The inhibitory effects of m-nisoldipine on the 5-hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells via Ca2+ antagonism and antioxidant mechanisms.

The excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a critical role in the development of pulmonary arterial hypertension. Recent studies indicate that Ca(2+) and reactive oxygen species are critically involved in the process of smooth muscle cell proliferation stimulated by mitogens, such as 5-hydroxytryptamine (5-HT). Because m-nisoldipine, a Ca(2+) channel blocker of the dihydropyridine class, possesses some calcium antagonistic and antioxidant properties, we investigated the effect of m-nisoldipine on PASMC proliferation. The results indicated that m-nisoldipine inhibited 5-HT-induced PASMC proliferation, evaluated by BrdU incorporation and the MTT assay, and this effect was associated with a decreased expression of proliferating cell nuclear antigen (PCNA). Flow cytometry analysis showed that m-nisoldipine blocked 5-HT-induced cell-cycle progression by arresting the cells in the G(0)/G(1) phase. Next, the production of reactive oxygen species and the levels of [Ca(2+)](i) in PASMCs were measured by laser scanning confocal microscopy; m-nisoldipine pretreatment attenuated the [Ca(2+)](i) elevation and the production of reactive oxygen species induced by 5-HT. In addition, m-nisoldipine significantly decreased the 5-HT-induced activation of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) and the subsequent c-fos and c-jun mRNA expression. Meanwhile, results also showed that N-acetylcysteine (a reactive oxygen species scavenger) suppressed the proliferation and the ERK1/2 and JNK activation induced by 5-HT. In summary, this study demonstrated that m-nisoldipine effectively suppressed the 5-HT-induced PASMC proliferation, ERK1/2 and JNK activation and subsequent c-fos and c-jun mRNA expression, all of which might be associated with the Ca(2+) antagonistic and antioxidant properties of m-nisoldipine.

Characterization of the rapidly activating delayed rectifier potassium current, I (Kr), in HL-1 mouse atrial myocytes.

HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.

Effects of three metabolites of propiverine on voltage-dependent L-type calcium currents in human atrial myocytes.

The non-selective muscarinic receptor antagonist propiverine impairs L-type Ca(2+) currents (I(Ca,L)) in human detrusor smooth muscle cells and atrial cardiomyocytes. Here, we have investigated the effects of three metabolites of propiverine on human cardiac I(Ca,L). Propiverine reduced I(Ca)(,L) with a -logIC(50) [M] value of 4.1, M-5 only showed minor effect on I(Ca)(,L) at high concentrations, M-6 did not influence I(Ca)(,L) at all. Like the parent compound M-14 also reduced I(Ca)(,L) (-logIC(50) [M]=4.6). We conclude, that propiverine and M-14 reduce cardiac I(Ca)(,L) at higher concentrations than in detrusor cells and therefore preferentially reduce I(Ca)(,L) in the urinary bladder than in the heart.

m-Nisoldipine attenuates monocrotaline-induced pulmonary hypertension by suppressing 5-HT/ERK MAPK pathway.

Effect of new calcium antagonist m-nisoldipine (m-Nis) on MCT-induced PH in rats and its mechanisms were investigated. Rats were injected with a single dose (60 mg x kg(-1)) of MCT subcutaneously to induce PH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. PCNA, ERK1 and p-ERK expressions were analyzed by Western blotting. The expressions of 5-HT and PCNA were observed with immunohistochemistry. Results suggested that the PAP, right ventricular index and the degree of muscularization of small pulmonary artery were elevated markedly in MCT group, which was attenuated by m-Nis treatment. A significant reduction in MDA production and an increase in the SOD activity in the serum were also observed in all three m-Nis groups. The number of PCNA and 5-HT positive smooth muscle cells increased significantly in MCT group, and m-Nis treatment attenuated the expression obviously. Western blotting results suggested that the protein expression of PCNA and the ratio of p-ERK/ ERK1 increased markedly in MCT group and decreased by m-Nis. In conclusion, m-Nis protected against MCT-induced PH by decreasing PAP, right ventricular index, PAMSCs proliferation and pulmonary artery remodelling, which may be related to the reduction of 5-HT and the suppression of the ERK/MAPK signal pathway.

Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts.

Cardiac voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive CaV1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant CaV1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+ current (ICa) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC50 of 66 nM and complete block at 250 nM. In contrast, ICa in cells infected with AdCaV1.2DHPi was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 microM diltiazem. In order to study CaV1.2DHPi in isolation, myocytes infected with AdCaV1.2DHPi were incubated with nisoldipine. Under this condition the cells expressed a large ICa (12 pA/pF) and displayed Ca2+ transients during field stimulation. Furthermore, addition of 2 microM forskolin and 100 microM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased IBa in the AdCaV1.2DHPi-infected cells. A Cd2+-sensitive IBa was also recorded in cardiac fibroblasts infected with AdCaV1.2DHPi. Thus, expression of CaV1.2DHPi will provide an important tool in studies of cardiac myocyte and fibroblast function.

Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells.

Capacitative Ca2+ entry (CCE) has been speculated to contribute to Ca2+ influx during hypoxic pulmonary vasoconstriction (HPV). The aim of the present study was to directly test if acute hypoxia causes intracellular Ca2+ concentration ([Ca2+]i) rises through CCE in canine pulmonary artery smooth muscle cells (PASMCs). In PASMCs loaded with fura-2, hypoxia produced a transient rise in [Ca2+]i in Ca2+-free solution, indicating Ca2+ release from the intracellular Ca2+ stores. Subsequent addition of 2 mm Ca2+ in hypoxia elicited a sustained rise in [Ca2+]i, which was partially inhibited by 10 microm nisoldipine. The dihydropyridine-insensitive rise in [Ca2+]i was due to increased Ca2+ influx, because it was abolished in Ca2+-free solution and hypoxia was shown to significantly enhance the rate of Mn2+ quench of fura-2 fluorescence. The dihyropyridine-insensitive rise in [Ca2+]i and the increased rate of Mn2+ quench of fura-2 fluorescence were inhibited by 50 microm SKF 96365 and 500 microm Ni2+, but not by 100 microm La3+ or 100 microm Gd3+, exhibiting pharmacological properties characteristic of CCE. In addition, predepletion of the intracellular Ca2+ stores inhibited the rise in [Ca2+]i induced by hypoxia. These results provide the first direct evidence that acute hypoxia, by causing Ca2+ release from the intracellular stores, activates CCE in isolated canine PASMCs, which may contribute to HPV.

Signaling pathways transduced through the elastin receptor facilitate proliferation of arterial smooth muscle cells.

In this report we demonstrate that soluble peptides, elastin degradation products stimulate proliferation of arterial smooth muscle cells. We show that these effects are due to generation of intracellular signals transduced through the cell surface elastin receptor, which consists of peripheral 67-kDa elastin-binding protein (EBP) (spliced variant of beta-galactosidase), immobilized to the transmembrane sialidase and the protective protein. We found that elastin receptor-transduced signaling triggers activation of G proteins, opening of l-type calcium channels, and a sequential activation of tyrosine kinases: FAK, c-Src, platelet-derived growth factor-receptor kinase and then Ras-Raf-MEK1/2-ERK1/2 phosphorylation cascade. This, in turn, causes an increase in expression of cyclins and cyclin-dependent kinases, and a consequent increase in cellular proliferation. The EBP-transduced signals also induce tyrosine kinase-dependent phosphorylation of beta-tubulin, LC3, microtubule-associated protein 1, and alpha-actin and troponin-T, which could be linked to reorganization of cytoskeleton. We have also disclosed that induction of these signals can be abolished by anti-EBP antibody or by galactosugars, which cause shedding of EBP from the cell surface. Moreover, elastin-derived peptides did not induce proliferation of EBP-deficient cells derived from patients bearing a nonsense mutation of the beta-galactosidase gene or sialidase-deficient cells from patients with congenital sialidosis.

