Calcium wave evoked by activation of endogenous or exogenously expressed receptors in Xenopus oocytes.

The mRNA encoding the cloned substance K receptor was microinjected into Xenopus laevis oocytes. After expression of the mRNA, Ca2+ was imaged in the oocytes with a digital imaging fluorescence microscopy system using the Ca2(+)-sensitive dyes fura-2 and fluo-3. Application of substance K caused a dose-related wave of Ca2+ mobilization to spread from a focus and to elevate the Ca2+ concentration in the oocyte. Activation of endogenous muscarinic or angiotensin II receptors in noninjected oocytes evoked a similar response. The Ca2+ rise in oocytes induced by substance K was due to internal Ca2+ mobilization and was independent of external Ca2+, since it occurred in Ca2(+)-free medium fortified with 2 mM EGTA. The Ca2+ imaging was well correlated with ion current measurements of voltage-clamped oocytes. Imaging, in addition to detecting the spatial spread of Ca2+ across the cell, was at least as sensitive as voltage clamping and much faster when screening oocytes for the expression of receptor mRNAs that stimulate Ca2+ mobilization. While it is known that fertilization of Xenopus eggs causes a spreading wave of Ca2+ mobilization, we found that activation of either native or newly expressed receptors in oocytes causes a similar change in Ca2+ distribution.     

Expression of striatal D1 dopamine receptors coupled to inositol phosphate production and Ca2+ mobilization in Xenopus oocytes.

Expression of central nervous system receptors for dopamine was examined by injection of poly(A)+ RNA (mRNA) from rat striatum into oocytes from Xenopus laevis. Electrophysiological measurements in mRNA-injected oocytes indicated that addition of 100 microM dopamine induced an inward current (40-100 nA) that was consistent with the activation of endogenous Ca2(+)-dependent Cl- channels. This current was also elicited by addition of the selective D1 agonist SKF 38393 but not by the selective D2 agonist quinpirole. Prior addition of the dopaminergic antagonist cis-piflutixol completely abolished dopamine-induced currents but had no effect on currents produced by serotonin. Using 45Ca2+ efflux assays, addition of 100 microM dopamine to injected oocytes stimulated efflux 2- to 3-fold. This increase was mimicked by SKF 38393 and was blocked by the D1-selective antagonist (+)SCH 23390 but not by the D2-selective antagonist domperidone. No increase in 45Ca2+ efflux was seen with 100 microM quinpirole. Size fractionation of striatal mRNA yielded a single peak (2.5-3.0 kilobases) of D1 receptor-mediated 45Ca2+ efflux activity in injected oocytes. In addition, dopamine stimulation of oocytes injected with peak fractions and prelabeled with myo-[3H]inositol caused a 3-fold increase in [3H]inositol 1,4,5-triphosphate [( 3H]InsP3) formation. No effect on [3H]InsP3 production or 45Ca2+ efflux was observed, however, in injected oocytes incubated with 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate. Thus, in addition to D1 receptors that stimulate adenylyl cyclase, rat striatum contains D1 receptors that can couple to InsP3 formation and mobilization of intracellular Ca2+.     

Activation of protein kinase C differentially modulates neuronal Na+, Ca2+, and gamma-aminobutyrate type A channels.

Xenopus oocytes were used to study the interaction of neuronal quisqualate receptors with neuronal ion channels. Total mRNA was isolated from chick forebrain and injected into Xenopus oocytes. This technique led to the expression of functional voltage-gated Na+ and Ca2+ channels, of ligand-gated gamma-aminobutyrate and kainate receptor channels, and of quisqualate receptors that could activate endogenous chloride channels by means of inositol trisphosphate-mediated Ca2+ release. Exposure of the oocytes to quisqualate decreased the amplitude of the Na+ current and of the gamma-aminobutyrate type A-gated current and increased the amplitude of the Ba2+ current through Ca2+ channels. This modulation of neuronal ion channels by quisqualate could be mimicked by the protein kinase C activator phorbol 12-myristate 13-acetate and the diacylglycerol analogue 1,2-oleoylacetylglycerol. The kainate-gated channel was not affected by these agents. Phorbol esters that do not activate protein kinase C, alpha-phorbol 12-myristate 13-acetate and alpha-phorbol, were without effect. The inhibitor of protein kinase C, tamoxifen, prevented the modulatory effects of phorbol 12-myristate 13-acetate. The present evidence suggests that the activity of the neuronal Na+ and Ca2+ channels and the ligand-gated gamma-aminobutyrate type A receptor channel are under the control of protein kinase C and that neurotransmitters that activate protein kinase C could profoundly affect neuronal signaling.     

Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons.

The role of intracellular Ca2+ concentration ([Ca2+]i) in the muscarinic suppression of Ca2+ current and M-type K+ current has been investigated in isolated rat sympathetic neurons using the whole-cell patch-clamp technique and fura-2 fluorescence measurements. Muscarinic stimulation suppressed currents without raising [Ca2+]i. Nonetheless, intracellular bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) (11-12 mM), a Ca2+ chelator, reduced Ca2(+)-current suppression from 82 to 15%. For the latter, we explain the BAPTA action by a requirement for a certain minimum [Ca2+]i for continued operation of the pathway coupling muscarinic receptors to M-type K+ channels. The pathway coupling muscarinic receptors to Ca channels also showed some dependence on [Ca2+]i, but there may also be a blocking action of BAPTA that is independent of Ca2+ chelation.     

Actions of dopamine and dopaminergic drugs on cloned serotonin receptors expressed in Xenopus oocytes.

Using electrophysiological techniques, we studied interactions of dopamine and selected dopaminergic drugs with serotonin (5-hydroxytryptamine; 5-HT) receptors expressed in Xenopus oocytes by RNAs transcribed from cloned cDNAs. Oocytes showing strong expression of 5-HT1c and 5-HT2 receptors became weakly responsive to the neurotransmitter dopamine, which, like 5-HT, elicited Cl- currents through activation of the phosphatidylinositol/Ca2+ messenger pathway. The two types of 5-HT receptors showed similar sensitivity to dopamine; threshold responses were activated at concentrations as low as 1 microM. However, maximum dopamine responses were only 5-20% of maximum responses activated by 5-HT. The dopamine D1 receptor antagonist SCH 23390 was a potent agonist on 5-HT1c and 5-HT2 receptors. SCH 23390 elicited currents at concentrations as low as 1 nM, but maximum responses were again only 5-20% of those activated by 5-HT. Fenoldopam, a dopamine D1 receptor agonist, also interacted with 5-HT1c and 5-HT2 receptors, eliciting threshold responses between 10 and 20 nM. Our experiments raise the possibility that low micromolar concentrations of dopamine can cause weak activation and concomitant desensitization of serotoninergic systems in vivo and demonstrate that benzazepines can interact with 5-HT receptors at nanomolar concentrations.     

Serotonin inhibits voltage-gated K+ currents in pulmonary artery smooth muscle cells: role of 5-HT2A receptors, caveolin-1, and KV1.5 channel internalization

Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) and voltage-gated K+ (KV) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of KV channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on KV channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hKV1.5 channels stably expressed in Ltk- cells. 5-HT reduced native KV and hKV1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on KV currents and contraction were markedly prevented by the 5-HT2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (G?6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent beta-cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and this was increased on stimulation with 5-HT. Moreover, KV1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT2A receptors inhibits native KV and hKV1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. KV channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH.     

Protein kinase C activation enhances the delayed rectifier potassium current in guinea-pig heart cells

The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na(+)-and K(+)-free external solution, the delayed rectifier K+ current (IK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol-13-acetate (TPA). The amplitude of the IK tail elicited by a return from a depolarizing pulse for 3 s at + 50 mV to a holding potential of -30 mV was increased by 32 +/- 4% (mean +/- S.E., (n = 6) after the external application of 1 nM TPA, and by 60 +/- 17% (n = 5) after 10 nM. The increase in IK produced by 1 nM TPA was abolished by the inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 microM). In addition, the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG, 125 microM) also increased IK (58 +/- 9%, n = 3). PKC purified from bovine brain remarkably increased IK (151 +/- 101%, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve of IK for the intracellular concentration of Ca2+ was shifted to the left by 1 nM TPA, suggesting a Ca2(+)-dependent action of PKC and/or altered Ca2(+)-sensitivity of IK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 +/- 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 +/- 5% in outward component, and 3 +/- 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase IK without affecting the other two currents.     

