Effects of endothelin-1 on Ca2+ signaling in guinea-pig ventricular myocytes: role of protein kinase C

The effects of ET-1 on contraction, Ca2+ transient and L-type Ca2+ current (ICa.L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanism of the positive inotropic effect during endothelin receptor stimulation by focusing on the role of PKC. ET-1 at concentrations of 5 and 10 nM produced a biphasic pattern of inotropism: a first decrease in contraction by 34.4 +/- 2.5% of the control followed by a sustained increase in contraction by 66.6 +/- 8.4% (mean +/- SEM, n = 9). The Ca2+ transient decreased by 13.5 +/- 1.0% during the negative inotropic phase, while it increased by 58.1 +/- 8.4% (n = 10) during the positive inotropic phase. Using the whole-cell voltage-clamp technique with conventional microelectrodes, the application of ET-1 (5 nM) increased the ICa.L by 32.6 +/- 5.1% (n = 10), which was preceded by a short-lived decrease in ICa.L. Incubation of myocytes with pertussis toxin (PTX, at 2 micrograms/ml for > 3 h at 35 degrees C) failed to block the ET-1-induced enhancement of ICa.L. The increases in contraction, Ca2+ transient, and ICa.L by ET-1 were inhibited by pretreatment with 5-N-methyl-N-isobutyl amiloride (MIA; 10 microM), an amiloride analog, and a novel selective Na+/H+ exchange inhibitor HOE694 (10 microM). To determine whether activation of protein kinase C (PKC) is responsible for the enhancement of ICa.L by ET-1, we tested a PKC inhibitor, GF109203X, and found that it does exert an inhibitory effect on the ET-1-induced ICa.L increase. Our study suggests that during ET receptor stimulation an increase in ICa.L due to stimulation of Na+/H+ exchange via PKC activation causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture

Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type ICa) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type ICa, without affecting T-type ICa. L-type ICa was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type ICa is halved by 0.1 microM nicardipine, 3.0 microM diltiazem, 0.6 microM verapamil, and 0.1 microM flunarizine, whereas T-type ICa is halved by the same drugs at 0.6, 30, 30, and 0.1 microM, respectively. Diltiazem and verapamil accelerated the decay of L-type ICa and cumulatively blocked L-type ICa during repetitive step depolarizations elicited every 30 seconds ("use-dependent block"). Diltiazem and verapamil neither changed the decay of T-type ICa nor showed a use-dependent block of T-type ICa. Nicardipine and flunarizine blocked both L- and T-type ICa from the first depolarization step after drug treatment ("tonic block") and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively) were smaller than those for the resting state of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 microM) of nicardipine initially potentiated T-type ICa and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.     

Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein

Effects of concentrations of intracellular calcium, [Ca2+]i, on the voltage-dependent Ca2+ current (ICa) recorded from dispersed single smooth muscle cells of the rabbit portal vein were studied, using a whole cell voltage clamp method combined with an intracellular perfusion technique. Outward currents were minimized by replacement of Cs+ -rich solution in the pipette and 20 mM tetraethylammonium in the bath. The ICa was evoked by command pulses of above -30 mV, and the maximum amplitude was obtained at about 0 mV. This ICa was dose dependently inhibited by increases in the [Ca2+]i above 30 nM. The Kd value of the [Ca2+]i required to inhibit the ICa was about 100 nM. The Ba2+ current was also inhibited by increases in the [Ca2+]i. Conversely, perfusion of Ba2+ into the cell up to 100 microM did not suppress the ICa. Changes in the [Ca2+]i did not modify the steady-state inactivation curve. The inhibition of the ICa evoked by the test pulse is most prominent when the preceding influx of Ca2+ during the conditioning pulse was large, as estimated using a double pulse protocol. This inhibition was proportionally reduced by increases in the concentration of the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Therefore, the Ca2+ -dependent inactivation of the Ca2+ channel may contribute toward regulating [Ca2+]i in smooth muscle cells of the rabbit portal vein.     