Developmental changes of Ca(2+) handling in mouse ventricular cells from early embryo to adulthood.

Transplant of immature cardiomyocytes is recently attracting a great deal of interest as a new experimental strategy for the treatment of failing hearts. Full understanding of normal cardiomyogenesis is essential to make this regenerative therapy feasible. We analyzed the molecular and functional changes of Ca(2+) handling proteins during development of the mouse heart from early embryo at 9.5 days postcoitum (dpc) through adulthood. From the early to the late (18 dpc) embryonic stage, mRNAs estimated by the real time PCR for ryanodine receptor (type 2, RyR2), sarcoplasmic reticulum (SR) Ca(2+) pump (type 2, SERCA2) and phospholamban (PLB) increased by 3-15 fold in the values normalized to GAPDH mRNA, although Na(+)/Ca(2+) exchanger (type 1, NCX1) mRNA was unchanged. After birth, there was a further increase in the mRNAs for RyR2, SERCA2 and PLB by 18-33 fold, but a 50% decrease in NCX1 mRNA. The protein levels of RyR2, SERCA2, PLB and NCX1, which were normalized to total protein, showed qualitatively parallel developmental changes. L-type Ca(2+) channel currents (I(Ca-L)) were increased during the development (1.3-fold at 18 dpc, 2.2-fold at adult stage, vs. 9.5 dpc). At 9.5 dpc, the Ca(2+) transient was, unlike adulthood, unaffected by the SR blockers, ryanodine (5 microM) and thapsigargin (2 microM), and also by a blocker of the Ca(2+) entry via Na(+)/Ca(2+) exchanger, KB-R 7943 (1 microM). The Ca(2+) transient was abolished after application of nisoldipine (5 microM). These results indicate that activator Ca(2+) for contraction in the early embryonic stage depends almost entirely on I(Ca-L).

Protective effects of dihydropyridine Ca-blockers against endothelial cell oxidative injury due to combined nitric oxide and superoxide.

Nitric oxide (NO) and superoxide anions (*O(2)(-) ), which are known to be generated by inflammatory cells under certain pathological conditions, may be cytotoxic to the endothelial cells (ECs) due to peroxynitrite formation. We reported previously that certain lipophilic dihydropyridine (DHP) Ca-blockers exhibit antioxidant activities. In the present study, the extent of antioxidant protection by nisoldipine against combined NO/*O(2)(-) or peroxynitrite-mediated EC injury was assessed and compared with nicardipine, nifedipine and Trolox (water-soluble vitamin E). When confluent bovine aortic ECs were exposed to combined NO/*O(2)(-) (generated from 0.25 mM SIN-1), dramatic loss of cell GSH (53 +/- 8%) occurred in 60 min; cell survival/viability, determined 24 h later by the tetrazolium MTT assay, decreased by 45 +/- 6%. NO alone or O(2)(-) alone were ineffective. Nisoldipine pretreatment (30 min) of the cells concentration dependently (0.3-10 micro M) attenuated the SIN-1-induced GSH loss: the EC(50)value was 4.7 micro M and the corresponding values for nicardipine and nifedipine were 7.8 micro M and >20 micro M, respectively, and that for Trolox was 5.2 micro M. These agents (10 micro M) also protected against the loss of cell viability: nisoldipine, 86 plus minus 8%; nicardipine, 60 +/- 7 %; nifedipine, 35 +/- 5 %, and Trolox, 78 +/- 9%. In addition, significant losses of GSH and viability could be induced by incubation of the EC monolayers with purified peroxynitrite (25 micro M). Attenuation of these peroxynitrite-mediated GSH and viability losses was observed with the following order of efficacy: nisoldipine > or = Trolox > nicardipine >> nifedipine. In a cell-free system containing 0.05 mM GSH, none of the agents (10 micro M) were able to inhibit SIN-1- (0.25 mM) or peroxynitrite- (25 micro M) induced depletion (approximately 50%) of GSH. However, with a purified microsomal membrane system, all four agents inhibited the SIN-1- (or peroxynitrite-) induced lipid peroxidation (TBARS) with the following IC(50)values: nisoldipine, 6.3 micro M; nicardipine, 10.6 micro M; nifedipine, >20 micro M, and Trolox, 6.5 micro M. In conclusion, nisoldipine, a vascular selective DHP calcium channel-blocker, demonstrated the greatest protection against the EC injury induced either by SIN-1 or peroxynitrite. The protective mechanism against the cytotoxicity is most likely through a lipophilic 'chain-breaking' antiperoxidative action against the 'OH-like species' released from peroxynitrite or SIN-1.

