Effects Of L-type Ca2 Research Articles

Sodium-Mediated Plateau Potentials in Lumbar Motoneurons of Neonatal Rats

The development and the ionic nature of bistable behavior in lumbar motoneurons were investigated in rats. One week after birth, almost all (∼80%) ankle extensor motoneurons recorded in whole-cell configuration displayed self-sustained spiking in response to a brief depolarization that emerged when the temperature was raised >30°C. The effect of L-type Ca(2+) channel blockers on self-sustained spiking was variable, whereas blockade of the persistent sodium current (I(NaP)) abolished them. When hyperpolarized, bistable motoneurons displayed a characteristic slow afterdepolarization (sADP). The sADPs generated by repeated depolarizing pulses summed to promote a plateau potential. The sADP was tightly associated with the emergence of Ca(2+) spikes. Substitution of extracellular Na(+) or chelation of intracellular Ca(2+) abolished both sADP and the plateau potential without affecting Ca(2+) spikes. These data suggest a key role of a Ca(2+)-activated nonselective cation conductance ((CaN)) in generating the plateau potential. In line with this, the blockade of (CaN) by flufenamate abolished both sADP and plateau potentials. Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a common activator of thermo-sensitive vanilloid transient receptor potential (TRPV) cation channels, promoted the sADP. Among TRPV channels, only the selective activation of TRPV2 channels by probenecid promoted the sADP to generate a plateau potential. To conclude, bistable behaviors are, to a large extent, determined by the interplay between three currents: L-type I(Ca), I(NaP), and a Na(+)-mediated I(CaN) flowing through putative TRPV2 channels.

Constitutively active calcium channels in plasma membrane of T-lymphocytes

Compensated influx and efflux of calcium ions maintain the constancy of Ca2+ concentration in cytoplasm of quiescent cells under variable external conditions. In cell plasma membrane there exist several types of Ca2+ channels with different properties, regulation mechanisms, and pharmacology. Using fluorescent Ca2+-sensitive probes, we have shown here that in T-lymphocytes under resting conditions, Ca2+ influx occurs through special constitutively active Ca2+ channels, permeable to Ni2+ and Mn2+. These channels differ from the receptor-activated SOC channels, from Ca2+ channels activated by arachidonic acid, and from calmidazolium-activated channels. Ca2+ influx rate in quiescent cells increases with a rise in temperature (Q10 =1.9). The strong dependence of the constitutively active channel activity on temperature coincided with the plasma membrane Ca2+-ATPase dependence, indicating that intracellular enzymes regulate the channel activity. To identify the constitutively active channel, we analyzed the effects of L-type Ca2+ channels, SOC channels, Ca2+-independent phospholipase A2, and calmodulin inhibitors. Of all inhibitors listed only dihydropyridine blocker of L-type voltage-dependent Ca2+ channels, isradipin, at a concentration of 1.5 μM completely suppressed calcium influx. However, the channels did not exhibit sensitivity to changes in membrane potential. Our observations testify to the existence of a new nonselective Ca2+ channel in T-lymphocyte plasma membrane and characterize the new channels pharmacologically. The results obtained are important for understanding the regulation mechanisms of Ca2+ channels in plasma membrane of non-excitable cells.

Effects of L-type Ca2+ channel antagonists on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate

The inhibitory effects of L-type Ca2+ channel antagonists on Na cholate-induced in vitro excystment (CIIE) of Paragonimus ohirai metacercariae were studied. At concentrations of 10 microM, nicardipine and nimodipine inhibited CIIE completely and by approximately 92%, respectively. Nitrendipine and (+/-)-verapamil inhibited CIIE by about one half and one third, respectively. Nifedipine and diltiazem did not inhibit CIIE significantly. At higher concentrations, nitrendipine at 20 microM completely inhibited CIIE, and (+/-)-verapamil at 40 microM inhibited CIIE by 93%. Nifedipine and diltiazem inhibited CIIE only slightly and little, respectively, even at 40 microM. Complete inhibition by nicardipine at 10 microM required preincubation of metacercariae with the antagonist for 15 min. The inhibitory effects of nicardipine and nimodipine were reversible, and most of the nimodipine-treated metacercariae could excyst within 1 h after being washed, but the nicardipine-treated ones started to excyst 1 h after washing. Nicardipine suppressed the active movement of encysted juveniles evoked by Na cholate, whereas nimodipine did not suppress this significantly. These results suggested that L-type Ca2+ channels appeared to be involved in CIIE of P. ohirai metacercariae and that the inhibitory effect of the channels was due primarily to factors other than the inhibition of muscular activity, probably involving the secretion and release of enzymes lytic against the metacercarial cyst wall.