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways

Ghrelin is an endogeneous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is determined by Ca²⁺ influx and release from intracellular Ca²⁺ storage sites. Ca²⁺ influx is via voltage-gated Ca²⁺ channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K⁺ channels. The present study investigates the in vitro effect of ghrelin on membrane voltage-gated K⁺ channels in the GH₃ rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K⁺ currents under voltage-clamp conditions. In the presence of Co²⁺ (1 mM, Ca²⁺ channel blocker) and tetrodotoxin (1 μM, Na⁺ channel blocker) in the bath solution, two types of voltage-gated K⁺ currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K⁺ current (I _A) represented a significant proportion of total K⁺ currents in some cells, whereas delayed rectifier K⁺ current (I _K) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both I _A and I _K currents to 48% and 64% of control, respectively. Application of apamin (1 μM, SK channel blocker) or charybdotoxin (1 μM, BK channel blocker) did not alter the K⁺ current or the response to ghrelin. The ghrelin-induced reduction in K⁺ currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K⁺ current response to ghrelin. These results suggest that ghrelininduced reduction of voltage-gated K⁺ currents in GH₃ cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K⁺ currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.     

Neurotransmitter receptors and voltage-dependent Ca2+ channels encoded by mRNA from the adult corpus callosum.

The presence of mRNAs encoding neurotransmitter receptors and voltage-gated channels in the adult human and bovine corpus callosum was investigated using Xenopus oocytes. Oocytes injected with mRNA extracted from the corpus callosum expressed functional receptors to glutamate, acetylcholine, and serotonin, and also voltage-operated Ca2+ channels, all with similar properties in the two species studied. Acetylcholine and serotonin elicited oscillatory Cl- currents due to activation of the inositol phosphate-Ca2+ receptor-channel coupling system. Glutamate and its analogs N-methyl-D-aspartate (NMDA), kainate, quisqualate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) induced smooth currents. The non-NMDA responses showed a strong inward rectification at positive potentials and were potently blocked by 6,7-dinitroquinoxaline-2,3-dione, as observed for the AMPA/kainate glutamate receptors GLUR1 and GLUR3. Furthermore, in situ hybridization experiments showed that GLUR1 and GLUR3 mRNAs are present in corpus callosum cells that were labeled with antiserum to glial fibrillary acid protein and that, in primary cell cultures, had the morphology of type 2 astrocytes. These results indicate that glial cells in the adult corpus callosum possess mRNA encoding functional neurotransmitter receptors and Ca2+ channels. These molecules may provide a mechanism for glial-neuronal interactions.     

Characterization of muscarinic receptor subtypes inhibiting Ca2+ current and M current in rat sympathetic neurons.

Muscarinic receptors mediating suppression of Ca2+ current and of M-type K+ current in rat superior cervical ganglion neurons were subclassified pharmacologically by using the muscarinic receptor antagonists pirenzepine and himbacine. Our voltage clamp experiments previously distinguished fast and slow intracellular signaling pathways coupling muscarinic receptors to calcium channels. We now establish that the fast, pertussis toxin-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M4 subtype, whereas the slow, bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA)-sensitive suppression of Ca2+ current is mediated primarily by muscarinic receptors of the M1 subtype. Both actions on Ca2+ current are blocked by guanosine 5'-[beta-thio]diphosphate. Muscarinic suppression of M current is slow, BAPTA-sensitive, and mediated by receptors of the M1 subtype. Hence the two muscarinic pathways use different receptors and different guanine nucleotide binding proteins to produce different actions on channels.     