Effects of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle.

In the present study, effects of different types of K+ channel modulators on the spontaneous rhythmic contractile activity were examined in guinea-pig urinary bladder smooth muscle (UBSM). Guinea-pig UBSM exhibited myogenic rhythmic contraction in the presence of atropine (1 microM), phentolamine (1 microM), propranolol (1 microM), suramin (10 microM) and tetrodotoxin (1 microM). Nisoldipine (100 nM) or diltiazem (10 microM) substantially diminished UBSM contractile activity. Nisoldipine-resistant component of UBSM rhythmic contraction was further inhibited by gadolinium (200 microM). Iberiotoxin (50 nM), a selective blocker of large-conductance, voltage-gated Ca2+-activated K+ (K(Ca)) (BK) channel, dramatically increased both contraction amplitude and frequency whereas NS-1619 (30 microM), which increases BK channel activity, decreased them. Apamin (100 nM), a selective blocker of small-conductance, K(Ca) (SK) channel, increased contraction amplitude but decreased frequency. A blocker of voltage-gated K+ (Kv) channel, 4-aminopyridine (100 microM), significantly increased contraction frequency. E-4031, a blocker of a novel inwardly rectifying K+ channel, i.e. the human ether-a-go-go-related gene (HERG) K+ channel, significantly increased contraction amplitude. Glibenclamide (1-10 microM) (K(ATP) channel blocker) and Ba2+ (10 microM) (conventional K(ir) channel blocker) did not exhibit conspicuous effects on spontaneous contractile activity of UBSM. These findings imply that two types of K(Ca) (BK and SK) channels have prominent roles as negative feedback elements to limit extracellular Ca2+ influx-mediated guinea-pig UBSM contraction by regulating both amplitude and frequency. It was also suggested that both non-K(Ca) type of K+ (Kv and HERG-like K+) channels may contribute to the regulation of UBSM myogenic rhythmic contraction.

Membrane depolarization, elevated Ca(2+) entry, and gene expression in cerebral arteries of hypertensive rats.

Elevated intracellular Ca(2+) ([Ca(2+)](i)) has been implicated in contractile and phenotypic changes in arterial smooth muscle during hypertension. This study examined the role of membrane potential and [Ca(2+)](i) in altered gene expression in cerebral arteries of a rat (Dahl) genetic model of salt-sensitive hypertension. Cerebral arteries from hypertensive animals (Dahl salt-sensitive) exhibited a tonic membrane depolarization of approximately 15 mV compared with normotensive (Dahl salt-resistant) animals. Consistent with this membrane depolarization, voltage-dependent K(+) currents were decreased in cerebral artery myocytes isolated from hypertensive animals. Arterial wall Ca(2+) was elevated in cerebral arteries from hypertensive animals, an effect reversed by diltiazem, a blocker of voltage-dependent Ca(2+) channels. This depolarization-induced increase in [Ca(2+)](i) was associated with increased activation of the transcription factor, cAMP response element binding protein, and increased expression of the immediate early gene c-fos, both of which are reversed by acute exposure to the voltage-dependent Ca(2+) channel blocker nisoldipine. This study provides the first information linking altered Ca(2+) handling to changes in gene expression in cerebral arteries during hypertension.

Decrease in the Ca2+-activated K+ current of pulmonary arterial smooth muscle in pulmonary hypertension rats.

