Genetic risk of neuropsychological impairment in schizophrenia: a study of monozygotic twins discordant and concordant for the disorder

We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30-40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 +/- 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

Single L-type calcium channels in smooth muscle cells from resistance arteries of spontaneously hypertensive rats

The amplitude of the whole-cell L-type Ca2+ channel current recorded from vascular smooth muscle cells is reportedly greater in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). However, no study has examined properties of single Ca2+ channels in arterial cells from these strains. To further test the hypothesis that activation of L-type Ca2+ channels in arterial smooth muscle cells would be enhanced in SHR, we recorded single Ca2+ channel currents in resistance mesenteric artery cells from SHR and WKY (8 to 9 weeks of age) using a cell-attached patch clamp technique. With 50 mmol/L Ba2+ in the recording pipette, the depolarizing pulse from a holding potential of -40 mV evoked the single L-type Ca2+ channel current. Opening of the single channels was more frequent in cells from SHR than from WKY. Single-channel conductance (20 pS) and open time (1 ms at 0 mV) did not differ in the two strains. The results suggest that an increased amplitude of the whole-cell current can be attributed to the enhanced opening of single Ca2+ channels in the arterial smooth muscle cells from SHR compared with WKY.     

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.     

Vasopressin stimulates Ca2+ spiking activity in A7r5 vascular smooth muscle cells via activation of phospholipase A2

[Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd approximately 2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is approximately 5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 approximately 0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2-loaded A7r5 cells. Frequency of CA2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(beta-mercapto-beta,beta,-pentamethylenepropionic acid),2-0-methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca(2+)-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 mumol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 mumol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca(2+)-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.     

Flanking proline residues identify the L-type Ca2+ channel binding site of calciseptine and FS2

Calciseptine and FS2 are 60-amino acid polypeptides, isolated from venom of the black mamba (Dendroaspis polylepis polylepis), that block voltage-dependent L-type Ca2+ channels. We predicted that these polypeptides contain an identical functional site between residues 43 and 46 by searching for proline residues that mark the flanks of protein-protein interaction sites [Kini, R. M., and Evans, H. J. (1966) FEBS Lett. 385, 81-86]. The predicted Ca2+ channel binding site also occurs in closely related toxins, C10S2C2 and S4C8. Therefore, it is likely that these toxins also will block L-type Ca2+ channels. To test the proposed binding site on calciseptine and FS2, an eight-residue peptide, named L-calchin (L-type calcium channel inhibitor), was synthesized and examined for biological activity. As expected for an L-type Ca2+ channel blocker, L-calchin reduced peak systolic and developed pressure in isolated rat heart Langendorff preparations without affecting diastolic pressure or heart rate. Furthermore, L-calchin caused a voltage-independent block of L-type Ca2+ channel currents in whole-cell patch-clamped rabbit ventricular myocytes. Thus the synthetic peptide exhibits the L-type Ca2+ channel blocking properties of the parent molecules, calciseptine and FS2, but with a lower potency. These results strongly support the identification of a site in calciseptine and FS2 that is important for binding to L-type Ca2+ channels and reinforce the importance of proline brackets flanking protein-protein interaction sites.     

Activin A and all-trans-retinoic acid cooperatively enhanced the functional activity of L-type Ca2+ channels in the neuroblastoma C1300 cell line

Activin, a member of the transforming growth factor-beta superfamily, regulates various physiological functions. In the present study, we investigated the effect of activin on neuronal differentiation, particularly the functional activity of voltage-dependent Ca2+ channels, in murine neuroblastoma C1300 cells. A slight K(+)-induced increase in the intracellular free Ca2+ ([Ca2+]i) was observed in C1300 cells untreated and treated with either activin A or all-trans-retinoic acid, while treatment with both agents significantly enhanced the increase. The [Ca2+]i increases potentiated by activin A and all-trans-retinoic acid were nearly abolished in the presence of 1.0 mM nickel or in the absence of extracellular Ca2+. Nifedipine (0.1 microM) and omega-conotoxin (1.0 microM), inhibitors of L- and N-type Ca2+ channels, respectively, partially inhibited these responses, however the inhibitory effects of these compounds were not additive. In addition, Bay K 8644, an activator of L-type Ca2+ channels, enhanced the K(+)-induced [Ca2+]i increase. These findings indicated that depolarization evoked the Ca2+ influx, at least in part, through L-type Ca2+ channels in C1300 cells treated with both activin A and all-trans-retinoic acid.     

