Farnesol inhibits L-type Ca2+ channels in vascular smooth muscle cells

Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.     

Voltage-dependent Ca2+ channels in arterial smooth muscle cells

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.     

Effects of isoflurane on receptor-operated Ca2+ channels in rat aortic smooth muscle

OBJECTIVE: Map-guided procedures have been the accepted standard for ventricular tachycardia surgery. However, promising results of visually guided resections without mapping have been reported. The goal of this study was to evaluate the efficacy of large encircling cryoablation without mapping for ventricular tachycardia after anterior myocardial infarction. METHODS: Between 1985 and 1996, this procedure, along with aneurysmectomy, was performed on 38 patients for malignant ventricular tachycardia. The mean interval between the operation and myocardial infarction was 59.2 months; 7 patients (18.4%) were operated on within 1 month of myocardial infarction. The mean patient age was 62.1 +/-7.3 years and the mean left ventricular ejection fraction was 29.0% +/-7.2%. RESULTS: Hospital mortality was 2.6% (1 patient). The electrical success rate based on postoperative electrophysiologic studies was 94.5%. Overall electrical success rate was 89.1%. Freedom from ventricular tachycardia was 77% (95% CI 61%-94%) at both 5 and 7 years. Freedom from sudden cardiac death was 91% (95% CI 80%-100%) at both 5 and 7 years, with overall actuarial survivals at 5 and 7 years of 63% (95% CI 47%-80%) and 42% (95% CI 22%-63%), respectively. The main cause of late death was congestive heart failure in 62.6% of these patients. CONCLUSIONS: One can achieve good results without intraoperative mapping in the treatment of patients with ventricular tachycardia after anterior myocardial infarction by using large encircling cryoablation.     

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas the cytokines alone were without effect. After 48 h, 40% of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70% and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42%. Within a few minutes after addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3-0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

Differential effects of Ca2+ channel blockers on Ca2+ transients and cell cycle progression in vascular smooth muscle cells

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV > Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.     

Inhibitors of spasmogen-induced Ca2+ channel suppression in smooth muscle cells from small intestine

OBJECTIVE: To examine relationships between anti-beta2-glycoprotein (beta2-GPI) antibodies and other antiphospholipid antibody (aPL) tests (aPL ELISA and the lupus anticoagulant or LAC) and the associations of each of these aPL tests with individual clinical manifestations of the antiphospholipid antibody syndrome (APS). METHODS: IgG and IgM anti-beta2-GPI antibodies were determined by ELISA in 281 patients with SLE, primary APS, or other connective tissue diseases. Frequencies, sensitivities, specificities, and predictive values and correlations of anti-beta2-GPI were compared to the aPL ELISA (IgG and IgM) and LAC for individual (and combined) features of APS. RESULTS: Among 139 patients with positive aPL ELISA and/or LAC tests, 57 (41%) had anti-beta2-GPI antibodies (IgG and/or IgM) compared to 11% of patients with SLE negative for these tests (p = 0.00001). In 130 patients with APS, anti-beta2-GPI occurred in 42% and tended to be more specific but less sensitive than the aPL ELISA or LAC. When all 3 aPL tests were combined, the best sensitivities and negative predictive values were achieved; however, specificity and positive predictive values remained low. Anti-beta2-GPI antibodies occurred more frequently in primary APS (58%) vs secondary antiphospholipid syndromes (33%) (p = 0.008, OR = 2.9). Among 79 patients with SLE negative by both aPL ELISA and LAC, 9 (11 %) were positive for anti-beta2-GPI, 7 of whom had clinical features consistent with APS (representing 5% of all with APS). Stepwise multiple logistic regression analysis revealed beta2-GPI to be most strongly associated with neurological syndromes other than stroke, deep venous thrombosis, and recurrent fetal loss, while LAC was most strongly correlated with stroke and thrombocytopenia. IgM aPL antibodies also were independently associated with neurological syndromes and recurrent fetal loss. CONCLUSION: Testing for beta2-GPI antibodies may be clinically useful in the diagnosis of APS but cannot supplant other aPL ELISA or LAC. Multivariate analyses suggest that anti-beta2-GPI antibodies may play a more central role in certain clinical manifestations of APS than antibodies detected by the aPL ELISA or LAC.     