Pulmonary hypertension exhibits acute elevation of vascular tone and hyperreactivity of pulmonary vasculature, which are closely related to patient mortality. In the present study, we investigated the characteristics of membrane currents of isolated pulmonary artery smooth muscle cells taken from rats with monocrotaline-induced pulmonary hypertension. Male Wistar rats were given a single subcutaneous injection of monocrotaline or saline, and then sacrificed between 18 to 21 days after the injection. The membrane currents in the smooth muscle cells from both groups of rats were compared using the whole-cell patch clamp technique. With 0.1 mM EGTA in the pipette, the densities of outward currents in monocrotaline-injected rats were smaller than those in control rats. When EGTA in patch pipettes was increased to 10 mM, the densities of the outward currents in monocrotaline-injected rats were equal to those of control rats. The Ca2+-activated K+ channel blockers (TEA, iberiotoxin) and nisoldipine were less effective on the outward currents of monocrotaline-injected rats. In the current clamp mode, a depolarization of membrane potential induced by 4-aminopyridine was greater in monocrotaline-injected rats than in control rats because of the reduced activity of the Ca2+-activated K+ channels. The Ca2+-activated K+ channels were decreased in pulmonary hypertension. The reduced activity of the currents may be related to the vascular hyperreactivity in pulmonary hypertension.

Selectivity of blocking of low- versus high-voltage activated calcium currents by the dihydropyridine derivatives Bay E5759 and Bay A4339 in neuroblastoma--glioma NG 108-15 cells.

Beneficial therapeutic effects of dihydropyridine derivatives in cardiovascular and neurological disorders are often associated with selective L-type Ca(2+)channel blockade. Here the new dihydropyridine derivatives Bay E5759 (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid ethyl-1-methylethyl ester) and Bay A4339 (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl-ester) were tested for their potency and selectivity of blocking of Ba(2+)currents mediated by low-(LVACC)vs high-voltage activated Ca(2+)channels (HVACC) in neuroblastoma-glioma hybrid cells. Nisoldipine and mibefradil served as reference compounds. Bay E5759 and Bay A4339 blocked HVACC at low nanomolar concentrations, whereas LVACC was hardly reduced at up to 10 microM. The order of potency for blockade of HVACC was Bay E5759 (IC(50): 0.4 nM) > Bay A4339 (2.5 nM) approximately = nisoldipine (4 nM) >> mibefradil (3.8 microM). Thus Bay E5759 and Bay A4339 are highly potent and selective blockers of HVACC, presumably L-type Ca(2+)channels.

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Ca(2+) channels.

Limited information is available regarding the effects of protein kinase C (PKC) isozyme(s) in the regulation of L-type Ca(2+) channels due to lack of isozyme-selective modulators. To dissect the role of individual PKC isozymes in the regulation of cardiac Ca(2+) channels, we used the recently developed novel peptide activator of the epsilonPKC, epsilonV1-7, to assess the role of epsilonPKC in the modulation of L-type Ca(2+) current (I(Ca,L)). Whole cell I(Ca,L) was recorded using patch-clamp technique from rat ventricular myocytes. Intracellular application of epsilonV1-7 (0.1 microM) resulted in a significant inhibition of I(Ca,L) by 27.9 +/- 2.2% (P < 0.01, n = 8) in a voltage-independent manner. The inhibitory effect of epsilonV1-7 on I(Ca,L) was completely prevented by the peptide inhibitor of epsilonPKC, epsilonV1-2 [5.2 +/- 1.7%, not significant (NS), n = 5] but not by the peptide inhibitors of cPKC, alphaC2-4 (31.3 +/- 2.9%, P < 0.01, n = 6) or betaC2-2 plus betaC2-4 (26.1 +/- 2.9%, P < 0.01, n = 5). In addition, the use of a general inhibitor (GF-109203X, 10 microM) of the catalytic activity of PKC also prevented the inhibitory effect of epsilonV1-7 on I(Ca,L) (7.5 +/- 2.1%, NS, n = 6). In conclusion, we show that selective activation of epsilonPKC inhibits the L-type Ca channel in the heart.