Testosterone acts as a coronary vasodilator by a calcium antagonistic action

T acts as a vasodilator in vitro and in vivo. Supplemental T therapy in humans with angina improves symptoms and reduces objective measures of ischemia. In left anterior descending coronary arteries taken from adult male Wistar rats, T abolishes 100+/-4.2% of calcium-dependent contraction induced by potassium chloride, 82.3+/-6.1% of the mostly calcium-dependent contraction induced by prostaglandin-F-2-alpha, but only 45.3+/-3.4% of the contraction induced by phorbol-12,13-dibutyrate (PDBu) in the presence of extracellular calcium, and 54.5+/-4.5% of the contraction induced by PDBu in the absence of extracellular calcium. These findings suggest that T is primarily inhibiting the calcium-dependent elements of vascular contraction.     

Developmental changes in the beta-adrenergic modulation of calcium currents in rabbit ventricular cells.

We studied the developmental changes in the beta-adrenergic modulation of L-type calcium current (ICa) in enzymatically isolated adult (AD) and newborn (NB, 1-4-day-old) rabbit ventricular cells using the whole-cell patch-clamp method. ICa was measured as the peak inward current at a test potential of +15 mV by applying a 180-450-msec pulse from a holding potential of -40 mV with Cs(+)-rich pipettes and a K(+)-free bath solution at room temperature. In control, ICa density (obtained by normalizing ICa to the cell capacitance) was significantly higher in AD cells (5.5 +/- 0.2 [mean +/- SEM] pA/pF, n = 65) than in NB cells (2.6 +/- 0.1 pA/pF, n = 60). Isoproterenol (ISO, 1 nM-30 microM) increased ICa in a dose-dependent manner for both groups. The maximal effect (Emax) of ISO, expressed as percent increase in ICa over control levels, and the concentration for one half of the maximal effect (EC50) were 203% and 51 nM, respectively, for AD cells and 111% and 81 nM, respectively, for NB cells. The effect of ISO (1 microM) on ICa was decreased as the test potential was increased from -10 to +40 mV. However, the ratio of the percent increase in ICa for AD versus NB cells was almost constant (2.09-2.45) at each test potential. Dose-response curves of forskolin (FOR, 0.3-50 microM) gave Emax and EC50 of 268% and 0.74 microM, respectively, for AD cells and 380% and 1.15 microM, respectively, for NB cells. After stimulating ICa by 10 microM ISO, the addition of 10 microM FOR produced a further increase in ICa of only 12 +/- 2% in AD cells (n = 4) but a further increase of 140 +/- 41% in NB cells (n = 6). FOR (10 microM) did not produce any increase in ICa for AD and NB cells after stimulating ICa by intracellular application of 200 microM cAMP. ICa density stimulated by 10 microM ISO (17.8 +/- 1.1 pA/pF, n = 7), 10 microM FOR (21.0 +/- 1.3 pA/pF, n = 8), or 200 microM cAMP (18.0 +/- 1.3 pA/pF, n = 5) was equivalent in AD cells, whereas ICa density stimulated by 10 microM ISO (5.8 +/- 0.6 pA/pF, n = 9) was significantly lower than that stimulated by either 10 microM FOR (13.8 +/- 1.5 pA/pF, n = 7) or 200 microM cAMP (13.4 +/- 0.7 pA/pF, n = 7) in NB cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Possible involvement of protein kinase C in gonadotropin and growth hormone release from dispersed goldfish pituitary cells