Postnatal maturation of excitation-contraction coupling in rat ventricle in relation to the subcellular localization and surface density of 1,4-dihydropyridine and ryanodine receptors.

To better understand excitation-contraction coupling in cardiac muscle, we investigated the main Ca2+ channels involved in that process in adult and neonatal rat ventricle. Voltage-dependent (L-type) Ca2+ channels and sarcoplasmic reticulum Ca2+ release channels were labeled by means of [3H] (+)-PN200-110 and [3H]ryanodine, respectively. The number of [3H]ryanodine binding sites (per gram tissue) increased more than that of [3H] (+)-PN200-110 binding sites over the postnatal period (2.1-fold versus 1.35-fold, respectively). After equilibration of microsomal fractions in density gradient, ryanodine receptors were characterized by a heavy distribution pattern that did not change appreciably between days 1 and 30 after birth. In neonatal tissue, 1,4-dihydropyridine receptors were found mainly in low-density subfractions, together with other sarcolemmal constituents, whereas in adult tissue, they were recovered predominantly in high-density subfractions, together with ryanodine receptors. Thus, after birth, and in parallel with the development of T tubules, there was a progressive concentration of L-type Ca2+ channels in junctional structures of high equilibrium density, where they were situated close to the Ca2+ release channels of the sarcoplasmic reticulum. In adult ventricle, L-type channels were, on an average, threefold more abundant in T tubules than in external sarcolemma. In parallel mechanical studies, we found that the inhibitory action of ryanodine on systolic contraction was much more pronounced in adult than in neonatal right ventricle, and that, conversely, neonatal tissue was more sensitive that adult tissue to inhibitors of L-type channels. We conclude that, in view of the presumed mechanism of Ca2+ release from the sarcoplasmic reticulum, that is, Ca(2+)-induced Ca2+ release, the predominant localization in adult rat ventricle of the major Ca2+ entry pathway in the vicinity of the Ca2+ release pathway is of great functional significance. Furthermore, owing to the relative stoichiometry of Ca2+ entry and Ca2+ release channels in junctional structures (about 1:9), a physical link between these channels is not likely to be involved in the modulation of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle.     

Expressional potency of mRNAs encoding receptors and voltage-activated channels in the postmortem rat brain.

The stability and integrity of mRNAs encoding neurotransmitter receptors and voltage-activated channels in the postmortem rat brain was investigated by isolating poly(A)+ mRNA, injecting it into Xenopus oocytes, and then examining the expression of functional neurotransmitter receptors and voltage-activated channels in the oocyte membrane by electrophysiological recording. This approach was also used to assess the stability of mRNAs in brains that were incubated in oxygenated mammalian Ringer's solution for various lengths of time and from brains that were freshly frozen and then thawed at room temperature. Oocytes injected with mRNA from up to 21-hr postmortem brains gave large agonist- and voltage-activated responses, indicating that mRNAs encoding neurotransmitter receptors and voltage-activated channels are relatively stable in postmortem brain tissue. In contrast, oocytes injected with mRNA from brains incubated in Ringer's solution exhibited smaller responses, and oocytes injected with mRNA from tissue that was frozen and then thawed displayed very small or undetectable responses. Northern blot analysis using a nucleic acid probe for rat brain Na(+)-channel mRNA indicated that the size of the Na+ currents in injected oocytes reflected the levels of mRNA for Na+ channels in the different mRNA preparations. Thus, the expressional potency of mRNAs encoding neurotransmitter receptors and voltage-activated channels is quite stable in postmortem brains in situ, but it is reduced if the brains are kept in oxygenated saline, and freezing and thawing of tissue results in rapid degeneration of mRNA.     