Potentiating effects of L-type Ca2+ channel blockers on pentobarbital-induced hypnosis are influenced by serotonergic system

In order to elucidate the mechanism(s) behind the interactions between barbiturates and Ca(2+) antagonists, the effects of three structurally diverse types of Ca(2+) antagonists combined or not with 5-HT on pentobarbital-induced hypnosis in mice were investigated. The results showed that dihydropyridine derivative nifedipine (10.0 and 20.0 mg/kg, p.o.) and other types of Ca(2+) antagonist, verapamil (5.0 and 10.0 mg/kg, p.o.) and diltiazem (2.5, 5.0 and 10.0 mg/kg, p.o.) increased both the sleeping time in hypnotic dosage of pentobarbital (45 mg/kg, i.p.) treated mice and the rate of sleep onset in the sub-hypnotic dosage of pentobarbital (28 mg/kg, i.p.) treated mice in a dose-dependent manner, respectively, and these effects were significantly augmented by 5-hydroxytryptophan (5-HTP), the immediate precursor of 5-hydroxytryptamine (5-HT). Pretreatment with p-chlorophenylalanine (PCPA, 300 mg/kg, s.c.), an inhibitor of tryptophan hydroxylase, significantly decreased pentobarbital-induced sleeping time and nifedipine (10.0 mg/kg, p.o.), verapamil (5.0 mg/kg, p.o.) and diltiazem (2.5 mg/kg, p.o.) abolished this effect. From these results, it should be presumed that the augmentative effect of L-type Ca(2+) channel blockers on pentobarbital-induced sleep may be influenced by serotonergic system.

Effects of L-type Ca2+ channel modulation on direct myocardial effects of diazepam and midazolam in adult rat ventricular myocytes

Our objective was to determine whether an L-type Ca2+ channel modulation could alter myocardial depression induced by midazolam or diazepam in adult rat ventricular myocytes. Freshly isolated rat ventricular myocytes were loaded with fura-2/AM and field-stimulated (0.3 Hz) at 28 degrees C. Amplitude and timing of intracellular Ca2+ concentration ([Ca2+]i) and myocyte shortening were simultaneously monitored in individual cells. Midazolam (3-100 microM) caused a decrease in both peak [Ca2+]i and shortening. Diazepam (30, 100 microM) increased myocyte shortening and peak [Ca2+]i; however, higher concentration of diazepam (300 microM) decreased shortening and peak [Ca2+]i. Bay K 8644 (0.01-10 microM), an L-type Ca2+ channel agonist, caused dose-dependent increases in peak [Ca2+]i and shortening. In contrast, verapamil (0.1-50 microM), an L-type Ca(2+) channel antagonist, caused dose-dependent decreases in peak [Ca2+]i and shortening. Dose-response curves to benzodiazepines on peak [Ca2+]i and shortening were not affected by pretreatment with Bay K 8644 (0.1 microM) or verapamil (1 microM). Diazepam (30, 100 microM), but not midazolam (3-30 microM), increased shortening and [Ca2+]i in the presence or absence of L-type Ca2+ channel modulators. Diazepam (30 microM) and midazolam (10 microM) had no effect on peak [Ca2+]i of a caffeine-induced [Ca2+]i transient, which was used as a measure of SR Ca2+ content. Midazolam and diazepam have differential effects on cardiac E-C coupling. Diazepam, but not midazolam, enhances cardiac E-C coupling independent of L-type Ca2+ channel modulation.

Role of L-type calcium channels in pacing-induced short-term and long-term cardiac memory in canine heart.

We tested the hypothesis that ICa,L is important to the development of cardiac memory. The effects of L-type Ca2+ channel blockade and beta-blockade were tested on acutely anesthetized and on chronically instrumented, conscious dogs. Short-term memory (STM) was induced by 2 hours of ventricular pacing and long-term memory (LTM) by ventricular pacing for 21 days. STM dogs received placebo, nifedipine, or propranolol, and LTM dogs received placebo, atenolol, or amlodipine. AT1 receptor blockade (candesartan) and ACE inhibition (trandolapril) were also tested in LTM. Microelectrodes were used to record transmembrane potentials from isolated epicardial and endocardial slabs using a protocol simulating STM in intact animals. Left ventricular epicardial myocytes from LTM or sham control dogs were dissociated, and ICa,L was recorded (whole-cell patch-clamp technique). Evolution of STM and LTM was attenuated by ICa,L blockers but not beta-blockers. Neither AT1 receptor blockade nor ACE inhibition suppressed LTM. In microelectrode experiments, pacing induced an epicardial-endocardial gradient change mimicking STM that was suppressed by nifedipine. In patch-clamp experiments, peak ICa,L density in LTM and control were equivalent, but activation was more positive and time constants of inactivation longer in LTM (P<0.05). ICa,L blockade but not beta-adrenergic blockade suppresses cardiac memory. LTM evolution is unaffected by angiotensin II blockade and is associated with altered ICa,L kinetics.

