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PubMed1992

Calcium channels and nifedipine inhibition of serotonin-induced [3H]thymidine incorporation in cultured cerebral smooth muscle cells.

Cultures of smooth muscle cells were prepared from the basilar artery of adult guinea pigs. Passaged cultures (10-30 passages) that expressed serotonin receptors were studied using [3H]thymidine incorporation. When tested in quiescent medium, serotonin potently stimulated [3H]thymidine incorporation (EC50 of 31 nM) by as much as 400% at 24 h. The number of cells was not significantly increased at 24 or 48 h. At concentrations of 10(-8)-10(-5) M 5-HT, [3H]thymidine uptake was reduced 40-50% by the dihydropyridine Ca2+ channel blocker, nifedipine (1 microM). To demonstrate a possible mechanism for the sensitivity to nifedipine, Ca2+ currents were measured using the whole cell patch clamp technique. The cells expressed dihydropyridine-sensitive L-type Ca2+ channels, but not other subtypes of Ca2+ channels, as indicated by the kinetic and voltage-dependent characteristics of the current and by the stimulatory effect of Bay K 8644. The magnitude of the Ca2+ currents was related exponentially to the membrane surface area, measured as cell capacitance. These data support the association of dihydropyridine-sensitive Ca2+ channels with mitogenesis in vascular smooth muscle, and suggest an alternate mechanism of action for the beneficial effect of dihydropyridines in prophylaxis of cerebral vasospasm.

Animals↗
PubMed1992

Contribution of Ca(2+)-induced Ca2+ release to the [Ca2+]i transients in myocytes from guinea-pig urinary bladder.

1. Smooth muscle cells from guinea-pig urinary bladder were studied at an extracellular Ca2+ concentration ([Ca2+]o) of 3.6 mM and 36 degrees C. Fluorescence of Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transients) induced by step membrane depolarizations. 2. During a 6 s depolarization step from -60 to 0 mV [Ca2+]i increased from a resting 118 +/- 22 nM to 1150 +/- 336 nM and decayed to a sustained level of 295 +/- 62 nM. The experiments were designed to evaluate the contribution of the release of intracellularly stored Ca2+ to components of the depolarization-induced [Ca2+]i transient, i.e. 'phasic', which decayed during a maintained depolarization step, and 'tonic' which constituted the sustained elevation of [Ca2+]i above resting level. 3. A short (1 s) application of 10 mM caffeine mimicked the phasic component. After wash-out of caffeine, the subsequent depolarization induced a [Ca2+]i transient with reduced peak, the degree of suppression depending on the interval between wash-out of caffeine and depolarization. The phasic component of the depolarization and the caffeine-induced [Ca2+]i transients were not additive but saturative. 4. The phasic component was largely abolished in the continuous presence of 10 mM caffeine. It was also abolished by a 10 min cell dialysis of 10 microM ryanodine from the pipette solution and was strongly reduced by dialysis of 5 microM thapsigargin. Changes of the tonic component of the depolarization-induced [Ca2+]i transient were much less pronounced with all three interventions. 5. The tonic component of the depolarization-induced [Ca2+]i transient was increased when [Ca2+]o was elevated briefly before a depolarization close to 0 mV, whereas the phasic component was not significantly changed. Similarly, brief application of 1 microM Bay K 8644 increased the tonic component several-fold without modifying significantly the phasic component. 6. It is concluded that depolarization-induced influx of Ca2+ through L-type Ca2+ channels induces the release of Ca2+ from intracellular caffeine-sensitive stores which constitutes the major part of the phasic component. Ca2+ release superimposes on the effects of Ca2+ influx through L-type Ca2+ channels, the non-inactivating part of which constitutes the tonic component of the [Ca2+]i transient. Since the two processes interact, a dissection by simple subtraction is not possible.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

The long-term inhibitory effect of a Ca2+ channel blocker, nisoldipine, on cytosolic Ca2+ and contraction in vascular smooth muscle.