Static incubation with tumor-promoting 4 beta-phorbol esters, activators of the Ca2(+)- and phospholipid-dependent protein kinase C enzyme (PKC), caused dose-dependent increases in gonadotropin (GTH) and growth hormone (GH) secretion in primary cultures of dispersed goldfish pituitary cells. The estimated half-maximal effective doses (ED50) for stimulating GTH and GH release were 0.35 +/- 0.17 and 0.32 +/- 0.13 nM 12-O-tetradecanoyl phorbol 13 acetate (TPA), 3.71 +/- 1.30 and 1.37 +/- 0.76 nM 4 beta-phorbol 12,13-dibutyrate, 6.90 +/- 4.84 and 1.89 +/- 0.25 nM 4 beta-phorbol 12,13-dibenzoate, and 455 +/- 258 and 311 +/- 136 nM 4 beta-phorbol 12,13-diacetate, respectively. In contrast, treatments with up to 10 microM of the inactive 4 alpha-phorbol 12,13-didecanoate ester did not alter GTH and GH release. Additions of the synthetic diacylglycerol, dioctanoyl glycerol, also enhanced GTH and GH secretion in a dose-dependent manner and with ED50s of 1.73 +/- 0.83 and 1.73 +/- 1.19 microM, respectively. The GTH and GH responses to stimulation by TPA were attenuated by incubation with Ca2(+)-depleted medium containing EGTA or by treatment with the Ca2+ channel blocker verapamil. Coincubation with the PKC inhibitor H7 reduced the GTH and GH responses to TPA. As in previous studies, additions of salmon gonadotropin-releasing hormone (sGnRH) or chicken GnRH-II (cGnRH-II) induced GTH and GH release; these hormone responses to sGnRH and cGnRH-II were also decreased by the addition of H7. These results indicate that activation of PKC may stimulate GTH and GH release in goldfish and suggest that sGnRH and cGnRH-II actions on goldfish pituitary GTH and GH secretion are also mediated, at least partially, by PKC.     

Dopamine inhibits two characterized voltage-dependent calcium currents in identified rat lactotroph cells

The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the whole-cell configuration, two types of Ca2+ currents, L and T, were characterized on the basis of their kinetics, voltage sensitivity, and pharmacology. The L component had a threshold of -25 mV, showed little inactivation during a 150-msec voltage step, and was maximal at +10 mV. Cadmium ions (100 microM) significantly reduced its amplitude (75%). The T component was activated at a membrane potential close to -50 mV, was maximal at -10 mV, and showed a voltage-dependent inactivation between -90 and -30 mV. It was quickly inactivated during a maintained depolarization (time constant, 27 ms at -30 mV) and was strongly reduced (80%) by nickel ions (100 microM). Bath application of DA (10 nM) caused a markedly general depression of inward Ca2+ currents, acting differently on the T- and L-type currents. DA application shifted the voltage-dependence of the L-type current activation toward depolarization values (8 mV) without modifying its time- and voltage-dependent inactivation. In contrast, DA enhanced the inactivation of the T-type current by accelerating its time-dependent inactivation (25% decrease in the time constant of inactivation) and by shifting the voltage-dependence of the T-type current inactivation toward hyperpolarizing values (-63 mV in control vs. -77 mV in the presence of DA). These effects of DA were dose-dependent and involved the activation of a D2 receptor type. They were mimicked by bromocriptine application (10 nM), whereas sulpiride (100 nM) blocked the DA-evoked response. The D1 antagonist SCH 23390 was ineffective up to 100 microM. All of these DA-induced modifications in Ca2+ currents were abolished using a GTP-free pipette solution or after pretreatment of cells with pertussis toxin, suggesting that DA can regulate the function of Ca2+ channels through GTP-binding proteins (G-proteins). Our results show that DA acts simultaneously by reducing both voltage-dependent Ca2+ currents on lactotroph cells. Thus, DA reduces the entry of Ca2+ ions across the surface membrane and thereby influences electrical activity and the cytosolic free Ca2+ concentration involved in both basal and evoked PRL release.     

Orexin-B augments voltage-gated L-type Ca(2+) current via protein kinase C-mediated signalling pathway in ovine somatotropes

