Bay K8644及nifedipine对大鼠下丘脑神经元L—型Ca^2＋通道特性的影响

采用神经元急性分离和膜片箍技术以及细胞贴附式方式记录通道活动,探讨DHP类Ca^2 通道激动剂Bay K8644及拮抗剂nifedipine对下丘脑神经元L－型Ca^2 通道的影响,结果显示,在Bay K8644作用下,通道开放形式发生变化,明显可见多级开放;通道平均开放时间,平均开放概况显著增加,但单通道电导无明显变化。nifedipine的作用与Bay K8644相反。结果提示,Bay K8644对下丘脑神经元L－型Ca^2 通道有明显激动作用 nifedipine有显著抑制作用。     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.     

Single calcium channel behavior in native skeletal muscle

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.     

Novel 1,4-Dihydropyridine (Bay K 8644) Facilitates Calcium-Dependent [3H]Noradrenaline Release from PC 12 Cells

The effects of the novel 1,4-dihydropyridine Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration-dependent manner evoked 3H-transmitter release with an EC50 of 50-56 mM. Bay K 8644 at 30 nM potentiated the K+-evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644-potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol X mg-1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a Ki of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a "calcium agonist" to mediate calcium-dependent cellular events such as catecholamine release from PC 12 cells.     

Calcium channels in isolated cells and strips of longitudinal muscle of rat myometrium

The properties of Ca2+ channels in strips and single muscle cells of longitudinal muscle of estrogen-dominated rat myometrium were studied under the effects of elevation of K+ concentration, the partial channel agonist Bay K 8644, and nitrendipine. In isolated strips in 0.5 mM Ca2+, Bay K 8644 (pD2 = 7.8-8.0) lowered the threshold for and enhanced the contractions in response to an elevation of K+ concentration, including the maximum response to K+ elevation alone. Bay K 8644 alone in concentrations up through 10(-6) M did not initiate contractions in 0.5 mM Ca2+ solutions. At higher concentrations (10(-5) M), Bay K 8644 behaved as an antagonist to contractions induced by elevation of K+. In isolated cells 10(-7) M Bay K 8644 enhanced the shortenings to elevated K+ and lowered the threshold K+ concentration required. Also no significant contraction occurred with 10(-7) M Bay K 8644 at normal K+ concentration. In contrast with its effect in isolated strips, no significant increase in maximum shortening (to 60 mM K+) was observed, possibly because cells without a mechanical load were maximally shortened by K+ alone. From these studies, we conclude that Ca2+ channels of isolated strips and cells of rat myometrium behave similarly and have similar properties to those of other smooth muscles in their interactions with elevation of K+, nitrendipine, and Bay K 8644.     

The calcium agonist Bay K 8644 stimulates secretion from a pituitary cell line

The dihydropyridine calcium agonist Bay K 8644 acts in a dose-dependent manner to increase prolactin secretion from the GH₄C₁ pituitary cell line. Enhanced secretion was observed at agonist concentrations as low as 10 nM. In the continued presence of Bay K 8644 secretion remained elevated for at least 30 min. The effect of the agonist was Ca²⁺-dependent and competitively antagonized by dihydropyridine antagonists. Apparently Bay K 8644 acts at the dihydropyridine binding site associated with GH₄C₁ Ca²⁺ channels to enhance Ca²⁺ influx and stimulate secretion from these cells. This is the first report demonstrating that the newly discovered Ca²⁺ agonist can, by itself, stimulate secretion from a cell.     

Bay K8644及nifedipine对大鼠下丘脑神经元L-型Ca~(2 )通道特性的影响

采用神经元急性分离和膜片箝技术以及细胞贴附式方式记录通道活动 ,探讨DHP类Ca2 通道激动剂BayK8644及拮抗剂nifedipine对下丘脑神经元L 型Ca2 通道的影响。结果显示 ,在BayK8644作用下 ,通道开放形式发生变化 ,明显可见多级开放 ;通道平均开放时间、平均开放概率显著增加 ,但单通道电导无明显变化。nifedipine的作用与BayK8644相反。结果提示 ,BayK8644对下丘脑神经元L 型Ca2 通道有明显激动作用 ,nifedip ine有显著抑制作用     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

Opposing actions of Bay K 8644 enantiomers on calcium current, prolactin secretion, and synthesis in pituitary cells.