The mechanism of the long-term inhibitory effect of a dihydropyridine Ca2+ channel blocker, nisoldipine, on contraction and cytosolic Ca2+ level ([Ca2+]i) was examined in isolated rat aorta. Nisoldipine inhibited the [Ca2+]i and muscle tension induced by high K+. The inhibitory effects were antagonized by a Ca2+ channel activator, 100 nM Bay k8644, and by a high concentration of Ca2+ (6.5 mM). Ultraviolet light, which has been shown to decompose dihydropyridines, attenuated the effects of nisoldipine. After nisoldipine had been removed from muscle bath, the inhibitory effect faded away slowly. The residual inhibitory effects on [Ca2+]i and muscle tension were antagonized by Bay k8644, high Ca2+ and ultraviolet light. These results suggest that the inhibitory effect of nisoldipine is caused by a decrease in [Ca2+]i as a result of inhibition of L-type Ca2+ channels, and that the residual inhibitory effects are caused by the same mechanism as the inhibitory effects of nisoldipine, namely the tight binding of nisoldipine to Ca2+ channels even after washout.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Synergism between cAMP and ATP in signal transduction in cardiac myocytes.

ATP transiently increases the intracellular Ca2+ concentration in cardiac myocyte suspensions. Pretreatment with norepinephrine (NE) greatly potentiates the ATP response. We performed experiments on adult rat myocyte suspensions loaded with fura-2 to investigate the mechanism of NE potentiation. We found that forskolin (an activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase), and permeative adenosine 3',5'-cyclic monophosphate (cAMP) analogues potentiate the increase in cytosolic Ca2+ concentration induced by ATP. NE, forskolin, and 8-(4-chlorophenylthio)-cAMP all increase Vmax of the Ca2+ response curve of ATP. Measurement of cAMP by radioimmunoassay confirmed that the changes in the ATP response were accompanied by an increase in cAMP. These results suggest that the noradrenergic potentiation of the ATP-induced Ca2+ mobilization involves cAMP as a second messenger. Patch-clamp studies of isolated myocytes showed that neither NE nor forskolin alters the inward current elicited by ATP, but rather they increase the duration of secondary slow action potentials elicited by ATP. NE also increases the Ca2+ current through L-type Ca2+ channels in the myocytes. We conclude that NE potentiates the ATP-induced Ca2+ transient by increasing cAMP levels and that one of the early events is the increase of the inward Ca2+ current during the action potential.

Adenosine Triphosphate↗
PubMed1992

Voltage-dependent ionic currents in solitary horizontal cells isolated from cat retina.

1. Horizontal cells of the cat retina were isolated by enzymatic dissociation. Two types of horizontal cells were identified: the axonless (A-type) horizontal cell having four to six thick, long (approximately 100 microns) dendrites, and the short-axon (B-type) horizontal cell having many (> 5) fine, short (approximately 30 microns) dendrites. 2. Membrane properties of isolated horizontal cells were analyzed under current-clamp and voltage-clamp conditions. In the A-type cell, the average resting potential was -55 mV and the mean membrane capacitance was 110 pF, whereas values in the B-type cell were -58 mV and 40 pF, respectively. The A-type cell showed long-lasting Ca spikes, but B-type cells had no Ca spikes. 3. Five types of voltage-dependent ionic currents were recorded: a sodium current (INa), a calcium current (ICa), and three types of potassium currents. Potassium currents consisted of potassium current through the inward rectifier (Ianomal), transient outward potassium current (IA), and potassium current through the delayed rectifier (IK(v)). INa was recorded only from A-type cells. Other currents were recorded from both types of cells. 4. INa activated when cells were depolarized from a holding potential (Vh) of -95 mV, and it was maximal at -25 mV. This current was blocked by tetrodotoxin. Approximately half of the A-type cells had INa, but no B-type cell had this current. 5. L-type ICa, an inward-going sustained current, was activated with depolarization more positive than -25 mV. Current amplitude reached a maximal value near 15 mV and became smaller with further depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2026

Utero-placental calcium and magnesium ion channels: A systematic review of obstetric implications of their alterations.