Orexins, orexigenic neuropeptides, are secreted from lateral hypothalamus and orexin receptors are expressed in the pituitary. Since growth hormone (GH) secreted from pituitary is integrally linked to energy homeostasis and metabolism, we studied the effect of orexin-B on voltage-gated Ca(2+) currents and the related signalling mechanisms in primary cultured ovine somatotropes using whole-cell patch-clamp techniques. With a bath solution containing TEA-Cl (40 mM) and Tetrodotoxin (TTX) (1 microM), three subtypes of Ca(2+) currents, namely the long-lasting (L), transient (T), and N currents, were isolated using different holding potentials (-80 and -30 mV) in combination with specific Ca(2+) channel blockers (nifedipine and omega-conotoxin). About 75% of the total current amplitude was contributed by the L current, whereas the N and T currents accounted for the rest. Orexin-B (1-100 nM) dose-dependently and reversibly increased only the L current up to approximately 125% of the control value within 4-5 min. Neither a specific protein kinase A (PKA) blocker (H89, 1 microM) nor an inhibitory peptide (PKI, 10 microM) had any effect on the increase in L current by orexin-B. The orexin-B-induced increase in the L current was abolished by concurrent treatment with calphostin C (Cal-C, 100 nM), protein kinase C (PKC) inhibitory peptide (PKC(19-36), 1 microM), or by pretreatment with phorbol-12,13-dibutyrate (PDBu) (0.5 microM) for 16 h (a downregulator of PKC). Orexin-B also increased in vitro GH secretion in a dose-dependent manner. We conclude that orexin-B increases the L-type Ca(2+) current and GH secretion through orexin receptors and PKC-mediated signalling pathways in ovine somatotropes.     

Alterations in bidirectional transmembrane calcium flux occur without changes in protein kinase C levels in rat aorta during sepsis

A depression in aortic contractility has been previously demonstrated in rat intraperitoneal sepsis and during endotoxemia. In this study, we determined whether the mobilization of extracellular calcium (using 45Ca) and the release of intracellular calcium are altered in septic rat aorta when compared to sham-operated controls. The concentration of protein kinase C was also determined by using [3H] phorbol-12,13-dibutyrate (PDBu). We found that calcium influx was unaltered under basal conditions but that the ability of norepinephrine (NE) to augment influx was significantly depressed (P less than .05; [control vs. septic, 572 +/- 54 [SE] vs. 428 +/- 30 mumol Ca2+/kg dry wt. aorta]). Calcium influx stimulated by high K+ was unchanged in aortae between control and septic animals. In the presence of NE, calcium efflux (an indirect measurement of intracellular calcium release) was significantly diminished (P less than .001) in aortae from septic rats. The concentration of aortic protein kinase C as assessed by PDBu binding sites was unaltered in septic rats when compared with controls. In conclusion, we found that during sepsis alpha 1-adrenergic receptor activation of both calcium influx and efflux by NE is decreased; these alterations could be related to the depressed aortic contractility observed in sepsis.     

Omega 3 polyunsaturated fatty acid modulates dihydropyridine effects on L-type Ca2+ channels, cytosolic Ca2+, and contraction in adult rat cardiac myocytes.

The effect of docosahexaenoic acid (DHA; C22:6) on dihydropyridine (DHP) interaction with L-type Ca2+ channel current (ICa), cytosolic Ca2+ (Cai), and cell contraction in isolated adult rat cardiac myocytes was studied. The DHP L-type Ca(2+)-channel blocker nitrendipine (10 nM) reduced peak ICa (measured by whole-cell voltage clamp from -45 to 0 mV) and reduced the amplitude of the Ca2+ transient (measured as the transient in indo-1 fluorescence, 410/490 nm) and the twitch amplitude (measured via photodiode array) during steady-state electrical stimulation (0.5 Hz). The DHP L-type Ca2+ channel agonist BAY K 8644 (10 nM) significantly increased ICa, the amplitude of the Cai transient, and contraction. When cells were exposed to DHA (5 microM) simultaneously with either BAY K 8644 or nitrendipine, the drug effects were abolished. Arachidonic acid (C20:4) at 5 microM did not block the inhibitory effects of nitrendipine nor did it prevent the potentiating effects of BAY K 8644. DHA modulation of DHP action could be reversed by cell perfusion with fatty acid-free bovine serum albumin at 1 mg/ml. Neither DHA nor arachidonic acid alone (5 microM) had any apparent effect on the parameters measured. DHA (5 microM) had no influence over beta-adrenergic receptor stimulation (isoproterenol, 0.01-1 microM)-induced increases in ICa, Cai, or contraction. The findings that DHA inhibits the effect of DHP agonists and antagonists on Ca(2+)-channel current but has no effect alone or on beta-adrenergic-induced increases in ICa suggests that DHA specifically binds to Ca2+ channels at or near DHP binding sites and interferes with ICa modulation.     

