6. Gabapentin inhibition was voltage-dependent, producing an approximately 7 mV hyperpolarizing shift in current voltage properties and reducing a non-inactivating component of whole-cell current activated at relatively depolarized potentials. 7. The use of specific Ca(2+) channel antagonists revealed a mixed pharmacology of the gabapentin-sensitive current (N-, L- and P/Q-type), which is dominated by N-type current. 8. The present study is the first to demonstrate that gabapentin directly mediates inhibition of voltage-gated Ca(2+) influx in DRG neurones, providing a potential means for gabapentin to effectively mediate spinal anti-nociception.     

Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca(2+) entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG x neuroblastoma hybrid) cells. Gabapentin (2.5 microM) in contrast to GABA (10 microM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba(2+). Gabapentin attenuated high voltage-activated Ca(2+) channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca(2+) influx evoked by K(+) depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA(B) receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca(2+) channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca(2+) channels to gabapentin. We analysed the Ca(2+) channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca(2+) channel beta(2) and alpha(2)delta subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca(2+) current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca(2+) channel beta(2) subunit type as critically important to drug sensitivity and interactions with alpha(1) and alpha(2)delta subunits may be implicated in antihyperalgesic therapeutic action for this compound.     

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.     

Inhibition of neuronal Ca(2+) influx by gabapentin and pregabalin in the human neocortex.

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.     

On the specificity of verapamil as a calcium channel-blocker

The stimulated uptake of 45Ca2+ into incubated cerebrocortical synaptosomes caused by veratrine (75 microM) was blocked by low concentrations of verapamil (0.5-30 microM) which did not prevent or reduce depolarization as judged by efflux of potassium (K+). However, verapamil did not prevent amino acid neutrotransmitter release at these low concentrations and this is discussed in terms of mobilization of internal calcium (Ca2+) stores. At higher concentrations (30-200 microM) verapamil appeared to act additionally at sodium (Na+) channels since both depolarization-induced K+ efflux and neurotransmitter release were reduced or prevented. When K+, at a high concentration (56 mM), was used as the depolarizing agent, both 45Ca2+ influx and neurotransmitter release were prevented by verapamil across a wide concentration range (0.5-200 microM). The data are discussed in terms of the specificity of action of verapamil on Ca2+ channels.     

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca(2+) channels and adenylyl cyclase. Through Ca(2+) imaging and immunocytochemistry, we have recently shown that adenosine A(1), GABA(B) and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca(2+) channels by analysing this process in nerve terminals from mice lacking the alpha(1B) subunit (Ca(v) 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca(2+) channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABA(B) and adenosine A(1). Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A(1), GABA(B) and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca(2+) channel type but rather, it is associated to functional mGlu7 receptors.     

Reduced verapamil inhibition of endothelin-1-constricted rabbit basilar artery due to enhanced non L-type Ca(2+)-channel-dependent constriction.

This study tested whether (1) L-type Ca(2+) channel blockade and extracellular Ca(2+) removal prior to endothelin-1, as compared to during the endothelin-1 constriction, resulted in lesser inhibition, and (2) the reduced inhibition due to prior L-type Ca(2+) channel blockade resulted from enhanced non L-type Ca(2+)-channel-dependent constriction. Pretreatment of rabbit basilar artery in vitro with 1 microM verapamil, an L-type Ca(2+) channel blocker, inhibited 3, 10, 30, and 100 nM endothelin-1 constrictions to a lesser extent than verapamil addition during the plateau endothelin-1 constriction. Ni(2+) (0.03 and 0.1 mM), a nonselective cation channel blocker, relaxed the plateau endothelin-1 constrictions in vessels pretreated with verapamil to greater magnitudes than vessels unexposed to verapamil. Extracellular Ca(2+) removal prior to 10, 30, and 100 nM endothelin-1 also inhibited the endothelin-1 constrictions to smaller magnitudes than Ca(2+) removal during the plateau endothelin-1 constrictions. These results suggest that the reduced inhibition of the endothelin-1 constriction following pretreatment with L-type Ca(2+) channel blocker or Ca(2+)-free solution, as compared to addition of these agents during the endothelin-1 constriction, is the result of non L-type Ca(2+) channel opening and enhanced Ca(2+)-independent constriction, respectively.     