Involvement of T-type calcium channels in the mechanism of action of 5-HT in rat glomerulosa cells: a novel signaling pathway for the 5-HT7 receptor

We have previously demonstrated that, in rat, the stimulatory effect of 5-HT on aldosterone secretion is mediated through a 5-HT7 receptor subtype. The aim of the present study was to characterize the transduction mechanisms associated with activation of native 5-HT7 receptors. 5-HT induced a dose-dependent increase in cAMP production in rat glomerulosa cells. Pretreatment of cells with the adenylyl cyclase (AC) inhibitor SQ 22536 or the protein kinase A (PKA) inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Administration of 5-HT in the vicinity of glomerulosa cells induced a robust increase in cytosolic calcium concentration ([Ca2+]i) and this effect was abrogated by the T-type calcium channel blocker mibefradil. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. H-89 attenuated both the [Ca2+]i response and the activation of T-type calcium current induced by 5-HT. Reduction of extracellular calcium concentration in the medium or administration of mibefradil caused a marked reduction of the maximum effect (Emax) of 5-HT on aldosterone secretion. These data demonstrate that activation of native 5-HT7 receptors stimulates cAMP formation, which in turn provokes calcium influx through T-type calcium channels. Both the activation of the AC/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.     

Estrogens act in rat hippocampus and frontal cortex to produce rapid, receptor-mediated decreases in serotonin 5-HT(1A) receptor function

Previously our laboratory has shown that 17beta-estradiol in vivo rapidly decreases R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding (a measure of the initial biochemical event in the intracellular signaling pathway associated with 5-HT(1A) receptors) in the hippocampus, frontal cortex and amygdala. Studies were designed to determine if 17beta-estradiol also acts in vitro on estrogen receptors in the hippocampus and frontal cortex to decrease 5-HT(1A) receptor function. Hippocampus and frontal cortex were dissected from ovariectomized rats and incubated for up to 3 h with various estrogens and antiestrogens; membrane homogenates were prepared for R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding assays. 17beta-Estradiol (10(-6) M) decreased the maximal response in the R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding assay in a time-dependent manner (observed at 30, 60 and 120 min) in both hippocampus and frontal cortex. The hormone, however, did not alter the EC(50) of R(+)-8-OH-DPAT. When hippocampus and frontal cortex were incubated in graded concentrations of 17beta-estradiol for 1 h, the calculated EC(50) was approximately 2.5 x 10(-8) M in both brain regions. The nonestradiol estrogen diethylstilbestrol also decreased 5-HT(1A) receptor function while the less potent estrogens 17alpha-estradiol and estriol were inactive at 5 x 10(-8) M. The estrogen receptor antagonist ICI 182,780 potently and completely blocked the effects of 17beta-estradiol on 5-HT(1A) receptor function with an apparent K(B) of approximately 10(-9) M. These data demonstrate clearly that estrogens can act on estrogen receptors located in hippocampus and frontal cortex of ovariectomized rats to produce rapid heterologous decreases in 5-HT(1A) receptor function.     

Repetitive increases in cytosolic Ca2+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels.

Many signal-transduction processes in higher plant cells have been suggested to be triggered by signal-induced opening of Ca2+ channels in the plasma membrane. However, direct evidence for activation of plasma-membrane Ca2+ channels by physiological signals in higher plants has not yet been obtained. In this context, several lines of evidence suggest that Ca2+ flux into the cytosol of guard cells is a major factor in the induction of stomatal closing by abscisic acid (ABA). ABA closes stomatal pores, thereby reducing transpirational loss of water by plants under drought conditions. To directly investigate initial events in ABA-induced signal transduction in guard cells, we devised an experimental approach that allows simultaneous photometric measurements of cytosolic Ca2+ and patch-clamp recordings of ion currents across the plasma membrane of single Vicia faba guard cells. Using this approach, we found that the resting cytosolic Ca2+ concentration was 0.19 +/- 0.09 microM (n = 19). In responsive guard cells, external exposure to ABA produced transient repetitive increases in the cytosolic free Ca2+ concentration. These Ca2+ transients were accompanied by concomitantly occurring increases in an inward-directed ion current. Depolarization of the membrane terminated both repetitive elevations in cytosolic Ca2+ and inward-directed ion currents, suggesting that ABA-mediated Ca2+ transients were produced by passive influx of Ca2+ from the extracellular space through Ca2(+)-permeable channels. Detailed voltage-clamp measurements revealed that ABA-activated ion currents could be reversed by depolarizations more positive than -10 mV. Interestingly, reversal potentials of ABA-induced currents show that these currents are not highly Ca2(+)-selective, thereby permitting permeation of both Ca2+ and K+. These results provide direct evidence for ABA activation of Ca2(+)-permeable ion channels in the plasma membrane of guard cells. ABA-activated ion channels allow repetitive elevations in the cytosolic Ca2+ concentration, which, in turn, can modulate cellular responses promoting stomatal closure.     