Calcium current is increased in isolated adult myocytes from hypertrophied rat myocardium

To study the effects of myocardial hypertrophy resulting from chronic pressure overload on excitation-contraction coupling, the cardiac transmembrane L-type calcium current (ICa) was investigated in the Goldblatt renovascular hypertensive (HBP) rat. ICa was measured in single myocytes enzymatically isolated from control (CTRL) and HBP rat hearts using the whole-cell, patch-clamp method. The peak ICa and ICa density (obtained by normalizing ICa to the average cell capacitative surface area) were larger in HBP cells (n = 15) than in CTRL cells (n = 10) at membrane potentials of -20 to 50 mV (p less than 0.01). The maximal peak ICa increased from 0.9 +/- 0.5 nA (mean +/- SD) in CTRL cells to 2.8 +/- 1.0 nA in HBP cells (p less than 0.001). The corresponding ICa density increased from 5.3 +/- 2.7 to 16.2 +/- 6.0 microA/cm2 (p less than 0.001). There was no shift in the current-voltage relation between CTRL and HBP cells. The time course of decay of HBP ICa in response to clamp steps to the plateau range of the action potential (membrane potential, Vm = -10 to 30 mV) was delayed when compared with that of CTRL ICa. The inactivation time constants (biexponential) for the maximal ICa were 6.9 +/- 1.9 and 36.0 +/- 9.3 msec for CTRL cells and 6.7 +/- 1.4 and 49.5 +/- 12.9 msec for HBP cells (p less than 0.05 for the slower component of the maximal ICa). There was no difference in the steady-state inactivation of ICa (f infinity) for the CTRL and HBP cells. From the maximal peak ICa, cytoplasmic free Ca2+ was estimated to reach a pCa of 6.95 +/- 0.07 for CTRL cells and 6.64 +/- 0.13 for HBP cells. It is concluded that ICa is increased with myocardial hypertrophy. The lengthening of the action potential in hypertrophied rat myocardium is due to an increase in peak current density and to the slower inactivation of the maximal ICa. The increased transmembrane flux of Ca2+ via ICa in HBP cells is inadequate to achieve a myoplasmic free Ca2+ level sufficient for direct partial activation of the contractile myofilaments. However, in the scheme of the calcium-triggered calcium release hypothesis such an increase could provide an increased amount of activator calcium and/or serve to amplify the release of Ca2+ from sarcoplasmic reticulum, thereby contributing to preserved peak developed tension in hypertrophied rat myocardium.     

Inhibitory effects of carvedilol on calcium channels in vascular smooth muscle cells

Carvedilol has hypotensive effects and inhibits agonist-induced cell proliferation of vascular smooth muscle and then prevents vascular remodeling. However, the basic mechanisms have not been clarified. We examined the effects of carvedilol on [Ca2+]i mobilization and voltage-dependent L-type Ca2+ current (ICa.L) in vascular smooth muscle cells, and compared them with metoprolol. [Ca2+]i was measured using fura-2 AM and patch clamp techniques in rat embryonic aortic smooth muscle cells (A7r5). In the presence of extracellular Ca2+, vasopressin and endothelin-1 increased [Ca2+]i due first to the Ca2+ release from store sites, and subsequently Ca2+ entry. Carvedilol did not inhibit the Ca2+ release, but significantly suppressed the sustained rise due to Ca2+ entry concentration-dependently. Nilfedipine and nicardipine (10 microM) partly inhibited the sustained rise, but carvedilol inhibited it more effectively than the Ca2+ channel blockers. Under voltage clamp conditions, carvedilol (0.2-10 microM) reversibly inhibited the ICa.L concentration-dependently without any changes in the current-voltage relationships of ICa.L. Carvedilol shifted the steady-state inactivation for ICa.L to more negative potentials and inhibited ICa.L in a voltage-dependent manner. In addition, carvedilol did not inhibit Ca2+ release from store sites induced by thapsigargin, but significantly inhibited the sustained rise due to capacitative Ca2+ entry unrelated to ICa.L. In contrast, metoprolol did not mimic these effects of carvedilol. These results provide evidence that carvedilol inhibits ICa.L and may also inhibit the channels for agonist (vasopressin and endothelin-1)-induced Ca2+ entry in vascular smooth muscle cells, which might contribute to the vasorelaxing and antiproliferative effects of carvedilol.     