Mechanisms of the antinociceptive action of gabapentin

Gabapentin, a gamma-aminobutyric acid (GABA) analogue anticonvulsant, is also an effective analgesic agent in neuropathic and inflammatory, but not acute, pain systemically and intrathecally. Other clinical indications such as anxiety, bipolar disorder, and hot flashes have also been proposed. Since gabapentin was developed, several hypotheses had been proposed for its action mechanisms. They include selectively activating the heterodimeric GABA(B) receptors consisting of GABA(B1a) and GABA(B2) subunits, selectively enhancing the NMDA current at GABAergic interneurons, or blocking AMPA-receptor-mediated transmission in the spinal cord, binding to the L-alpha-amino acid transporter, activating ATP-sensitive K(+) channels, activating hyperpolarization-activated cation channels, and modulating Ca(2+) current by selectively binding to the specific binding site of [(3)H]gabapentin, the alpha(2)delta subunit of voltage-dependent Ca(2+) channels. Different mechanisms might be involved in different therapeutic actions of gabapentin. In this review, we summarized the recent progress in the findings proposed for the antinociceptive action mechanisms of gabapentin and suggest that the alpha(2)delta subunit of spinal N-type Ca(2+) channels is very likely the analgesic action target of gabapentin.     

Variable, voltage-dependent, blocking effects of nitrendipine, verapamil, diltiazem, cinnarizine and cadmium on adrenomedullary secretion.

1. Catecholamine release from cat adrenal glands perfused at a high rate (4 ml min-1) at 37 degrees C with modified Krebs solutions lacking Ca and containing 1.2 mM K (hyperpolarizing solution) or 118 mM K (depolarizing solution) was triggered by 10-s pulses of Ca (0.5 mM) in the presence of 118 mM K. Hyperpolarized glands released 1280 +/- 135 ng per pulse and depolarized glands 831 +/- 98 ng per pulse (n = 29). 2. While the dihydropyridine Ca channel blocker nitrendipine inhibited secretion in hyperpolarized glands with an IC50 of 214 nM, in depolarizing conditions the drug was much more potent (IC50 = 0.99 nM). In contrast, the inorganic Ca channel blocker cadmium inhibited secretion with the same potency both in hyperpolarized or depolarized glands. 3. Cinnarizine, diltiazem and verapamil exhibited intermediate degrees of voltage-dependence in blocking secretion. The IC50 ratios between hyperpolarized and depolarized glands were 215, 36, 19, 8 and 0.76 respectively for nitrendipine, cinnarizine, diltiazem, verapamil and cadmium. Because the experimental design (strong depolarization in the absence of Ca) favours the highest opening probability of Ca channels, it seems that these drugs bind preferentially to their receptors when these channels are in their open state. 4. Variable voltage-dependent effects of the five Ca channel blockers on adrenomedullary catecholamine release suggests different sites and mechanisms of action on, or near L-type Ca channels in chromaffin cells. In addition, these findings might help to explain why these drugs exhibit tissue selectivity and why they act differently in normal polarized as compared to ischaemic depolarized cells.     

Inhibitory mechanisms of gabapentin, an antiseizure drug, on platelet aggregation

Gabapentin (Neurontin) is an analogue of gamma-aminobutyric acid (GABA) that is effective against partial seizures. Gabapentin has been reported to modulate serotonin release from platelets, but the effects of gabapentin on platelet activation have not been explored. In this study, gabapentin concentration-dependently (60-240 microM) inhibited platelet aggregation in washed platelets stimulated by collagen (1 microg mL(-1)), ADP (20 microM) and arachidonic acid (60 microM). Gabapentin (120 and 240 microM) also concentration-dependently inhibited collagen (1 microg mL(-1))-induced phosphoinositide breakdown, intracellular Ca(2+) mobilization, thromboxane A(2) formation, and p38 MAPK phosphorylation in human platelets. In conclusion, the most important findings of this study suggest that gabapentin inhibits platelet aggregation, at least in part, through the phospholipase C-inositol 1,4,5-trisphosphate-thromboxane A(2)-Ca(2+) pathway. Thus, it is possible that gabapentin treatment, alone or in combination with other antiplatelet drugs, may induce or potentiate inhibition of platelet aggregation, which may affect haemostasis in-vivo.     