Despite the established roles of calcium (Ca2+) and magnesium (Mg2+) in placental function and uterine contractility, limited information exists on how dysregulation of major ion channels contributes to poor pregnancy outcomes. We synthesized data on the consequences of Ca2+ and Mg2+ channelopathies in uterine and placental functions. Using PubMed, Wiley Online, AJOL, and Web of Science databases for article search, a systematic review of forty-nine papers published between 2000 and March 2026 was carried out and reported in accordance with the PRISMA 2020 guideline. Based on the PICO framework, eligible studies involving human, animal, and in vitro designs were chosen and subjected to narrative analysis. L-type and T-type voltage-gated Ca2+ channels, together with transient receptor potential channels, emerged as principal mediators of placental Ca2+ transport and myometrial contractility. Mechanosensitive Piezo1 channels mediate stretch-activated Ca2+ influx, while store-operated Ca2+ entry pathways involving STIM1-Orai1 sustain intracellular Ca2+ homeostasis. Potassium-Ca2+ coupling channels modulated membrane hyperpolarization and anti-labor effects, and intracellular regulators such as PMCA and RYR1 fine-tuned Ca2+ homeostasis. The Mg2+ transporters are essential for preserving Mg2+ homeostasis and regulating Ca2+-dependent excitability. Dysregulation of these ion channel systems was consistently linked to abnormal uterine contractility, preterm birth, preeclampsia, fetal growth restriction, and adverse pregnancy outcomes. Both Ca2+ and Mg2+ ion channelopathies represent both a potential therapeutic target and a mechanistic factor underlying key obstetric complications.

Female↗
PubMed1992

Caffeine-induced release and reuptake of Ca2+ by Ca2+ stores in myocytes from guinea-pig urinary bladder.

1. Voltage-clamped isolated smooth muscle cells from guinea-pig urinary bladder were studied with 3.6 mM extracellular Ca2+ at 36 degrees C. The fluorescence of the Ca(2+)-sensitive dye Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transient). Fast application of caffeine (10 mM) to the cell was used to release the intracellular Ca2+ from a 'caffeine-sensitive Ca2+ store'. 2. At the holding potential -60 mV, a short (1 s) caffeine application increased [Ca2+]i within less than 1 s from the resting 118 +/- 22 nM to 1490 +/- 332 nM. Following the caffeine wash-out, [Ca2+]i fell from this peak to a subresting level of 47 +/- 12 nM, i.e. an 'undershoot' of [Ca2+]i occurred. Subsequent caffeine-induced [Ca2+]i transients had attenuated peaks suggesting that the caffeine-sensitive Ca2+ store had lost a part of the releasable Ca2+. 3. In the continuous presence of caffeine, [Ca2+]i decayed from its peak to control resting [Ca2+]i values. The wash-out of caffeine following prolonged (10-30 s) treatment also resulted in [Ca2+]i undershoot. Subsequent caffeine-induced [Ca2+]i transients were largely abolished as if the caffeine-sensitive Ca2+ store had lost a large part of releasable Ca2+. During the undershoot, hyperpolarization to -100 mV did not affect [Ca2+]i. In most cells studied, recovery of [Ca2+]i from the undershoot to the resting level required depolarizations inducing Ca2+ influx through L-type Ca2+ channels. 4. Block of plasmalemmal Ca(2+)-ATPase (PMCa) with extracellular La3+ (3 mM) did not modify the decay of the [Ca2+]i transients induced by depolarization or by a 1 s caffeine application suggesting that decay rate of both is not limited by PMCa rate. La3+ abolished the undershoot of [Ca2+]i. In the continuous presence of caffeine, La3+ largely prevented the decay of [Ca2+]i. 5. When the depolarizing steps from -60 to 0 mV (160 ms duration) were applied during the period of [Ca2+]i undershoot, the half-time of decay of the corresponding [Ca2+]i transients was up to three times faster than in control. Repetitive depolarizations restored the rate of decay and [Ca2+]i recovered to the resting value. Both processes recovered along a similar time course. 6. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 0.1 mM) or of 8-Br-cAMP (0.1 mM) did not mimic the above caffeine effects suggesting that stimulation of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCa) by cAMP-dependent phosphorylation is not the underlying mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1992