Relation of human neutrophil phorbol ester receptor occupancy and NADPH- oxidase activity

Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50's for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.     

Effect of a novel class I antiarrhythmic agent, TYB-3823, on the calcium current in single guinea pig ventricular myocytes.

The effect of TYB-3823 on Ca2+ current (ICa) of guinea pig ventricular myocytes was investigated by means of whole-cell patch-clamp technique. TYB-3823 (100-1,000 microM) caused a concentration-dependent decrease in ICa. Furthermore, a reduction of ICa induced by TYB-3823 (1,000 microM) was progressively accentuated by repetitive membrane depolarization, indicating a rate-dependent block of ICa. However, the inhibitory potency on ICa was approximately 1/1000 of a Ca2+ antagonistic agent, verapamil hydrochloride. Considering evidence that 3-30 microM TYB-3823 decreased the maximum upstroke velocity of the action potential of guinea pig ventricular muscles, it is indicated that the drug does not show its Ca2+ antagonistic property in the usual concentration range as a class I antiarrhythmic agent.     

Catalytic and receptor-binding properties of the calcium-sensitive phospholipid-dependent protein kinase (protein kinase C) in swine luteal cytosol

We have evaluated the catalytic and receptor-binding properties of protein kinase C in swine luteal cytosol using two complementary approaches: assay of catalytic activity assessed as the enzymatic transfer of radiolabeled phosphate to histone III-s acceptor protein in the presence of specific phospholipid, diacylglycerol, or phorbol ester and ionic calcium; and, the high-affinity binding of [3H]phorbol-12,13-dibutyrate ([3H]PDB) to the protein kinase C receptor. Catalytic properties of pig luteal protein kinase C included: absolute dependence on calcium ions for maximal activation (approximate ka = 0.5 microM); synergistic activation by 1,2-sn-diolein, phospholipid and calcium ions; and rank order of specific phospholipid activational potency: phosphatidylserine greater than phosphatidic acid greater than phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. The enzyme was also activated by specific phorbol esters at the following half-maximally effective (ED50) concentrations: 12-O-tetradecanoylphorbol-13-acetate (TPA) 11 nM; phorbol-12,13-dibenzoate (PDBe) 26 nM; phorbol-12,13-dibutyrate (PDBu) 33 nM; mezerein 65 nM; and phorbol-12,13-diacetate (PDA) 130 nM. Phorbol-ester receptor properties of protein kinase C included specific, high-affinity (kd congruent to 19 nM), saturable, low-capacity (congruent to 44 pmol/mg protein) [3H]PDB binding sites. Moreover, the rank order of the equilibrium binding ID50s for various phorbol compounds was similar to that of catalytic ED50s: viz. 3 nM TPA; 8 nM PDBe; 16 nM PDBu; 19 nM mezerein; and 590 nM PDA. Thus, swine luteal cytosol contains catalytically active protein kinase C with specific phospholipid sensitivity, synergistic activation by diacylglycerol, phospholipid and calcium, and a strict dependence on ionic calcium concentrations that is influenced markedly by the presence of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous and corticotropin-releasing factor-induced cytosolic calcium transients in corticotrophs.