Delayed rectifier K currents have reduced amplitudes and altered kinetics in myocytes from infarcted canine ventricle.

OBJECTIVE: The rapid (I(Kr)) and slow (I(Ks)) components of delayed rectifier currents play an important role in determining the cardiac action potential configuration. Abnormalities in their function may contribute to arrhythmogenesis under pathological conditions. We studied the effects of myocardial infarction on I(Kr) and I(Ks) in canine ventricular myocytes and their molecular basis. METHODS: Infarct zone myocytes (IZs) were isolated from a thin layer of surviving epicardium overlying an infarct 5 days after a total occlusion of the left anterior descending (LAD) coronary artery. Normal myocytes (NZs) were isolated from the corresponding region of control hearts for comparison. Currents were recorded under the whole-cell patch clamp conditions. RESULTS: Both I(Kr) and I(Ks) current densities were reduced in IZs versus NZs. Kinetic analysis further suggests an acceleration of I(Kr) activation and I(Ks) deactivation. RNase protection assays were used to quantify the mRNA levels of I(Kr) and I(Ks) channel subunits (dERG, dIsK and dKvLQT1) in tissue immediately adjacent to the region where myocytes were isolated. mRNA levels of all three subunits were reduced 2 days after LAD occlusion (by 48+/-9%, 68+/-5%, and 45+/-4% for dERG, dIsK and dKvLQT1, respectively, n=8 each). By day 5, the dKvLQT1 message returned to control while those of dERG and dIsK remained reduced (by 52+/-7% and 76+/-6%, respectively). CONCLUSIONS: The decrease in I(Kr) and I(Ks) amplitudes and changes in their kinetics in infarcted tissue might be due to a decrease in functional channels and/or changes in their subunit composition. Heterogeneous changes in I(Kr) and I(Ks) in infarcted hearts may impact on the effects of varying heart rate or neurohumoral modulation on repolarization.

Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway.

Previous studies have suggested that voltage-gated Ca(2+) influx in glomus cells plays a critical role in sensory transduction at the carotid body chemoreceptors. The purpose of the present study was to determine the effects of hypoxia on the Ca(2+) current in glomus cells and to elucidate the underlying mechanism(s). Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca(2+) current was monitored using the whole cell configuration of the patch-clamp technique, with Ba(2+) as the charge carrier. Hypoxia (pO(2) = 40 mmHg) augmented the Ca(2+) current by 24 +/- 3% (n = 42, at 0 mV) in a voltage-independent manner. This effect was seen in a CO(2)/HCO(3)(-)-, but not in a HEPES-buffered extracellular solution at pH 7.4 (n = 6). When the pH of a HEPES-buffered extracellular solution was lowered from 7.4 to 7. 0, hypoxia augmented the Ca(2+) current by 20 +/- 5% (n = 4, at 0 mV). Nisoldipine, an L-type Ca(2+) channel blocker (2 microM, n = 6), prevented, whereas, omega-conotoxin MVIIC (2 microM, n = 6), an inhibitor of N and P/Q type Ca(2+) channels, did not prevent augmentation of the Ca(2+) current by hypoxia, implying that low oxygen affects L-type Ca(2+) channels in glomus cells. Protein kinase C (PKC) inhibitors, staurosporine (100 nM, n = 6) and bisindolylmaleimide (2 microM, n = 8, at 0 mV), prevented, whereas, a protein kinase A inhibitor (4 nM PKAi, n = 10) did not prevent the hypoxia-induced increase of the Ca(2+) current. Phorbol 12-myristate 13-acetate (PMA, 100 nM), a PKC activator, augmented the Ca(2+) current by 20 +/- 3% (n = 8, at 0 mV). In glomus cells treated with PMA overnight (100 nM), hypoxia did not augment the Ca(2+) current (-3 + 4%, n = 5, at 0 mV). Immunocytochemical analysis revealed PKCdelta-like immunoreactivity in the cytosol of the glomus cells. Following hypoxia (6% O(2) for 5 min), PKCdelta-like immunoreactivity translocated to the plasma membrane in 87 +/- 3% of the cells, indicating PKC activation. These results demonstrate that hypoxia augments Ca(2+) current through L-type Ca(2+) channels via a PKC-sensitive mechanism.