Alternative splicing of the Ca2+ channel beta4 subunit confers specificity for gabapentin inhibition of Cav2.1 trafficking

Gabapentin is well established as an effective treatment for neuropathic pain; however, little is known about its mechanism of action. It binds with high affinity to Ca2+ channel alpha2delta subunits that are expressed in dorsal root ganglia. Mutation of a single alpha2delta amino acid, R217A, eliminates both gabapentin binding and analgesic efficacy. Gabapentin does not seem to have direct Ca2+ channel blocking properties but does affect overall levels of Ca2+channel surface expression in some circumstances. In this report, we examined gabapentin effects on trafficking and voltage-dependent gating properties of recombinant Ca(v)2.1 Ca2+ channel complexes transiently expressed in Xenopus laevis oocytes. We also determined electrophysiologically whether gabapentin causes displacement of beta subunits from Ca(v)2.1 complexes. Our principal findings are as follows: 1) gabapentin inhibits trafficking of recombinant Ca(v)2.1 Ca2+ channels in X. laevis oocytes; 2) gabapentin inhibition occurs in the presence of the Ca2+ channel beta4a subunit but not in the presence of beta4b; 3) gabapentin does not affect Ca(v)2.1 voltage-dependent gating parameters; 4) inhibition of Ca(v)2.1 trafficking is highly dependent on beta-subunit concentration; and 5) gabapentin inhibition of Ca(v)2.1 trafficking can be reversed by the alpha2delta R217A mutation. Overall, our results suggest that gabapentin reduces the number of beta4a-bound Ca(v)2.1 complexes that are successfully trafficked to the plasma membrane. This mechanism may help to explain why gabapentin is both effective and selective in the treatment of neuropathic pain states that involve up-regulation of alpha2delta subunits.     

Effect of calcium channel modulators on temperature regulation in ovariectomized rats

Clinical studies evaluating a calcium channel modulator, gabapentin, for the treatment of vasomotor symptoms have been reported. The present studies evaluated three calcium channel modulators in ovariectomized (OVX) rodent models of temperature regulation. Gabapentin, reported to interact with the alpha(2)delta subunit of voltage-sensitive calcium channels and the L-type voltage-gated calcium channel blockers, verapamil and nifedipine, were examined. These series of experiments demonstrated that orally administered gabapentin, verapamil and nifedipine all acutely and dose-dependently lower tail skin temperature in both models of OVX-induced thermoregulatory dysfunction. These compounds all had a rapid onset of action, however, the efficacy of all three calcium channel modulators is less than that observed following chronic estrogen treatment. Additionally, these compounds were also tested in a telemetric rat model measuring core body temperature to evaluate any temperature effects on internal core temperature. The present data suggests that gabapentin, verapamil and nifedipine all act to globally alter temperature regulation in steroid-dependent models of thermoregulatory function.     

Tetraethylammonium exacerbates ischemic neuronal injury in rat cerebrocortical slice cultures

We investigated potential contribution of K+ channel activity to regulation of ischemia-induced neuronal injury, using cerebrocortical slice cultures. Exposure of cultures to a glucose-free conditioning solution containing sodium azide and 2-deoxyglucose caused neuronal cell death as assessed by cellular uptake of propidium iodide, which was prevented by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist. Application of tetraethylammonium markedly exacerbated ischemic neuronal injury. Charybdotoxin, a blocker of large-conductance Ca(2+)-activated K+ (BK(Ca)) channels, also augmented ischemic injury, whereas AM 92016, a blocker of delayed rectifier K+ channels, and dequalinium, a blocker of small-conductance Ca(2+)-activated K+ channels, had no significant effect. In addition, tetraethylammonium and charybdotoxin were effective in augmenting NMDA-induced neuronal injury. These results present unprecedented evidence for the ability of tetraethylammonium to enhance ischemic neuronal death, and suggest that BK(Ca) channels constitute an endogenous system to protect cortical neurons from ischemic injury, via prevention of NMDA receptor over-activation.     