Modulation of evoked contractions in rat arteries by ryanodine, thapsigargin, and cyclopiazonic acid.

The contribution of sarcoplasmic reticulum (SR) Ca2+ release to evoked tension in rat arterial rings was studied by comparing the effects of ryanodine (an SR Ca2+ channel opener) and thapsigargin and cyclopiazonic acid (CPA) (two Ca(2+)-ATPase inhibitors). Isometric tension was evoked by serotonin (5-HT), 30-50 mM external K+, and 10 mM caffeine in rings of aorta and a small (second-order) branch of the superior mesenteric artery (SMA). Resting tension was unaffected by 10 microM ryanodine or 1-5 microM thapsigargin, but 20 microM CPA raised resting tension in aortic rings and evoked spontaneous contractions in some SMA rings. Ryanodine (10 microM) or 1-5 microM thapsigargin partially depleted the SR Ca2+ stores (indicated by reduced caffeine-evoked contractions) and attenuated 5-HT- and high K(+)-evoked contractions in aortic rings but augmented 5-HT- and high K(+)-evoked contractions in SMA. Caffeine completely emptied the SR Ca2+ stores in the presence of ryanodine but not thapsigargin in both the aorta and SMA; thus, thapsigargin may selectively affect one component of a heterogeneous SR. When the aortic Ca2+ stores were empty (i.e., caffeine contractions were abolished), the 5-HT- and high K(+)-evoked contractions in the aorta were also augmented. CPA rapidly emptied the SR Ca2+ stores in both the aorta and SMA. CPA augmented the 5-HT-evoked contractions in the SMA and in five of nine aortic rings but attenuated evoked contractions in the remaining aortic rings. The attenuation or abolition of the caffeine contractions implies that ryanodine, thapsigargin, and CPA all deplete the SR Ca2+ stores. The attenuated responses to 5-HT and high K+ observed when the aortic SR Ca2+ stores were only partially depleted are consistent with the idea that evoked SR Ca2+ release is a large component of the Ca2+ transient in the aorta. The augmentation of 5-HT- and high K(+P)-evoked responses after partial (SMA) or complete (aorta) depletion of the SR Ca2+ stores suggests that evoked release of SR Ca2+ normally regulates Ca2+ entry by negative feedback and/or that the SR normally buffers the evoked rise in cytosolic Ca2+.     

Block of Shaker potassium channels by external calcium ions

We describe an interaction between external Ca(2+) ions and Shaker K channels that is important in the gating of the channels. The interaction was first detected as a partial block of inward K(+) current in elevated Ca(2+), beginning near -40 mV and becoming stronger at more negative voltage. Surprisingly, the time course of the block can be resolved as a rapid decay of inward current magnitude following a repolarizing step. The rapid decay of current is shown to be the result of channel block by using a two-pulse procedure that monitors the time course of gate closing. As a result of block, the decay of the tail current after repolarization is two to three times faster than gate closing. With physiological values for voltage and calcium concentration, block is readily detectable from tail time course, implying that it occurs as a normal concomitant of gate closing in Shaker. The slight voltage dependence of block from -60 to -100 mV suggests that Ca(2+) is bound (with low affinity) near the outer mouth of the channel. Elevated calcium quickens the inward gating current recorded as Shaker channels are closing; this current approximately doubles in amplitude and has a faster time course and quicker rising phase. When combined, the results suggest that calcium accelerates the first step in closing of the channel gate, perhaps by changing the channel's ion-occupancy state.     

Reduction of Ca(2+) channel activity by hypoxia in human and porcine coronary myocytes.

OBJECTIVE: Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS: Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS: Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION: Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.