Cannabinoid CB1 receptor-mediated inhibition of prolactin release and signaling mechanisms in GH4C1 cells.

The GH4C1 cell line was used to study the cellular mechanisms of cannabinoid-mediated inhibition of PRL release. Cannabinoid CB1 receptor activation inhibited vasoactive intestinal polypeptide- and TRH-stimulated PRL release, but not its basal secretion. The cannabinoid-mediated inhibition of TRH-stimulated PRL release was reversed by the CB1 receptor-specific antagonist, SR141,716A, and was abolished by pertussis toxin pretreatment, indicating that G alpha subunits belonging to the G(i)alpha and G(o)alpha family were involved in the signaling. Photoaffinity labeling using [alpha-32P] azidoaniline GTP showed that cannabinoid receptor stimulation in cell membranes produced activation of four G alpha subunits (G(i)alpha2, G(i)alpha3, G(o)alpha1, and G(o)alpha2), which was also reversed by SR141,716A. The CB1 receptor agonists, WIN55,212-2 and CP55,940, inhibited cAMP formation and calcium currents in GH4C1 cells. The subtypes of calcium currents inhibited by WIN55,212-2 were characterized using holding potential sensitivity and calcium channel blockers. WIN55,212-2 inhibited the omega-conotoxin GVIA (Conus geographus)- and omega-agatoxin IVA (Aigelenopsis aperta)-sensitive calcium currents, but not the nisoldipine-sensitive calcium currents, suggesting the inhibition of N- and P-type, but not L-type, calcium currents. Taken together, the present findings indicate that CB1 receptors can couple through pertussis toxin-sensitive G alpha subunits to inhibit adenylyl cyclase and calcium currents and suppress PRL release from GH4C1 cells.

Coupling of Ca(2+) to CREB activation and gene expression in intact cerebral arteries from mouse : roles of ryanodine receptors and voltage-dependent Ca(2+) channels.

Pathological changes of the vasculature are characterized by changes in Ca(2+) handling and alterations in gene expression. In neurons and other cell types, [Ca(2+)](i) often drives changes in gene expression. However, the relationship between Ca(2+) signaling and gene expression in vascular smooth muscle is not well understood. This study examines the ability of Ca(2+) influx through voltage-dependent, L-type Ca(2+) channels (VDCCs) and Ca(2+) release through ryanodine receptors (RyRs) to activate the transcription factor, cAMP-responsive element binding protein (CREB), and increase c-fos levels in intact cerebral arteries. Membrane depolarization increased the fraction of nuclei staining for phosphorylated CREB (P-CREB) and levels of c-fos mRNA in intact mouse cerebral arteries. Ryanodine, which inhibits RyRs, increased P-CREB staining and c-fos levels. Forskolin, an activator of adenylyl cyclase, and sodium nitroprusside, an NO donor, increased P-CREB and c-fos levels. Nisoldipine, an inhibitor of VDCCs, reversed the effects of depolarization and ryanodine on P-CREB and c-fos levels, but not the effects of forskolin or sodium nitroprusside. Inhibition of Ca(2+)/calmodulin-dependent protein kinase (CaM kinase) blocked increases in P-CREB and c-fos levels seen with membrane depolarization, suggesting that CaM kinase has an important role in the pathway leading from Ca(2+) influx to CREB-mediated changes in c-fos levels. Our data suggest that membrane depolarization increases [Ca(2+)](i) through activation of VDCCs, leading to increased P-CREB and c-fos, and that RyRs have a profound effect on this pathway by indirectly regulating Ca(2+) entry through VDCCs. These results provide the first evidence of Ca(2+) regulation of CREB and c-fos in arterial smooth muscle.