Alpha-adrenergic inhibition of thyrotropin-releasing hormone-induced prolactin secretion in GH4C1 cells is associated with a depressed rise in intracellular Ca2+.

alpha-Adrenergic receptors are present on the plasma membrane of normal anterior pituitary cells and alpha-adrenergic agonists may play a role in the secretion of corticotropin (ACTH) and thyrotropin (TSH). However, alpha-adrenergic involvement in prolactin (PRL) secretion is uncertain. We have therefore examined this question in the PRL-secreting clonal rat pituitary tumor-derived GH4C1 cells. Norepinephrine (NE), an alpha-adrenergic agonist, had no effect on basal PRL secretion but abolished thyrotropin-releasing hormone (TRH)-induced PRL secretion in a dose-dependent manner (EC50 100 nM). NE also significantly suppressed the TRH-stimulated rise in [Ca2+]i. Phentolamine (PA), a non-selective alpha-adrenergic antagonist, reversed the inhibitory effect of NE on both the TRH-stimulated PRL secretion and [Ca2+]i rise. NE did not inhibit the rise in PRL secretion or [Ca2+]i induced by depolarizing 30 mM K+, 30% hyposmolarity or BAY K-8644, a specific L-type Ca2+ channel agonist. The inhibitory effect of NE on TRH-induced PRL and [Ca2+]i changes was also present when Ca2+ influx was prevented by removing medium Ca2+ or by blocking L-type Ca2+ channels with 2 microM nifedipine. The TRH-stimulated first-phase rise in [Ca2+]i in GH4C1 cells is believed to result primarily from release of sequestered Ca2+ from an intracellular pool through the activation of inositol 1,4,5-trisphosphate (IP3) and this [Ca2+]i spike stimulates PRL secretion. Our data thus suggest that GH4C1 cells have alpha-adrenergic receptors and that alpha-adrenergic agonists either suppress IP3 generation or block IP3 release of sequestered intracellular Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms.

Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.

Animals↗
PubMed1992

Calcium channel currents in Xenopus oocytes injected with rat skeletal muscle RNA.

1. Ba2+ currents (IBa) through voltage-dependent Ca2+ channels were studied in Xenopus laevis oocytes injected with heterologous RNA extracted from skeletal muscle (SkM) of young rats, using the two-electrode voltage clamp technique. 2. With 40 or 50 mM-extracellular Ba2+, native oocytes of most frogs displayed IBa between -5 and -20 nA at 0 mV. However, in 'variant' native oocytes of four frogs, IBa exceeded -30 nA and reached up to -100 nA. In oocytes injected with SkM RNA, IBa of up to -250 nA was observed. 3. In SkM RNA-injected oocytes and 'variant' native oocytes, the decay of IBa displayed two kinetic components. The faster component was selectively blocked by 40-100 microM-Ni2+ and thus was termed the Ni(2+)-sensitive IBa. The slower component was Ni2+ resistant, being inhibited only 10-20% by 100-200 microM-Ni2+. The half-activation and the half-inactivation voltages of the Ni(2+)-sensitive IBa were more negative (by 14.5 and 28.7 mV, respectively) than those of the Ni(2+)-resistant IBa. 4. Neither Ni(2+)-sensitive nor Ni(2+)-resistant IBa in native or SkM RNA-injected oocytes were affected by dihydropyridine antagonists nifedipine and (+) PN 200-110 (1-10 microM), by the dihydropyridine agonist (-)Bay K 8644 (0.01-2 microM), or by verapamil below 50 microM. IBa was blocked by diltiazem (half-block at about 500 microM). Thus, the pharmacology of IBa in SkM RNA-injected and in native oocytes was not characteristic of the L-type Ca2+ channel abundant in the skeletal muscle. 5. Destruction of the RNA coding for the channel-forming alpha 1-subunit of the SkM L-type Ca2+ channel using a hybrid arrest method failed to selectively suppress the appearance of either Ni(2+)-sensitive or Ni(2+)-resistant IBa in SkM RNA-injected oocytes. 6. Our results suggest that the appearance of large voltage-dependent Ba2+ currents in SkM RNA-injected oocytes is not due to the expression of the alpha 1-subunit of the SkM L-type Ca2+ channel. The possibility that the expression of a channel-forming subunit of another Ca2+ channel type underlies one of these currents cannot be rejected. However, since the Ba2+ currents in SkM RNA-injected oocytes resemble those observed in native oocytes, we suggest that their appearance may be the result of an enhanced activity of the native Ca2+ channels, possibly due to the expression of the 'auxiliary' subunits of the SkM Ca2+ channel that form complexes with a native alpha 1-subunit.