Dissociation of intracellular Ca2+ release and Ca2+ entry response to 5-hydroxytryptamine in cultured canine tracheal smooth muscle cells

The relationship between the agonist-sensitive Ca2+ pool and those discharged by the Ca2+ -ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca2+ ([Ca2+]i), followed by a sustained plateau phase that was dependent on extracellular Ca2+. In such cells, TG produced a concentration-dependent increase in [Ca2+]i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca2+. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca2+]i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca2+ -ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca2+ -influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca2+ stores is sufficient for activation of Ca2+ influx. Some characteristics of the Ca2+ -influx activated by depletion of internal Ca2+ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca2+ influx was inhibited by La3+, membrane depolarisation, and the novel Ca2+ -influx blocker 1-?beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl?-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca2+ influx by TG also was blocked by La3+, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca2+ stores in TSMCs is sufficient for activation of Ca2+ influx, and (2) the agonist-activated Ca2+ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool are indistinguishable.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca2+-activated K+ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 microM) had no effect in the absence of intracellular adenosine 5'-triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 microM) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5'-[beta gamma-methylene]-triphosphate (AMP-PCP). NPY inhibited Ca2+-activated K+ channel activity when ATP was replaced by adenosine 5'-O-(3-thiotriphosphate) (ATP [gamma-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 microM), a specific inhibitor of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 microM) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca2+-activated K+ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.     

Capacitative Ca2+ entry and the regulation of smooth muscle tone

In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.     

Intracellular alkalinization by NH4Cl increases cytosolic Ca2+ level and tension in the rat aortic smooth muscle

Intracellular pH (pHi) is elucidated to be an important regulator of various cell functions, but the role of pHi in smooth muscle contraction remains to be clarified. The purpose of the present study is to examine the effects of cell alkalinization by exposure to NH4Cl on cytosolic Ca2+ level ([Ca2+]i) and on muscle tone. We attempted simultaneous measurements of both [Ca2+]i and contractile force in rat isolated thoracic aorta from which the endothelium was removed. NH4Cl (10-80 mM) increased both [Ca2+]i and muscle tone in the presence of external Ca2+. These responses were reproducible. The removal of Ca2+ from the nutrient solution partially inhibited the rise in [Ca2+]i and the smooth muscle contraction induced by NH4Cl. In addition, the Ca2+ channel blocker verapamil also partially attenuated the responses to NH4Cl. The NH4Cl-induced responses were gradually reduced as NH4Cl was repeatedly added in a Ca(2+)-free solution. Norepinephrine (NE, 1 microM) induced a transient increase in [Ca2+]i and sustained contraction in the absence of external Ca2+, and the subsequent application of NE had little effect on [Ca2+]i. After internal Ca2+ stores were depleted by exposure to NE, the subsequent application of NH4Cl induced increases in [Ca2+]i and tension of the aorta in a Ca(2+)-free solution. These results suggest that NH4Cl mainly evokes Ca2+ release from the internal Ca2+ stores that are not linked with adrenergic alpha-receptor and causes Ca2+ influx through voltage-dependent Ca2+ channels in the vascular smooth muscle.     

Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy. 2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA : 70% ETB), with a density of approximately 3400+/-280 ETA and 8000+/-610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. 3. Challenge of rat airway smooth muscle cells in culture with endothelin- 1 elicited a concentration-dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA-mediated [Ca2+]i increase was due primarily to the mobilization of IP3-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. 4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100+/-800 receptors cell(-1) (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca2+ from intracellular stores and a strong rise in [Ca2+]i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ETB receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ETB receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca2+]i, which appeared to be attributable to the influx of extracellular Ca2+. In contrast, the populations of ET receptors and their linkage to [Ca2+]i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.     

Localization of 17-kDa myosin light chain isoforms in cultured aortic smooth muscle cells

Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.     

Muscarinic cation current and suppression of Ca2+ current in guinea pig ileal smooth muscle cells

Cationic current (Icat) and inhibition of the voltage-dependent Ca2+ current (ICa) evoked by muscarinic receptor activation with carbachol were studied using whole-cell patch clamp technique in smooth muscle cells isolated from longitudinal muscle of guinea pig small intestine. With low buffering of [Ca2+]i (0.1 mM BAPTA [1,2-bis-(2-aminophenoxy)-ethane-N,N, N', N'-tetraacetic acid] in pipette solution) Icat and ICa inhibitory responses had a rapid onset to an initial peak followed by a sustained phase. The sustained phase of ICa suppression was bigger than in the case when [Ca2+]i was clamped to 100 nM, but decreased with repeated stimulation. Upon repeated stimulation with 50 microM carbachol in cells where [Ca2+]i was clamped to 100 nM and when GTP was absent, Icat amplitude decreased strongly and more substantially compared to ICa inhibition, but both responses declined only slightly when 1 mM GTP was present in the pipette solution. GDP-betaS (1 or 5 mM) in pipette solution or pre-treatment of cells with pertussis toxin (6 microg/ml, for 4 h or longer) blocked Icat more than ICa suppression by carbachol, whereas L-NAME (N-omega-nitro-L-arginine methyl ester hydrochloride) (100 microM in pipette solution) affected neither of them significantly. We conclude that the cationic current and the suppression of the voltage-dependent Ca2+ current evoked by muscarinic receptor activation are mediated by pertussis toxin-sensitive G-protein(s) but the latter response was less sensitive to blockade by GDP-betaS and to GTP deficiency in the cell.     