Spontaneous and CRF-stimulated changes in the cytosolic free calcium concentration ([Ca2+]i) were studied in two types of corticotrophs: 1) cultured human ACTH-secreting pituitary adenoma cells (hACTH cells), and 2) identified small ovoid corticotrophs cultured from normal rat pituitaries. [Ca2+]i was monitored in individual corticotrophs by dual emission microspectrofluorimetry using indo-1 as the intracellular fluorescent Ca2+ probe. In hACTH cells, [Ca2+]i measurements were carried out in combination with electrophysiological recordings obtained using whole cell patch-clamp techniques. It was shown that a single spontaneous Ca(2+)-dependent action potential led to a marked transient increase in [Ca2+]i in human tumoral corticotrophs. Spontaneous fluctuations in [Ca2+]i were also observed in unpatched corticotrophs whether derived from human pituitary tumors or normal rat tissue. Based on their striking kinetic features and their sensitivity to external Ca2+, we suggest that these spontaneous [Ca2+]i transients were the consequence of action potential firing. Under separate voltage-clamp (patch-clamp) conditions, tumor corticotrophs showed two Ca2+ current components: a low threshold, rapidly inactivating (T-type) current, and a higher threshold, slowly inactivating (L-type) current. The dihydropyridine Ca2+ channel blocker PN 200-110 (100 nM) abolished the L-type current without affecting the T-type current, while the inorganic Ca2+ channel blocker Cd2+ (200 microM) suppressed both Ca2+ currents. The Na+ channel blocker tetrodotoxin (5 microM) did not affect inward currents in tumor corticotrophs. Both L- and T-type voltage-gated Ca2+ channels were involved in controlling [Ca2+]i transients in both tumor and normal corticotrophs, inasmuch as Cd2+ (200 microM) abolished [Ca2+]i) transients, while PN 200-110 (100 nM) greatly diminished, but did not completely abolish, [Ca2+]i transients. The latter did not appear to depend on a voltage-dependent Na+ influx, since they were unaffected by tetrodotoxin (5 microM). Corticotrophs generate [Ca2+]i transients in response to the hypothalamic secretagogue CRF by acting on their membrane excitability. Indeed, we demonstrated in combined fluorescent and electrophysiological experiments that CRF (100 nM) had a coordinate action on human tumoral corticotrophs comprised of a modest depolarization and an increase in the frequency of both action potentials and subsequent [Ca2+]i transients. A coincident increase in the peak amplitude of the [Ca2+]i transient and after hyperpolarization was also observed in some CRF-stimulated cells. CRF (100 nM) evoked qualitatively similar [Ca2+]i patterns in human tumoral and normal rat corticotrophs not subjected to patch-clamping.(ABSTRACT TRUNCATED AT 400 WORDS)     

T-type Ca2+ current contribution to Ca2+-induced Ca2+ release in developing myocardium

In normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electrically stimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.     

Long-term and immediate effect of testosterone on single T-type calcium channel in neonatal rat cardiomyocytes

In the cardiovascular system, T-type calcium channels play an important role for the intracellular calcium homeostasis and spontaneous pacemaker activity and are involved in the progression of structural heart diseases. Androgens influence the cardiovascular physiology and pathophysiology. However, their effect on native T-type calcium currents (I(Ca,T)) remains unclear. To test the chronic effect of testosterone on the cardiac I(Ca,T), cultured neonatal rat ventricular cardiomyocytes were treated with testosterone (1 nM-10 microM) for 24-30 h. Current measurements were performed after testosterone washout to exclude any acute testosterone effects. Testosterone (100 nm) pretreatment significantly increased whole-cell I(Ca,T) density from 1.26 +/- 0.48 pA/pF (n = 8) to 5.06 +/- 1.75 pA/pF (n = 7; P < 0.05) and accelerated beating rate. This was attributed to both increased expression levels of the pore-forming subunits Ca(v)3.1 and Ca(v)3.2 and increased T-type single-channel activity. On single-channel level, the increase of the ensemble average current by testosterone vs. time-matched controls was due to an increased availability (58.1 +/- 4.2 vs. 21.5 +/- 4.0%, P < 0.01) and open probability (2.78 +/- 0.29 vs. 0.85 +/- 0.23%, P < 0.01). Cotreatment with the selective testosterone receptor antagonist flutamide (10 mum) prevented these chronic testosterone-induced effects. Conversely, acute application of testosterone (10 microM) decreased T-type single-channel activity in testosterone pretreated cells by reducing the open probability (0.78 +/- 0.13 vs. 2.91 +/- 0.38%, P < 0.01), availability (23.6 +/- 3.3 vs. 57.6 +/- 4.5%, P < 0.01), and peak current (-20 +/- 4 vs. -58 +/- 4 fA, P < 0.01). Flutamide (10 microM) did not abolish the testosterone-induced acute block of T-type calcium channels. Our results indicate that long-term testosterone treatment increases, whereas acute testosterone decreases neonatal rat T-type calcium currents. These effects seem to be mediated by a genomic chronic stimulation and a nongenomic acute inhibitory action.     