Animals↗
PubMed1992

A contraction-related component of slow inward current in dog ventricular muscle and its relation to Na(+)-Ca2+ exchange.

1. The slow inward current component related to contraction (Isic) was studied in voltage clamp experiments on canine ventricular trabeculae at 30 degrees C with the aims of (a) estimating its relation to electrogenic Na(+)-Ca2+ exchange and (b) comparing it with similar currents as reported in cardiac myocytes. 2. Isic may be recorded under conditions of augmented contractility in response to depolarizing pulses below the threshold of the classic slow inward current (presumably mediated by L-type Ca2+ channels). In responses to identical depolarizing clamp pulses the peak value of Isic is directly related to the amplitude of contraction (Fmax). Isic peaks about 60 ms after the onset of depolarization and declines with a half-time of about 110 ms. 3. The voltage threshold of Isic activation is the same as the threshold of contraction. The positive inotropic clamp preconditions shift both thresholds to more negative values of membrane voltage, i.e. below the threshold of the classic slow inward current. 4. Isic may also be recorded as a slowly decaying inwardly directed current 'tail' after depolarizing pulses. In this representation the peak value of Isic changes with duration of the depolarizing pulses, again in parallel with Fmax. In response to pulses shorter than 100 ms both variables increase with depolarization time. If initial conditions remain constant, further prolongation of the pulse does not significantly influence either one (tail currents follow a common envelope). 5. Isic differs from classic slow inward current by: (a) its direct relation to contraction, (b) the slower decay of the current tail on repolarization, (c) slower restitution corresponding to the mechanical restitution, (d) its relative insensitivity to Ca(2+)-blocking agents (the decrease of Isic is secondary to the negative inotropic of Ca(2+)-blocking agents (the decrease of Isic is secondary to the negative inotropic effect) and (e) its disappearance after Sr2+ substitution for Ca2+. 6. The manifestations of Isic in multicellular preparations do not differ significantly from those reported in isolated myocytes (in contrast to calcium current). 7. The analysis of the correlation between Isic and Fmax transients during trains of identical test depolarizing pulses at variable extra- and intracellular ionic concentrations (changes of [Ca2+]o, 50% Li+ substitution for Na+, strophanthidin) indicate that the observed effects conform to the predictions based on a quantitative model of Na(+)-Ca2+ exchange. 8. It is concluded that Isic is activated by a transient increase of [Ca2+]i, in consequence of the release from the reticular stores.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Effects of atrionatriuretic factor on Ca2+ current and Cai-independent transient outward K+ current in human atrial cells.

The effect of 10 nM atrial natriuretic peptide (ANF) on macroscopic L-type calcium current, ICa, and calcium-independent outward potassium current, Ilo, were studied in myocytes isolated from human atrial trabeculae using the whole-cell-recording patch-clamp technique. When cells were dialysed with pipette media containing 0.2 mM GTP, ANF reduced ICa by 37.81% +/- 5.4% at +20 mV and Ilo by 21.72% +/- 3.68% at +60 mV in a reversible manner. When ICa was increased by beta-adrenoreceptor stimulation (0.1 microM isoproterenol) or by the phosphodiesterase inhibitor isobutylmethylxanthine (10 microM) ANF reduced ICa by 24.99 +/- 3.4% and by 39.9 +/- 6.3% respectively. In cells dialysed with GTP-free pipette media, ANF increased ICa markedly (39.8% +/- 7%) and reversibly, whereas it still depressed Ilo (18.92% +/- 2%). Addition of 0.2 mM GTP[gamma S] to the pipette solution in the absence of GTP increased ICa, decreased Ilo and suppressed the effect of ANF on both ICa and Ilo. It is suggested that activation of the ANF receptor in human atrial cells reduces ICa via guanylate-cyclase-dependent cGMP production, increases ICa via Gs protein activation and decreases Ilo via Gi protein activation.