Dihydropyridine (DHP) Ca2+ channel modulators were used to explore the relationship between voltage-gated Ca2+ channels and PRL secretion, synthesis, and mRNA in PRL-secreting pituitary cells. Optical isomers of the Ca2+ channel agonist Bay K 8644 produced stereospecific and opposing effects on L-type Ca2+ current, PRL release, and synthesis in GH3 and GH4C1 cells. (-)-Bay K 8644 (R5417) behaved as a pure agonist, enhancing Ca2+ current several-fold while shifting the current-voltage curve 10-15 mV in the hyperpolarizing direction. The agonist effect was independent of holding potential, but decreased during prolonged Ba2+ or Ca2+ entry. R5417 produced a concentration-dependent increase in acute PRL release and enhanced PRL production by GH cells several-fold during a 72-h period. (+)-Bay K 8644 (R4407) behaved as a weak Ca2+ channel antagonist, inhibiting L-type Ca2+ current, KCl-stimulated PRL secretion, and PRL production at concentrations of 0.5-5 microM. These two isomers produced similar effects on PRL production by normal rat pituitary cells in dispersed culture. R5417 (500 nM) increased PRL produced in 72 h to 233 +/- 8% of the control value. R4407 reduced this quantity by 36 +/- 9%. The effects of the DHPs on PRL mRNA levels were consistent with the effects observed for acute secretion and hormone production. The agonist R5417 increased PRL mRNA 147 +/- 5% over a 30-h period, and the potent DHP Ca2+ channel blocker nimodipine inhibited PRL mRNA production 2-fold. These results demonstrate that racemic Bay K 8644 interacts with L-type Ca2+ channels in normal and transformed pituitary cells as a mixed agonist-antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Bay K 8644 on renal function and renin secretion in anesthetized rats

Our previous study on kidney cortical slices showed that Bay K 8644, a dihydropyridine calcium channel agonist, produced a dose-dependent inhibitory action on the release of renin. The present study was performed to examine the effect of Bay K 8644 on renal function and renin secretion in vivo. When Bay K 8644 was directly infused into the renal artery of anesthetized rats, 2 micrograms/kg/min had no effect on renal blood flow (RBF) and glomerular filtration rate (GFR), but decreased urine flow (UF), urinary sodium excretion (UNaV) and fractional sodium excretion (FENa) by about 30%, 55% and 35%, respectively, thereby suggesting that Bay K 8644 enhanced the tubular reabsorption of water and sodium. When 10 micrograms/kg/min were infused, RBF, GFR, UF, UNaV and FENa decreased to about 95%, 70%, 35%, 35% and 30% of each control value. The administration of Bay K 8644 at 10 micrograms/kg/min did not influence the basal levels of plasma renin activity (PRA) and renin secretion rate (RSR), but did inhibit significantly isoproterenol-induced increasing effects on PRA and RSR. These results indicate that the activation of voltage-dependent calcium channels with Bay K 8644 influences the control of renal function and renin secretion in vivo.     

Ca2+ channel modulation and kinase-C activation in a pituitary cell line: induction of immediate early genes and inhibition of proliferation.

We have studied the interaction between dihydropyridine (DHP) Ca2+ modulators and the phorbol ester phorbol 12-myristate 13-acetate (PMA) on whole cell Ca2+ currents, 45Ca2+ uptake, immediate early gene (IEG) expression, and proliferation in the rat pituitary GH4C1 cell line. When short (3- to 5-msec) depolarizing voltage clamp steps were used to activate L-type Ca2+ channels, the DHP Ca2+ agonist (-)Bay K 8644 markedly enhanced Ca2+ entry by slowing channel closing upon repolarization. In contrast, the Ca2+ agonist induced only small and inconsistent increases in c-fos mRNA and did not measurably increase NGFI-A. Ca2+ channel activation by depolarization with 50 mM KCl in the presence of (-)Bay K 8644 induced large increases in 45Ca2+ uptake, but failed to markedly induce either of the IEGs. The phorbol ester PMA did not alter T- or L-type Ca2+ current or 45Ca2+ uptake by GH4C1 cells, but triggered large increases in both c-fos and NGFI-A mRNA. In combination, PMA and (-)Bay K 8644 acted synergistically to increase mRNAs for both IEGs. The effect of the DHPs was stereospecific; (+)Bay K 8644, a Ca2+ antagonist, inhibited PMA-induced increases in c-fos and NGFI-A mRNAs. Both PMA and (-)Bay K 8644 inhibited the proliferation of GH4C1 cells, measured by cell count or [3H]thymidine incorporation. The inhibition by the Ca2+ agonist was stereoselective and approximately additive to that of PMA. These results indicate that the expression of c-fos IEG and that of NGFI-A IEG are differentially regulated by separate second messenger pathways in GH4C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

K+-Evoked Depolarization Stimulates Cyclic AMP Accumulation in Photoreceptor-Enriched Retinal Cell Cultures: Role of Calcium Influx Through Dihydropyridine-Sensitive Calcium Channels

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.     