Contrasting effects of phorbol esters on serotonin- and vasopressin-evoked contractions in rat aorta and small mesenteric artery.

Phorbol esters, which activate protein kinase C, modulate vasoconstrictor-induced tension in vascular smooth muscle. We examined the effects of phorbol esters (phorbol 12,13-dibutyrate [PDBu] and 12-O-tetradecanoylphorbol 13-acetate [TPA]) on receptor agonist (serotonin [5-HT] and arginine vasopressin [AVP])-, high K(+)-, and caffeine-induced contractions in rings of rat aorta and a small (second-order) branch of the superior mesenteric artery (SMA). PDBu and TPA significantly augmented agonist-evoked contractions in aorta but diminished those in SMA. For example, 30 nM PDBu increased 5-HT- and AVP-evoked contractions 2.0-2.5-fold in aorta (p less than 0.01) but decreased 5-HT- and AVP-induced contractions by 40-60% in SMA (p less than 0.01). In contrast, PDBu and TPA amplified high K(+)- and 10 mM caffeine-induced contractions in both aorta and SMA. Augmentation of agonist-induced contractions by PDBu was greater in endothelium-denuded aorta than in intact aorta. Two protein kinase C antagonists, H-7 and staurosporine, inhibited 5-HT-evoked contractions in the absence as well as in the presence of PDBu in both types of arteries. The augmentation of contractile responses to caffeine and K+ by phorbol esters in both types of arteries suggests that the phorbols increase the sensitivity of the contractile apparatus to Ca2+, probably by activating protein kinase C. However, the inhibitory effects of phorbols on 5-HT- and AVP-evoked responses in SMA suggest that under these conditions the dominant effect of the phorbols is a marked reduction in the availability of Ca2+ in the SMA but not in the aorta.     

Calcium current restitution in mammalian ventricular myocytes is modulated by intracellular calcium

Restitution of the conventional L-type calcium current (ICa) was studied in dog or guinea pig ventricular myocytes to understand its time course and regulation. Whole-cell ICa free of other overlapping currents was recorded with a suction pipette. The intracellular environment was varied by intracellular dialysis. The properties of ICa were similar in dog and guinea pig ventricular myocytes, except that the amplitude of ICa was larger in the latter (2.2 +/- 0.5 nA in guinea pig cells and 0.9 +/- 0.2 nA in dog cells, n = 8 for both). In both types of cells during restitution a holding voltage (Vh) negative to -50 mV induced a transient increase in ICa above the control level (ICa overshoot). This overshoot was inhibited by substituting barium for calcium, lowering [Ca]0, increasing intracellular calcium buffering capacity, ryanodine (1-2 microM), or caffeine (10 mM). The overshoot peaked 30-100 msec after repolarization from the conditioning depolarization and gradually declined over the following 2-3 seconds. During the overshoot, although the amplitude of ICa was larger its half-time of decay was longer than the control. The maximum overshoot occurred following a conditioning step to plateau voltages and it was decreased by prolonging the conditioning step from 50 to 100 or 500 msec. It is concluded that intracellular calcium regulates restitution of the L-type calcium channels in mammalian ventricular myocytes and that the sarcoplasmic reticulum is involved in this process.