Effect of KB-2796, a new diphenylpiperazine Ca2+ antagonist, on voltage-dependent Ca2+ currents and oxidative metabolism in dissociated mammalian CNS neurons

The effects of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4- trimethoxybenzyl)piperazine-2HCl, on the low- and high-voltage activated Ca2+ currents (LVA and HVA ICa, respectively) and on oxidative metabolism were studied in neurons freshly dissociated from rat brain. KB-2796 reduced the peak amplitude of LVA ICa in a concentration-dependent manner with a threshold concentration of 10(-7) M when the LVA ICa was elicited every 30 s in the external solution with 10 mM Ca2+. The concentration for half-maximum inhibition (IC50) was 1.9 x 10(-6) M. At 10(-5) M or more of KB-2796, a complete suppression of the LVA ICa was observed in the majority of neurons tested. There was no apparent effect on the current-voltage (I-V) relationship and the current kinetics. KB-2796 delayed the reactivation and enhanced the inactivation of the Ca2+ channel for LVA ICa voltage- and time-dependently, suggesting that KB-2796 preferentially binds to the inactivated Ca2+ channel. KB-2796 at a concentration of 3.0 x 10(-6) M also decreased the peak amplitude of the HVA ICa without shifting the I-V relationship. In addition, KB-2796 reduced the oxidative metabolism (the formation of reactive oxygen species) of the neuron in a concentration-dependent manner with a threshold concentration of 3 x 10(-6) M. It is suggested that the inhibitory action of KB-2796 on the neuronal Ca2+ influx and the oxidative metabolism, in combination with a cerebral vasodilatory action, may reduce ischemic brain damage.     

Inhibitory effects of diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells

BACKGROUND: Benzodiazepines have a direct bronchodilator action in airway smooth muscle, but the mechanisms by which these agents produce muscle relaxation are not fully understood. The current study was performed to identify the effects of the benzodiazepines diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells. METHODS: Whole-cell patch-clamp recording techniques were used to evaluate the effects of the benzodiazepines diazepam (10(-8) to 10(-3) M) and midazolam (10(-8) to 10(-3) M) on inward Ca2+ and outward K+ channel currents in dispersed canine tracheal smooth muscle cells. The effects of the antagonists flumazenil (10(-5) M) and PK11195 (10(-5) M) on these channels were also studied. RESULTS: Each benzodiazepine tested significantly inhibited Ca2+ currents in a dose-dependent manner, with 10(-6) M diazepam and 10(-5) M midazolam each causing approximately 50% depression of peak voltage-dependent Ca2+ currents. Both benzodiazepines promoted the inactivated state of the channel at more-negative potentials. The Ca2+-activated and voltage-dependent K+ currents were inhibited by diazepam and midazolam (> 10(-5) M and > 10(-4) M, respectively). Flumazenil and PK11195 had no effect on these channel currents or on the inhibitory effects of the benzodiazepines. CONCLUSIONS: Diazepam and midazolam had inhibitory effects on voltage-dependent Ca2+ channels, which lead to muscle relaxation. However, high concentrations of these agents were necessary to inhibit the K+ channels. The lack of antagonized effects of their antagonists is related to the non-gamma-aminobutyric acid-mediated electrophysiologic effects of benzodiazepines on airway smooth muscle contractility.     

Dihydropyridine Ca2+ channel antagonists inhibit the salvage pathway for DNA synthesis in human vascular smooth muscle cells

Using electroencephalographic methods, rats learning or not learning a two-way active avoidance task were found to differ significantly in the structure of sleep determined the day before training. The main differences concerned (i) synchronized sleep episodes followed by wakefulness, which were longer and fewer in learning rats; (ii) paradoxical sleep episodes, which were longer in learning rats. Significant correlations were present between the number and/or the average duration of synchronized sleep episodes followed by wakefulness or by paradoxical sleep and the number of avoidances or escapes scored in the training session. Power spectral analysis indicated that the relative output in the 6-7-Hz region was higher in learning rats, notably during short episodes of synchronized sleep followed by paradoxical sleep. As two-way active avoidance training induces comparable modifications in postacquisition sleep (Ambrosini et al., Physiol. Behav., 51, 217-226, 1992), the features of preacquisition sleep which prevail in learning rats might directly determine their capacity to learn. Alternatively, they might reflect the existence of a genetic determinant independently conditioning the ability to learn.