The effect of dihydropyridine calcium agonists and antagonists on neuronal voltage sensitive calcium channels

The effect of dihydropyridine agonists and antagonists on neuronal voltage sensitive calcium channels was investigated. The resting intracellular calcium concentration of synaptosomes prepared from whole brain was 110 +/- 9 nM, as assayed by the indicator quin 2. Depolarisation of the synaptosomes with K+ produced an immediate increase in [Ca2+]i. The calcium agonist Bay K 8644 and antagonist nifedipine did not affect [Ca2+]i under resting or depolarising conditions. In addition, K+ stimulated 45Ca2+ uptake into synaptosomes prepared from the hippocampus was insensitive to Bay K 8644 and PY 108-068 in normal or Na+ free conditions. In neuronally derived NG108-15 cells the enantiomers of the dihydropyridine derivative 202-791 showed opposite effects in modulating K+ stimulated 45Ca2+ uptake. (-)-R-202-791 inhibited K+ induced 45Ca2+ uptake with an IC50 of 100 nM and (+)-S-202-791 enhanced K+ stimulated uptake with an EC50 of 80 nM. These results suggest that synaptosomal voltage sensitive calcium channels either are of a different type to those found in peripheral tissues and cells of neural origin or that expression of functional effects of dihydropyridines requires different experimental conditions to those used here.     

Dihydropyridine Modulation of Voltage-Activated Calcium Channels in PC12 Cells: Effect of Pertussis Toxin Pretreatment

In this study, we report the effect of pertussis toxin pretreatment on dihydropyridine modulation of voltage-sensitive calcium channels in PC12 cells. The rise in intracellular calcium concentration caused by potassium depolarization is not affected significantly by pertussis toxin pretreatment. Nicardipine, a dihydropyridine derivative, added either before or after potassium-induced depolarization, reduces the resultant elevation in cytosolic calcium level both in control and in pertussis toxin-treated cells. The dihydropyridine agonist Bay K 8644, when added before potassium, is able to enhance the potassium-induced spike of cytosolic calcium levels, an effect significantly reduced by pertussis toxin pretreatment. Moreover, the addition of Bay K 8644 after potassium holds the intracellular calcium concentration at a cytosolic sustained level during the slow inactivating phase of depolarization. This effect of Bay K 8644 is inhibited by nicardipine. Pertussis toxin pretreatment slightly weakens the effect of Bay K 8644 when added after potassium-induced depolarization, whereas it significantly reduces the nicardipine inhibition of cytosolic calcium rise stimulated by potassium and Bay K 8644, but not by potassium alone. In conclusion, our findings suggest that a pertussis toxin-sensitive guanine nucleotide regulatory protein could be involved in the interaction between dihydropyridine derivatives and voltage-dependent calcium channels.     

The endothelium-derived vasoconstrictor peptide endothelin inhibits renin release in vitro

The effect of endothelin, a newly identified endothelium-derived vasoconstrictor peptide, on renin release from rat kidney cortical slices was examined. Endothelin produced a concentration-dependent inhibition of renin release and this inhibitory effect was dependent on extracellular calcium. The dihydropyridine calcium channel blockers nifedipine and nicardipine did not antagonize the inhibitory effect induced by endothelin. On the other hand, nifedipine completely antagonized the extracellular high potassium- or Bay K 8644-induced inhibition of renin release. The endothelin-induced inhibition of the release was markedly blocked by the addition of Co2+. Similar blocking effects of Co2+ were also observed with extracellular high potassium or Bay K 8644. Thus, endothelin exerts an inhibitory action on renin release in vitro, in a calcium-dependent manner. This inhibition may be mediated by the increased calcium influx through dihydropyridine-insensitive calcium channels.     

Bay K 8644 induce enhancement of K+ current in both single heart cell and smooth muscle cell

The effects of the Ca2+ agonist Bay K 8644 on outward potassium currents have been studied in single ventricular cells of chick embryo and aortic single cells of rabbit using the whole-cell patch clamp technique. Bay K 8644 was found to increase 1K in both heart and aortic single cells. This effect of Bay K 8644 on both muscle was reversed by Mn2+ and blocked by 20 mM TEA. The Bay K 8644 potassium I/V curve of single heart cell had a N shape, which is Ca2+ dependent. These data strongly suggest that Bay K 8644 increases a gK(Ca) in both aortic and heart muscle.     

Effects of (+) and (-) enantiomers of calcium channel agonist, Bay K 8644, on mechanical and electrical responses of frog skeletal muscle

The effects of (+) and (-) enantiomers of Bay K 8644, a Ca2+ channel agonist, on the mechanical and electrical properties of frog skeletal muscle fibers were investigated. In the concentration range of 10(-6) to 10(-5) M, both (+) and (-) enantiomers of Bay K 8644 significantly increased the maximum amplitudes of twitch responses. Both (+) and (-) enantiomers of Bay K 8644, at higher concentrations such as 10(-4) M, greatly depressed the amplitudes of twitches. Potentiating and depressing effects of (-) enantiomer of Bay K 8644 on twitch responses were significantly greater than those of the (+) enantiomer. At all concentrations used, both (+) and (-) enantiomers of Bay K 8644 significantly decreased the area under the tetanic force x time curve. In intracellular recordings, it was found that the depressing effects of both (+) and (-)-Bay K 8644 on tetanic contractions and twitch responses were due to the inhibition of action potentials. The inhibitory effect of (-) enantiomer of Bay K 8644 on action potentials also was significantly greater than that of the (+) enantiomer. In conclusion, present results suggest that, in contrast with cardiac muscle fibers, (+) and (-) enantiomers of Bay K 8644 have similar inhibitory effects on the electrical and mechanical properties of frog skeletal muscle fibers.     

Reductions in calcium uptake induced in rat brain synaptosomes by ionizing radiation

Gamma irradiation (60Co) reduced KCl-stimulated voltage-dependent 45Ca2+ uptake in whole-brain, cortical, and striatal synaptosomes. The time course (3, 10, 30, and 60 s) of calcium uptake by irradiated (3 Gy) and nonirradiated synaptosomes, as well as the effect of KCl (15-65 mM), was measured in whole-brain synaptosomes. The fastest and highest rate of depolarization-dependent calcium uptake occurred at 3 s with 65 mM KCl. Irradiation reduced calcium uptake at all incubation times and KCl concentrations. Bay K 8644 enhancement of KCl-stimulated calcium influx was also reduced by radiation exposure. Nimodipine binding to dihydropyridine (DHP) L-type calcium channel receptors was not altered following radiation exposure. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive calcium channels in rat brain synaptosomes that are not mediated by DHP receptors.     

Bay K 8644 induce enhancement of K+ current in both single heart cell and smooth muscle cell

Summary The effects of the Ca²⁺ agonist Bay K 8644 on outward potassium currents have been studied in single ventricular cells of chick embryo and aortic single cells of rabbit using the whole-cell patch clamp technique. Bay K 8644 was found to increase l_K in both heart and aortic single cells. This effect of Bay K 8644 on both muscle was reversed by Mn²⁺ and blocked by 20 mM TEA. The Bay K 8644 potassium I/V curve of single heart cell had a N shape, which is Ca²⁺ dependent. These data strongly suggest that Bay K 8644 increases a g_K(ca) in both aortic and heart muscle.     

Inhibition of Ca2+ and K+ channels in sympathetic neurons by neuropeptides and other ganglionic transmitters.

Neuropeptides are known to modulate the excitability of frog sympathetic neurons by inhibiting the M-current and increasing the leak current, but their effects on Ca2+ channels are poorly understood. We compared effects of LHRH, substance P, epinephrine, and muscarine on Ca2+, K+, and leak currents in dissociated frog sympathetic neurons. At concentrations that inhibit M-current, LHRH and substance P strongly reduced N-type Ca2+ current and induced a leak conductance that may contribute to slow EPSPs. In contrast, muscarine produced little reduction of Ca2+ current, even in cells in which it strongly suppressed the M-current. We find that peptidergic inhibition of Ca2+ channels involves G proteins, but does not require protein kinases. In addition, it leads to reductions in Ca2(+)-activated K+ current and catecholamine release.