Mechanism of apoptosis induced by diazoxide,a K<Superscript>+</Superscript> channel opener,in HepG2 Human hepatoma cells

The effect of diazoxide, a K+ channel opener, on apoptotic cell death was investigated in HepG2 human hepatoblastoma cells. Diazoxide induced apoptosis in a dose-dependent manner and this was evaluated by flow cytometric assays of annexin-V binding and hypodiploid nuclei stained with propidium iodide. Diazoxide did not alter intracellular K+ concentration, and various inhibitors of K+ channels had no influence on the diazoxide-induced apoptosis; this implies that K+ channels activated by diazoxide may be absent in the HepG2 cells. However, diazoxide induced a rapid and sustained increase in intracellular Ca(2+) concentration, and this was completely inhibited by the extracellular Ca(2+) chelation with EGTA, but not by blockers of intracellular Ca(2+) release (dantrolene and TMB-8). This result indicated that the diazoxide-induced increase of intracellular Ca(2+) might be due to the activation of a Ca(2+) influx pathway. Diazoxide-induced Ca(2+) influx was not significantly inhibited by either voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), or by inhibitors of Na+, Ca(2+)-exchanger (bepridil and benzamil), but it was inhibited by flufenamic acid (FA), a Ca(2+)-permeable nonselective cation channel blocker. A quantitative analysis of apoptosis by flow cytometry revealed that a treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the diazoxide-induced apoptosis. Taken together, these results suggest that the observed diazoxide-induced apoptosis in the HepG2 cells may result from a Ca(2+) influx through the activation of Ca(2+)-permeable non-selective cation channels. These results are very significant, and they lead us to further suggest that diazoxide may be valuable for the therapeutic intervention of human hepatomas.     

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A₂ receptor-mediated extracellular Ca²⁺ influx in rat aorta

An involvement of signal transduction other than phosphatidylinositol turnover in thromboxane A(2) receptor (TP receptor)-mediated vascular contraction was investigated in rat aorta. The contraction induced by U46619, a TP receptor agonist, at low concentrations (≤ 30 nM) was partially inhibited by verapamil, an inhibitor of voltage-dependent Ca(2+) channels (VDCC), and was further diminished in Ca(2+)-free solution. Twenty nanomolar of U46619 induced contraction and elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)), which were consisted of two phases; slowly developing first phase followed by quickly rising second phase. The second phase was inhibited by verapamil, and all the [Ca(2+)](i) response was abolished in Ca(2+)-free solution. The contraction and [Ca(2+)](i) elevation induced by 20 nM U46619 were not inhibited by U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or GF109203X, a protein kinase C inhibitor, but were abolished by D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). However, D609 had no effect on those induced by 1 μM phenylephrine. The U46619-induced responses were also partially inhibited by cation channel blockers, 2-APB and LOE908. The inhibition by LOE908 was abolished in the presence of verapamil, suggesting that LOE908-sensitive cation channels lead to the activation of VDCC by depolarizing plasma membrane. In contrast, 2-APB further diminished the U46619-induced [Ca(2+)](i) elevation in the presence of verapamil. In conclusion, TP receptor stimulation is suggested to be coupled with PC-PLC. Diacylglycerol produced by PC-PLC seems to activate two types of cation channels independently of PKC, which in turn leads to VDCC-dependent and independent Ca(2+) influx, thereby eliciting contraction.     