Activation of calcium sparks by angiotensin II in vascular myocytes

Contraction in smooth muscle is triggered by an increase in cytoplasmic free calcium ([Ca2+]i) which depends on both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from the sarcoplasmic reticulum (SR). Two mechanisms have been shown to be involved in SR Ca2+ release, one is stimulated by Ca2+ and involved ryanodine-sensitive Ca2+-release channels; the other is stimulated by an increase in inositol 1,4,5-trisphosphate (InsP3) generation induced by various mediators and involved InsP3-sensitive Ca2+ release channels. Here, we examined the effects of angiotensin II on [Ca2+]i in single rat portal vein myocytes using both the whole cell patch-clamp method and a laser scanning confocal microscope. Elementary Ca2+ release events (Ca2+ sparks) were obtained spontaneously or in response to L-type Ca2+ channel current activation, and resulted from activation of ryanodine-sensitive Ca2+-release channels in the SR. We show that angiotensin AT1 receptors stimulate Ca2+ sparks through activation of L-type Ca2+ channels without involving InsP3-induced Ca2+ release. This novel transduction pathway may be a common mechanism for vasoconstrictors which do not stimulate generation of chemical second messengers.     

Ethanol acutely decreases calcium transients in cultured human myotubes

Ethanol consumption frequently leads to a number of skeletal muscle disorders, including acute and chronic alcoholic myopathy. Ethanol has been found to interfere with signal transduction mechanisms in cardiac and smooth muscle cells. We studied the effects of ethanol on the intracellular calcium ([Ca2+]i) transients responsible for excitation-contraction coupling in human myotubes from chronic alcoholic patients and healthy controls. Cultured myotubes were loaded with the fluorescent Ca2+ indicator fura-2 and evaluated on a single-cell basis. Following electrical stimulation, ethanol caused a significant reversible dose-dependent reduction in [Ca2+]i transient amplitude, achieving a mean decrease of 36+/-5% at 300 mM ethanol (p < 0.01), without modifying the basal [Ca2+]i. This acute effect of ethanol was similar in myotubes obtained from chronic alcoholics and controls. Similarly, ethanol caused a dose-dependent reduction of [Ca2+]i transient amplitude in control samples when depolarization was elicited by 100 mM KCl (p < 0.01). Several potential mechanisms of ethanol action were studied in control muscle samples. Sarcolemmal Ca2+ entry was measured indirectly by monitoring Mn2+-quenching of intracellular fura-2 via the nitrendipine-sensitive Ca2+ channels during electrical pacing. Ethanol at doses of 100 mM and greater caused a dose-dependent reduction in the rate of quench (p < 0.01). In addition, the intracellular pool of Ca2+ releasable by caffeine was found to be reduced at 300 mM ethanol (p < 0.05). We conclude that ethanol reduces the [Ca2+]i transients underlying excitation-contraction coupling in human myotubes, and that this occurs to a similar extent in cells obtained from chronic alcoholics and controls. This acute effect of ethanol was primarily due to an inhibitory effect of ethanol on sarcolemmal Ca2+ influx via voltage-operated Ca2+ channels, although there may also be an effect on the Ca2+ sarcoplasmic reticulum loading state.     

beta-Fructosidases: a new superfamily of glycosyl-hydrolases

Dihydropyridines (DHPs) block L-type Ca2+ channels more potently at depolarized membrane potentials, consistent with high affinity binding to the inactivated state. Nisoldipine (a DHP antagonist) blocks the smooth muscle channel more potently than the cardiac one, a phenomenon observed not only in native channels but also in expressed channels. We examined whether this tissue specificity was attributable to differences of inactivation in the two channel types. We expressed cardiac or smooth muscle alpha1C subunits in combination with beta2a and alpha2/delta subunits in human embryonic kidney cells, and used 2 mM Ca2+ as the permeant ion. This system thus reproduces the in vivo topology and charge carrier of the channels while facilitating comparison of the two alpha1C splice variants. Both voltage-dependent and isoform-specific sensitivity of 10 nM nisoldipine inhibition of the channel were demonstrated, with the use of -100 mV as the holding potential for fully reprimed channels and -65 mV to populate the inactivated state. Under drug-free conditions, we characterized fast inactivation (1-sec prepulses) and slow inactivation (3 min prepulses) in the two isoforms. Inactivation parameters were not statistically different in the two channel isoforms; if anything, cardiac channels tended to inactivate more than the smooth muscle channels at relevant voltages. Likewise, the voltage-dependent activation was identical in the two isoforms. We thus conclude that the more potent nisoldipine inhibition of smooth muscle versus cardiac L-type Ca2+ channels is not attributable to differences in channel inactivation or activation. Intrinsic, gating-independent DHP receptor binding affinity differences must be invoked to explain the isoform-specific sensitivity of the DHP block.     

Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca2+ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca2+-dependent rises of intracellular Ca2+ concentration ([Ca2+]i) evoked by ATP. These [Ca2+]i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+. Expression of the TRP5 cDNA, however, did not significantly affect [Ca2+]i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-dependent [Ca2+]i transients, whereas ATP suppressed [Ca2+]i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+. Omission of Ca2+ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were PCa:PNa:PCs = 14.3:1. 5:1. Our findings suggest that TRP5 directs the formation of a Ca2+-selective ion channel activated by receptor stimulation through a pathway that involves Ca2+ but not depletion of Ca2+ store in mammalian cells.     

Cardiovascular T-type calcium channels: physiological and pharmacological significance

CELLULAR CALCIUM REGULATION: A variety of Ca2+ control processes are responsible for Ca2+ homeostasis and signaling. Voltage-gated Ca2+ channels are dominant in the cardiovascular system. VOLTAGE-GATED Ca2+ CHANNELS: There are several distinct subclasses of Ca2+ channels, distinguished by location, biophysical, structural and pharmacological characteristics. They include both high- and low-voltage-activated channels. The long-lasting (L) type of high-voltage-activated channel is well characterized and is the site of action for the existing clinically available Ca2+ channel antagonists: nifedipine, verapamil and diltiazem. T-TYPE Ca2+ CHANNELS: The low-voltage-activated transient (T-type) channel is widespread in the cardiovascular system and in neurons. It serves pacemaking functions and supports Ca2+ signaling in secretory cells and vascular smooth muscle. The T-type channel also functions in cell growth processes under physiological and pathological conditions. MIBEFRADIL AS A T-TYPE Ca2+ CHANNEL ANTAGONIST: Mibefradil (Ro 40-5967) is a structurally novel Ca2+ antagonist with selectivity for T-type over L-type channels. This selectivity may underlie its vasodilating activity and heart rate depressive effect, its lack of negative inotropy and its cardioprotective properties.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

Effect of membrane hyperpolarization induced by a K+ channel opener on histamine-induced Ca2+ mobilization in rabbit arterial smooth muscle

1. The role of membrane hyperpolarization on agonist-induced contraction was investigated in intact and alpha-toxin-skinned smooth muscles of rabbit mesenteric artery by use of the ATP-sensitive K+ channel opener, (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2- dimethyl-2H-1-benzopyran-3-ol (Y-26763), and either histamine (Hist) or noradrenaline (NA). 2. Hist (3 microM) and NA (10 microM) both produced a phasic, followed by a tonic increase in intracellular Ca2+ concentration ([Ca2+]i) and force. Y-26763 (10 microM) potently inhibited the NA-induced phasic and tonic increase in [Ca2+]i and force. In contrast, Y-26763 attenuated the Hist-induced phasic increase in [Ca2+]i and force but had almost no effect on the tonic response. However, ryanodine-treatment of muscles in order to inhibit the function of intracellular Ca2+ storage sites altered the action of Y-26763 which now attenuated the Hist-induced tonic increase in [Ca2+]i and force in a concentration-dependent manner (at concentrations > 1 microM). Glibenclamide (10 microM) attenuated the inhibitory action of Y-26763. 3. Hist (3 microM) depolarized the smooth muscle cells to the same extent as NA (10 microM). In the absence of either agonist, Y-26763 (over 30 nM) hyperpolarized the membrane and glibenclamide inhibited this hyperpolarization. Y-26763 (10 microM) almost abolished the NA-induced membrane depolarization, but only slightly attenuated the Hist-induced membrane depolarization in which the delta (delta) value (the difference before and after application of Hist) was not modified by any concentration of Y-26763. In ryanodine-treated smooth muscle cells, Y-26763 hyperpolarized the membrane and potently inhibited the membrane depolarization induced by Hist. 4. In ryanodine-treated muscle, Y-26763 had no measurable effect on the Hist-induced [Ca2+]i-force relationship. Y-26763 also had no apparent effect on the myofilament Ca(2+)-sensitivity in the presence of Hist in alpha-toxin-skinned smooth muscles. 5. It is concluded that the membrane hyperpolarization induced by Y-26763 may not be enough to inhibit the Hist-activated Ca2+ influx. It is also suggested that Hist prevents the membrane hyperpolarization induced by Y-26763, activating an unknown mechanism which is thought to depend on the function of intracellular Ca2+ storage sites.     

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle

To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca2+] ([Ca2+]i) and phasic force showed that Wortmannin (1-4 microM) abolishes force with little or no change in [Ca2+]i and electrical activity. High-K+-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent - at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40-50%. It also significantly increased the delay between the Ca2+ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca2+ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca2+ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered.