Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture

The effect of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), a neuroprotective compound, on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCl (25 mM)-stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and omega-agatoxin IVA (omega-Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS-7 blocked KCl-stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS-7 on L-type Ca2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (+/-)-Bay K 8644, whereas NS-7 inhibited the maximal response without affecting the responses induced by low concentrations of (+/-)-Bay K 8644. The effects of NS-7 on L-type and P/Q-type Ca2+ channels involving KCl-stimulated cyclic GMP formation were subsequently examined. NS-7 suppressed the KCl-stimulated cyclic GMP formation measured in the presence of omega-Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS-7 had no influence on ionomycin-induced enhancement of cyclic GMP formation. Finally, NS-7 reversed KCl-induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS-7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L-type and P/Q-type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.     

Structural model of a synthetic Ca2+ channel with bound Ca2+ ions and dihydropyridine ligand.

Grove et al. have demonstrated L-type Ca2+ channel activity of a synthetic channel peptide (SCP) composed of four helices (sequence: DPWNVFDFLI10VIGSIIDVIL20SE) tethered by their C-termini to a nanopeptide template. We sought to obtain the optimal conformations of SCP and locate the binding sites for Ca2+ and for the dihydropyridine ligand nifedipine. Eight Ca2+ ions were added to neutralize the 16 acidic residues in the helices. Eight patterns of the salt bridges between Ca2+ ions and pairs of the acidic residues were calculated by the Monte Carlo-with-energy-minimization (MCM) protocol. In the energetically optimal conformation, two Ca2+ ions were bound to Asp-1 residues at the intracellular side of SCP, and six Ca2+ ions were arrayed in two files at the diametrically opposite sides of the pore, implying a Ca2+ relay mechanism. Nine modes of nifedipine binding to SCP were simulated by the MCM calculations. In the energetically optimal mode, the ligand fits snugly in the pore. The complex is stabilized by Ca2+ bound between two Asp-17 residues and hydrophilic groups of the ligand. The latter substitute water molecules adjacent to Ca2+ in the ligand-free pore and thus do not obstruct Ca2+ relay. The ligand-binding site is proximal to a hydrophobic bracelet of Ile-10 residues whose rotation is sterically hindered. In some conformations, the bracelet is narrow enough to block the permeation of the hydrated Ca2+ ions. The bracelet may thus act as a "gate" in SCP. Nifedipine and (R)-Bay K 8644, which act as blockers of the SCP, extend a side-chain hydrophobic moiety toward the Ile-10 residues. This would stabilize the pore-closing conformation of the gate. In contrast, the channel activator (S)-Bay K 8644 exposes a hydrophilic moiety toward the Ile-10 residues, thus destabilizing the pore-closing conformation of the gate.     

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)     

Unequal effects of renin-angiotensin system inhibitors in acute cardiac dysfunction induced by isoproterenol

Several clinical trials have demonstrated that angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor blocker (ARB) are equally effective in the treatment of chronic heart failure. However, this has not been confirmed for acute cardiac dysfunction. We examined whether ACEI or ARB prevents isoproterenol-induced acute left ventricular (LV) dysfunction in dogs. LV dysfunction induced by a large dose of isoproterenol (1 microg.kg(-1).min(-1), 3-h infusion) was compared in dogs treated with ACEI (temocaprilat) or ARB (olmesartan). Atrial pacing induced a constant heart rate and use of adjustable aortic banding provided a nearly constant afterload. LV systolic function (LV dP/dt, fractional shortening, and ejection fraction) and diastolic function (tau and LV end-diastolic pressure) were significantly deteriorated after isoproterenol infusion. The LV dysfunction was almost totally prevented by ARB but was only partially prevented by ACEI. The partial effect of ACEI was complemented by cotreatment with HOE-140, a bradykinin B2 receptor antagonist. At baseline, the response to low doses of isoproterenol was significantly attenuated by ACEI but not by ARB, and the ACEI-induced attenuation was totally abolished by cotreatment with HOE-140. The response to isoproterenol was significantly attenuated after 3 h of excess isoproterenol loading, and it was almost completely preserved by ARB but not by ACEI. In conclusion, acute LV dysfunction and beta-adrenergic desensitization induced by excess isoproterenol administration were almost totally prevented by ARB but only partially prevented by ACEI. These differences were attributable at least in part to bradykinin pathways activated by ACEI administration in acute LV dysfunction.     

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,β-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.     

Somatostatin, misoprostol and galanin inhibit gastrin- and PACAP-stimulated secretion of histamine and pancreastatin from ECL cells by blocking specific Ca2+ channels

The oxyntic mucosa is rich in ECL cells. They secrete histamine and chromogranin A-derived peptides, such as pancreastatin, in response to gastrin and pituitary adenylate cyclase-activating peptide (PACAP). Secretion is initiated by Ca2+ entry. While gastrin stimulates secretion by opening L-type and N-type Ca2+ channels, PACAP stimulates secretion by activating L-type and receptor-operated Ca2+ channels. Somatostatin, galanin and prostaglandin E2 (PGE2) inhibit gastrin- and PACAP-stimulated secretion from the ECL cells. In the present study, somatostatin and the PGE2 congener misoprostol inhibited gastrin- and PACAP-stimulated secretion 100%, while galanin inhibited at most 60-65%. Bay K 8644, a specific activator of L-type Ca2+ channels, stimulated ECL-cell secretion, an effect that was inhibited equally effectively by somatostatin, misoprostol and galanin (75-80% inhibition). Pretreatment with pertussis toxin, that inactivates inhibitory G-proteins, prevented all three agents from inhibiting stimulated secretion (regardless of the stimulus). Pretreatment with nifedipine (10 microM), an L-type Ca2+ channel blocker, reduced PACAP-evoked pancreastatin secretion by 50-60%, gastrin-evoked secretion by approximately 80% and abolished the response to Bay K 8644. The nifedipine-resistant response to PACAP was abolished by somatostatin and misoprostol but not by galanin. Gastrin and PACAP raised the intracellular Ca2+ concentration in a biphasic manner, believed to reflect mobilization of internal Ca2+ followed by Ca2+ entry. Somatostatin and misoprostol blocked Ca2+ entry (and histamine and pancreastatin secretion) but not mobilization of internal Ca2+. The present observations on isolated ECL cells suggest that Ca2+ entry rather than mobilization of internal Ca2+ triggers exocytosis, that gastrin and PACAP activate different (but over-lapping) Ca2+ channels, that somatostatin, misoprostol and galanin interact with inhibitory G-proteins to block Ca2+ entry via L-type Ca2+ channels, and that somatostatin and misoprostol (but not galanin) in addition block N-type and/or receptor-operated Ca2+ channels.     

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。     

Effect of massive sympathetic nervous system activation on coronary blood flow and myocardial energy pool

Our previous work indicates that myocardial ischemia could be the mechanism responsible for the left ventricular (LV) dysfunction that frequently develops after massive sympathetic nervous system (SNS) activation. In this study, coronary blood flow (CBF) and myocardial ATP, creatine phosphate, and lactate concentrations were measured after massively activating the SNS of anesthetized rabbits with an intracisternal injection of veratrine. CBF was measured at time 0 (baseline), and at 2, 10, and 20 min after SNS activation in one group, and at 0, 45, 90, and 150 min in a second group. Myocardial ATP, creatine phosphate, and lactate were measured at 0, 2, 10, 20, 90, and 150 min in separate groups of rabbits. SNS activation caused LV dysfunction in approximately 60% of the rabbits. SNS-related increases in CBF kept pace with the increases in myocardial energy demand as determined from the systolic pressure-heart rate product. The subendocardial-to-subepicardial blood flow ratio did not change significantly. Myocardial creatine phosphate concentration was depressed 2 min after SNS activation and remained depressed for at least 20 min. ATP fell continuously and was significantly lower than baseline by 20 min. Tissue lactate concentration was elevated at this time. By 90 min, the concentrations of all three metabolites had recovered. These results indicate that myocardial high-energy phosphate compounds fall after massive SNS activation, but ischemia does not appear to be the underlying mechanism.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.     

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.     

Angiotensin II inhibits bTREK-1 K+ channels in adrenocortical cells by separate Ca2+- and ATP hydrolysis-dependent mechanisms

Bovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (V(m)) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca2+- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca2+- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca2+-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca2+-dependent pathway was blocked by the AT1 receptor antagonist losartan, or by including guanosine-5'-O-(2-thiodiphosphate) in the pipette solution. The Ca2+-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca2+ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca2+ (5 or 10 microM) to the cytoplasmic membrane surface. The Ca2+ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca2+-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca2+ channels in these cells.     

Calcium channel activation in vascular smooth muscle by BAY K 8644

BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate) and CGP 28 392 (ethyl-4(2-difluoromethoxyphenyl)-1,4,5,7-tetrahydro-2-methyl-5-++ +oxofuro- [3,4-b]pyridine-3-carboxylate) are closely related in structure to nifedipine and other 1,4-dihydropyridine Ca2+ channel antagonists. However, both BAY K 8644 and CGP 28 392 serve as activators of Ca2+ channels. In the rat tail artery, responses to BAY K 8644 are dependent upon Ca2+ext and prior stimulation by K+ or by the alpha-adrenoceptor agonists, phenylephrine and BHT 920 (6-allyl-2-amino-5,6,7,8,-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). Responses are blocked noncompetitively by the Ca2+ channel antagonists D-600 [-)-D-600 greater than (+)-D-600) and diltiazem, but competitively by nifedipine (pA2 = 8.27). This suggests that activator and inhibitor 1,4-dihydropyridines interact at the same site. BAY K 8644 potentiates K+ responses and Ca2+ responses in K+-depolarizing media. The leftward shift of the K+ dose--response curve produced by BAY K 8644 suggests that this ligand facilitates the voltage-dependent activation of the Ca2+ channel. The pA2 value for nifedipine antagonism of BAY K 8644 responses is significantly lower than that for nifedipine antagonism of Ca2+ responses in K+ (25-80 mM) depolarizing media (9.4-9.6), suggesting that the state of the channel may differ according to the activating stimulus.     

Blocking of human T lymphocyte activation by channel antagonists

It has been established that early events in lymphocyte activation involve a rise in intracellular Ca++ as well as changes in the flux of other ions. Although a Ca++ channel has been postulated to participate in the early Ca++ rise, its presence in lymphocytes remains controversial. Also although yet undetected, electrophysiological data suggest the presence of a Ca++ activated K+ channel on human peripheral blood lymphocytes (HPBL). Here we report on the effect of specific channel blockers as an approach to the identification of these channels on HPBL. At 40 nM nifedipine, an inhibitor of voltage-gated Ca++ channels, fully inhibits the PHA-promoted activation of HPBL. This effect is concentration dependent with a half maximum effect at approximately 10 nM and is demonstrable whether the drug is added at the same time as or up to 18 h after the addition of the mitogen. This inhibition of activation is not seen if the lymphocytes are activated using IL-2 instead of PHA. Charybdotoxin a toxin which blocks a Ca++ activated K+ channel of muscle cells also blocks to almost 100 per cent the PHA-induced activation of HPBL. This inhibition can be demonstrated regardless of whether the blocker is added together with or up to 4 h after PHA. As opposed to nifedipine charybdotoxin shows no effect if added 18 h after the initiation of the activation process. When nifedipine and charybdotoxin were tested on mice splenocytes we found that nifedipine fully inhibits the LPS-promoted activation of these cells while charybdotoxin has no effect on their activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vitamin D receptor agonist elocalcitol upregulates L-type calcium channel activity in human and rat bladder

Human bladder contraction mainly depends on Ca2+ influx via L-type voltage-gated Ca2+ channels and on RhoA/Rho kinase contractile signaling, which is upregulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho kinase signaling in rat and human bladder. Since in the normal bladder from Sprague-Dawley rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness and increased sensitivity to the L-type Ca2+ channel antagonist isradipine, we investigated whether elocalcitol upregulated L-type Ca2+ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca2+], which was abrogated by the L-type Ca2+ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca2+ currents (I Ca), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist BAY K 8644. Addition of elocalcitol (10(-7) M) increased L-type I Ca size and specific conductance by inducing faster activation and inactivation kinetics than control and BAY K 8644, while determining a significant negative shift of the activation and inactivation curves, comparable to BAY K 8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10(-8) M, 48 h), which also showed increased mRNA and protein expression of pore-forming L-type alpha(1C)-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30 microg.kg(-1).day(-1), 2 wk). In conclusion, elocalcitol upregulated Ca2+ entry through L-type Ca2+ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.     

Modulation of cardiac Ca2+ channel by Gq-activating neurotransmitters reconstituted in Xenopus oocytes

L-type dihydropyridine-sensitive voltage dependent Ca(2+) channels (L-VDCCs; alpha(1C)) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, was G(q)- and PLC-independent and Ca(2+)-dependent, but not Ca(2+)-mediated, as only basal levels of Ca(2+) were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations.     

Resetting baroreceptors to a lower arterial pressure level by enalapril avoids baroreflex mediated activation of sympathetic nervous system by nifedipine.

Baroreceptor-unloading-mediated activation of sympathetic nervous system (SNS) by antihypertensive agents, such as dihydropyridine calcium channel blockers (CCB), has been considered to compromise the beneficial effects of the therapy and lead to unsatisfying clinical outcome. The present study was aimed at finding a novel way of using CCB without activating SNS. In anaesthetized Wistar rats, baroreceptor-unloading-mediated reflex activation of SNS, as indicated by tachycardia and increase of plasma catecholamines, was observed after mean arterial pressure (MAP) was decreased by 15 mmHg during 4-h administration of nifedipine, a CCB. However an angiotensin-converting enzyme inhibitor (ACEI), enalapril did not cause tachycardia or increase plasma catecholamine levels when it decreased MAP by 15 mmHg. After 100 min (supposedly baroreceptor resetting or adaptation to hypotension had occurred), enalapril infusion was gradually replaced by nifedipine infusion in 40 min. Nifedipine was infused for another 100 min, which kept the lowered MAP unchanged and did not activate SNS. In anaesthetized spontaneously hypertensive rats (SHR), baroreceptor-mediated reflex activation of SNS was observed after MAP was decreased by 25 mmHg during 4-h nifedipine administration. However enalapril did not cause tachycardia or increase plasma catecholamine levels when it decreased MAP by 25 mmHg. After 100 min, enalapril infusion was gradually replaced by nifedipine infusion in 40 min. Nifedipine was then infused for another 100 min, which kept the lowered MAP unchanged and did not activate SNS. The present study indicated that reflex activation of SNS caused by antihypertensive effect of CCB could be avoided if, prior to CCB administration, baroreceptors have been reset to a lower MAP by a drug that does not activate baroreceptor reflex.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

Influence of T-type Ca2+ (mibefradil) and Cl- (indanyloxyacetic acid 94) channel antagonists on alpha1-adrenoceptor mediated contractions in rat aorta

The effects of the T-type and L-type Ca2+ channel antagonists, mibefradil and nifedipine, respectively, and those of a Cl- channel antagonist, indanyloxyacetic acid 94, on mechanical responses elicited by selective activation of alpha1-adrenoceptors using cirazoline were examined in rat isolated aortic rings. The presence of mibefradil (300 nM), indanyloxyacetic acid, 94 (30 microM) and nifedipine (300 nM) alone inhibited mechanical responses elicited by cirazoline. The concentration-response curves to cirazoline were displaced to the right with significant increases in the EC50 and significant depressions of the maximal responses in the presence of the individual agents mibefradil, indanyloxyacetic acid 94, or nifedipine. A combination of mibefradil and indanyloxyacetic acid 94 further inhibited the mechanical activity produced by cirazoline. The further reduction in the maximal response to cirazoline, in the presence of mibefradil and nifedipine, was insignificant when compared with the effects of nifedipine alone. In addition, maximal mechanical responses produced by cirazoline were not significantly affected by a combination of nifedipine and indanyloxyacetic acid 94 when compared with either nifedipine alone or mibefradil and indanyloxyacetic acid 94 combined. Our current findings indicate that mibefradil, indanyloxyacetic acid 94, and nifedipine can inhibit cirazoline-induced contractions to a varying degree. Moreover, based on our present data it would be reasonable to suggest that the contribution of T-type versus L-type Ca2+ channels to contractile responses obtained with cirazoline are approximately 21% and 35%, respectively, of the Emax. It would appear that L-type Ca2+ channels play a greater role in processes that are involved in excitation-contraction coupling subsequent to stimulation of alpha1-adrenoceptors. In addition, Cl- channels also appear to be involved in the process of contraction following alpha1-adrenoceptor activation.