1-Methyl-3-isobutylxanthine↗
PubMed1992

P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells.

The pharmacological and single-channel properties of Ca2+ channels were studied in the somata and dendrites of adult cerebellar Purkinje cells. The Ca2+ channels were exclusively of the high threshold type: low threshold Ca2+ channels were not found. These high threshold channels were not blocked by omega-conotoxin GVIA and were inhibited rather than activated by BAY K 8644. They were therefore pharmacologically distinct from high threshold N- and L-type channels. Funnel web spider toxin was an effective blocker. The channels opened to conductance levels of 9, 14, and 19 pS (in 110 mM Ba2+). These slope conductances were in the range of those reported for N- and L-type channels. Our results are in agreement with previous reports suggesting that Ca2+ channels in Purkinje cells can be classified as P-type channels according to their pharmacology. The results also suggest that distinctions among Ca2+ channel types based on the single-channel conductance are not definitive.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

The beta subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca2+ channel.

The skeletal muscle (SKM) L-type Ca2+ channel is composed of a central subunit designated alpha 1, which contains the pore and the dihydropyridine (DHP) binding domains and three associated subunits, alpha 2/delta, beta, and gamma, which influence the activity of the SKM alpha 1. Coexpression of SKM alpha 1 and SKM beta in stably transfected mouse L cells results in a dramatic increase in DHP binding accompanied by fast gated Ba2+ currents. We report here that this "SKM alpha 1 beta-related phenotype" can be converted upon intracellular trypsin treatment into a slowly inactivating, DHP sensitive "SKM alpha 1 phenotype." These observations indicate that current amplitude, fast inactivation, and DHP sensitivity are modulated by an interaction of SKM alpha 1 and SKM beta on the internal side of the membrane.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists.

Myocardial cells have two types of Ca channels commonly called T-type and L-type. Whole cell Ca channel currents in guinea pig atrial myocytes can be separated and quantitated by analyzing channel closing kinetics after a brief depolarization (tail current analysis). L-type Ca channels deactivate rapidly when the membrane is repolarized and T-type Ca channels deactivate relatively slowly. Ca channel block by the therapeutically useful Ca channel antagonists is voltage dependent, so it is desirable to study block of both channel types over an extended voltage range. Tail current analysis allows this and was used to study block of both types of Ca channels under identical conditions. Amiodarone, bepridil, and cinnarizine block T-type Ca channels more potently than L-type Ca channels when binding equilibrates at normal diastolic potentials (approximately -90 mV). None of these drugs is a selective blocker of T-type Ca channels because block of L-type Ca channels is enhanced when cells are almost completely depolarized. Although weak block of T-type Ca channels by 1,4-dihydropyridines has usually been reported, we found that felodipine blocks these channels with high affinity. When most T-type Ca channels are inactivated, the apparent dissociation constant (KI) is 13 nM. Felodipine also blocks T-type Ca channels in GH3 cells (a cell line derived from rat anterior pituitary), but KI = 700 nM. Thus, T-type Ca channels in different cell types are pharmacologically distinct. Felodipine can block L-type Ca channels in atrial cells more potently than T-type Ca channels, but block of L-type Ca channels is potent only at depolarized potentials; block of both channel types is comparable at normal diastolic membrane potentials. Felodipine and the 1,4-dihydropyridines isradipine and (-)-202-791 are approximately equipotent at blocking T-type Ca channels, but differ substantially in potency for block of L-type Ca channels. Block of T-type Ca channels may account for some of the pharmacological effects of 1,4-dihydropyridines and for the antiarrhythmic activity of amiodarone and bepridil.

Animals↗
PubMed1992

Ca(2+)-activated K+ channels modulate muscarinic secretion in cat chromaffin cells.