Effects of L-type Ca(2+)-channel blockade, K(+)(ATP)-channel opening and nitric oxide on human uterine contractility in relation to gestational age and labour.

Relative uterine inactivity during pregnancy changes to vigorous rhythmic contractility during labour. We hypothesized that mechanisms involved in the regulation of uterine quiescence and contractility differ between term and preterm myometrium and in labour and non-labour states. Myometrial strips, prepared from biopsies taken at Caesarean section from labouring and non-labouring women preterm and at term, were mounted in organ chambers for isometric tension recording. Oxytocin (10(-9) mol/l) was added to maintain stable contractions, and effects of various inhibitors of uterine contractility were studied. The inhibitory effects of L-type Ca(2+)-channel blocker nifedipine and ATP-sensitive K(+)-channel opener pinacidil were greater in myometrium from the non-labour versus the labour group, both preterm and at term. In addition, pinacidil's effect was greater at term compared with preterm in the non-labour group. Mg(2+) and the nitric oxide donor sodium nitroprusside significantly inhibited contractility in all groups without significant differences with regard to labour or gestational age. Decreased inhibition of human uterine contractility by L-type Ca(2+)-channel blockers and K(+)(ATP)-channel openers in preterm and term labour may reflect changes in expression and activity of these channels. Effects of nitric oxide and Mg(2+) are not affected by gestational age or labour.

Overlap in the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

We have previously shown that elimination of buffer Ca2+ markedly reduced maximum 5-HT-induced contractions. We have now investigated the effect of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists on 5-HT- and K+-induced contractions in rat aorta to explore the possibility of a relationship between blockade of L-type Ca2+ channels and 5-HT2 receptor antagonism. Sodium nitroprusside, felodipine, nifedipine, diltiazem, cinnarizine, verapamil, ritanserin, cyproheptadine, ketanserin and mianserin inhibited 5-HT-induced contractions of rat aorta with mean IC50 values (concentration (M) resulting in 50% inhibition) of 2.2 x 10(-11), 6.6 x 10(-11), 1.5 x 10(-9), 1.7 x 10(-9), 3.2 x 10(-7), 5.4 x 10(-7), 9.7 x 10(-10), 1.9 x 10(-8), 5.0 x 10(-7) and 6.4 x 10(-7), respectively. The same compounds antagonized K+-induced rat aortic contractions with the rank order of potency (mean IC50, M): felodipine (7.0 x 10(-11)) > nifedipine (4.8 x 10(-9)) > sodium nitroprusside (4.1 x 10(-8)) > verapamil (5.5 x 10(-8)) > cyproheptadine (6.2 x 10(-8)) > diltiazem (4.1 x 10(-7)) > cinnarizine (1.3 x 10(-6)) > ritanserin (1.8 x 10(-6)) > ketanserin (9.0 x 10(-6)) > mianserin (2.0 x 10(-5)). These data are indicative of a highly significant correlation (r=0.81, P=0.03) between potency against 5-HT-induced contraction and that against contractile response to K+ depolarization, and suggest overlap of the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca2+ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca2+ ([Ca2+]i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca2+]i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D 600) and felodipine on [Ca2+]i during anoxia. A method was developed whereby [Ca2+]i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca2+]i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca2+]i were 118 +/- 2 (n = 98) and 662 +/- 22 (n = 160) nM, respectively. D 600 (1 microM), but not felodipine (10 microM), significantly reduced basal [Ca2+]i in normoxic incubations. During anoxia 1 microM and 100 microM D 600 significantly decreased anoxic [Ca2+]i levels by 22 and 63% respectively. Felodipine at 10 microM was as effective as 1 microM D 600. Removal of extracellular Ca2+ and addition of 0.1 mM La3+ completely abolished anoxia-induced increases in [Ca2+]i. We conclude that anoxia induces increases in [Ca2+]i in rabbit PT cells in primary culture, which results from Ca2+ influx. Since this Ca2+ influx is partially inhibited by low doses of CCBs, L-type Ca2+ channels may be involved.