Allosteric modulation of [(3)H]gabapentin binding by ruthenium red

Gabapentin is an anticonvulsant with an unknown mechanism of action. However, it has been proposed that gabapentin acts by binding to voltage-gated calcium channels. To further characterize the interaction of gabapentin with its endogenous binding site in cerebral cortex, we tested for competitive and allosteric interactions between [(3)H]gabapentin and a variety of calcium channel binding ligands. Most ligands for voltage- or ligand-gated calcium channels (verapamil, the omega-conotoxins MVIIC and GVIA, ryanodine, caffeine, capsaicin, MK-801) had no significant effect on [(3)H]gabapentin binding. However, ruthenium red, a relatively nonselective calcium channel ligand, was found to robustly modulate [(3)H]gabapentin binding. Ruthenium red slowed the association and dissociation kinetics of [(3)H]gabapentin while increasing the number of detectable binding sites. Spermine and MgCl(2), which also bind to calcium channels and modulate [(3)H]gabapentin binding, were found to act in a similar manner. These findings support the contention that the principal endogenous binding site for gabapentin is a calcium channel; they characterize the nature of the allosteric interaction of spermine, MgCl(2) and ruthenium red with this binding site; and they suggest possible mechanisms by which gabapentin may modulate calcium channel function and ultimately produce therapeutic actions.     

A comparative study of histamine and K+ effects on (Ca(2+)-Mg2+)-ATPase activity in synaptosomes.

Histamine (10(-4) M) and 60 mM K+, but not 60 mM Na+ or 60 mM choline+, increased the maximal synaptosomal (Ca(2+)-Mg2+)-ATPase activity by 15 and 36% respectively and decreased the extrasynaptosomal Ca2+ concentration necessary to reach it. Histamine and K+ enhanced the synaptosomal (Ca(2+)-Mg2+)-ATPase activity in a concentration-dependent manner. In synaptic plasma membranes histamine (10(-4) M) and 60 mM choline+ were not able to alter the enzymatic activity, however 60 mM K+ and 60 mM Na+ elevated (Ca(2+)-Mg2+)-ATPase activity by 20 and 15%, respectively, without altering the affinity for Ca2+. Histamine effects in synaptosomes were mediated by H2 receptor stimulation. 3-Isobutyl-1-methyl-xanthine (10(-4) M) potentiated (15%) the maximal histamine effect. The slow Ca2+ channel antagonists verapamil and diltiazem, both at 10(-6) M, completely inhibited K+ effects in synaptosomes, however histamine effects were only blocked by verapamil. The data suggest that K+ and histamine effects on synaptosomal (Ca(2+)-Mg2+)-ATPase activity are mediated by increases of intrasynaptosomal Ca2+ levels. Moreover, histamine effects on synaptosomal enzyme activity were mediated by cAMP.     

Antiproliferative effect of Ca2+ channel blockers on human epidermoid carcinoma A431 cells

The effects of Ca(2+) channel blockers on the proliferation of human epidermoid carcinoma A431 cells were investigated by microtiter tetrazolium (MTT) proliferation assay and bromodeoxyuridine (BrdU) incorporation assay. Dihydropyridine derivatives, such as amlodipine, nicardipine, and nimodipine inhibited A431 cell growth and the incorporation of BrdU into cells with IC(50) values of 20-30 microM, while verapamil, diltiazem and dihydropyridine nifedipine inhibited neither the cell growth nor BrdU incorporation at the same concentration. Though extracellular Ca(2+) is indispensable to the cell growth, an L-type Ca(2+) channel agonist, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl]pyridine-3-carboxylic acid methyl ester (200 nM), did not affect the antiproliferative action of amlodipine. Thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, inhibited itself the growth of A431 cells and also showed a synergistic effect with the antiproliferative action of amlodipine. In the fluorimetric measurement of intracellular free Ca(2+) concentration in fura-2 or fluo-3 loaded A431 cells, amlodipine blunted the thapsigargin- or cyclopiazonic acid-induced Ca(2+) release from endoplasmic reticulum and the ensuing Ca(2+) influx through Ca(2+)-permeable channels. The effect on the thapsigargin-induced Ca(2+) responses could be reproduced by nicardipine and nimodipine but not by nifedipine or verapamil, lacking antiproliferative potency. These findings suggest that the intracellular Ca(2+) control system responsible for thapsigargin- and cyclopiazonic acid-sensitive endoplasmic reticulum, but not L-type Ca(2+) channels, may be modulated by amlodipine, which results in the inhibition of A431 cell growth.     

Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.