1. This study was aimed at testing the hypothesis that Ca(2+)-dependent K+ channels regulate the release of catecholamines mediated by muscarinic stimulation of cat adrenal chromaffin cells. Two parameters were measured: the secretory response to brief pulses of methacholine (100 microM for 10 s) in intact cat adrenal glands perfused at a high rate with oxygenated Krebs solution; and the changes in cytosolic Ca2+ concentrations, [Ca2+]i, produced by puff applications of methacholine pulses (also 100 microM for 10 s) in isolated single cat adrenal chromaffin cells loaded with Fura-2. 2. A pulse of methacholine released 805 +/- 164 ng of catecholamines (mean of thirty-two pulses). d-Tubocurarine (DTC) increased the secretory response in a concentration-dependent manner. The maximum increase (around 1000 ng catecholamines over control values) was reached at 100 microM-DTC and the EC50 was around 10 microM. 3. The secretory responses to methacholine alone, or to the combination of methacholine plus DTC, were strongly dependent on the extracellular Ca2+ concentration, [Ca2+]o. Thus Ca2+o removal from the perfusing solution for 5-10 min abolished catecholamine release. 4. At 0.1 microM, isradipine (an L-type Ca2+ channel blocker) inhibited by 71% the secretory response to DTC plus methacholine. At 1 microM, Bay K 8644 (an L-type Ca2+ channel activator) increased 2-fold the secretory response to DTC plus methacholine (2746 ng of catecholamines). 5. Apamin (1 microM) increased 3.5-fold the secretory response to methacholine pulses (from 500 to 1800 ng of catecholamines). 6. Methacholine pulses enhanced [Ca2+]i from the resting level of 100 nM to a peak of 1000 nM which quickly declined to basal level. DTC (100 microM) enhanced by 20% the [Ca2+]i peak and substantially prolonged its duration. 7. Apamin (1 microM) increased by 60% the [Ca2+]i peak evoked by methacholine, and delayed the initiation of decline of the [Ca2+]i peak. 8. These results are compatible with the idea that muscarinic stimulation depolarizes the cat adrenal chromaffin cell through an unidentified mechanism. Depolarization is probably counteracted by activation of Ca2+i-dependent K+ channels. Therefore, inhibition of these channels enhances depolarization and firing of action potentials which activate voltage-dependent L-type Ca2+ channels to increase further the Ca2+i signal and the secretory response. Thus Ca2+i-dependent K+ channels, probably of the small-conductance type (SK), seem to be involved in the modulation of muscarinic-evoked catecholamine release responses in cat adrenal chromaffin cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Characterization of the effects of a new Ca2+ channel activator, FPL 64176, in GH3 cells.

We examined the effects of the benzolpyrrole-type Ca2+ channel activator FPL 64176 on voltage-dependent L-type Ca2+ channels in rat anterior pituitary (GH3) cells. FPL 64176 increased K(+)-dependent Ca2+ influx into GH3 cells with an EC50 value of 1.2 x 10(-7) M but had no effect on the binding of [3H]PN200-110 to GH3 cell membranes at concentrations up to 10(-6) M. Whole-cell patch-clamp electrophysiology revealed that FPL 64176 (1 microM) increased L-type Ca2+ channel current amplitude and shifted the current-voltage relationship in the hyperpolarizing direction. Furthermore, Ca2+ channel current activation and deactivation were prolonged. Single-channel analysis showed that FPL 64176 increased both the probability of channel opening and the mean channel open time. Interestingly, the effect of FPL 64176 on channel open time was highly voltage dependent, with much longer openings being observed at more hyperpolarized potentials. We conclude that FPL 64176 represents a new class of L-type Ca2+ channel activator with a novel site and mechanism of action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels, and it is suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). Figure 11 summarizes the possible mechanisms by which uterine contractility can be modulated. In contrast to vascular smooth muscle, neither ISO nor adenosine, which produce elevation of cyclic AMP, affected ICa and INa. Therefore, no arrow can be drawn between cA-PK/cG-PK and the Ca2+ slow channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