Differential remodeling of the left and right heart after norepinephrine treatment in rats: studies on cytokines and collagen.

Continuous intravenous infusion of norepinephrine norepinephrine (NE, 0.1 mg/kg/h) induced hypertrophy of the left ventricle (LV), but not of the right ventricle (RV) in rats, although RV systolic pressure (RVSP) was much more elevated than LVSP. After NE infusion, there was a time-dependent (20 min to 72 h) expression in the mRNA of interleukin (IL)-6 and IL-1 beta. The expression of IL-6 increased markedly and reached the maximum after 4 h with an 80-fold elevation. In the RV, the expression increased only 20-fold. The mRNA of IL-1 beta increased significantly after NE stimulation only in the LV and reached the maximum after 12 h with a 12-fold elevation. After 12 h of NE infusion, colligin mRNA was elevated for the first time with further progression until 72 h. The six-fold abundance of colligin mRNA seen after 72 h was significantly higher in the LV than in the RV. A similar increase was observed on the protein level (Western blotting). The expression of collagen I and III increased significantly after 24 h only in the LV. After 72 h, the mRNA expression of collagen I was increased 16-fold and that of collagen III 10-fold. This expression was significantly higher than that in the RV. Also the elevation in atrial natriuretic peptide (ANP) mRNA started earlier and was more pronounced in the LV than in the RV. The alpha- and beta-adrenergic receptor blocker carvedilol normalized all functional parameters after 6 h and 72 h and prevented the development of LV hypertrophy that occurred after 72 h. The NE-induced increased expression of the mRNAs studied was either prevented (IL-6, IL-1 beta ) or attenuated (colligin, collagen I and III, ANP) by combined alpha- and beta-receptor blockade. The elevation of afterload which was associated with the NE effect was normalized by the calcium-channel blocker nisoldipin, but NE-induced LV hypertrophy and the increase in ANP and collagen mRNA were not affected.

Calcium channel activation facilitated by nitric oxide in retinal ganglion cells.

We investigated the modulation of voltage-gated Ca channels by nitric oxide (NO) in isolated salamander retinal ganglion cells with the goals of determining the type of Ca channel affected and the signaling pathway by which modulation might occur. The NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 1 mM) and S-nitroso-cysteine (1 mM) induced modest increases in the amplitude of Ca channel currents recorded with ruptured- and permeabilized-patch techniques by causing a subpopulation of the Ca channels to activate at more negative potentials. The Ca channel antagonists omega-conotoxin GVIA and nisoldipine each reduced the Ca channel current partially, but only omega-conotoxin GVIA blocked the enhancement by SNAP. The SNAP-induced increase was blocked by oxadiazolo-quinoxaline (50 microM), suggesting that the NO generated by SNAP acts via a soluble guanylyl cyclase to raise levels of cGMP. The membrane-permeant cGMP analog 8-(4-chlorophenylthio) guanosine cyclic monophosphate also enhanced Ca channel currents and 8-bromo guanosine cyclic monophosphate (1 mM) occluded enhancement by SNAP. Consistent with these results, isobutyl-methyl-xanthine (IBMX, 10 microM), which can raise cGMP levels by inhibiting phosphodiesterase activity, increased Ca channel current by the same amount as SNAP and occluded subsequent enhancement by SNAP. Neither IBMX, the cGMP analogs, nor SNAP itself, led to activation of cGMP-gated channels. N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (2 microM), a broad spectrum inhibitor of protein kinase activity, KT5823 (1 microM), a specific protein kinase G (PKG) inhibitor, and a peptide inhibitor of PKG (200 microM) blocked SNAP enhancement, as did 5'-adenylylimidophosphate (1.5 mM), a nonhydrolyzable ATP analog that prevents protein phosphorylation. A peptide inhibitor of protein kinase A (10 nM) did not block the facilitory effects of SNAP. Okadaic acid (1 microM), a phosphatase inhibitor, had no effect by itself but increased the enhancement of Ca channel current by SNAP. These results suggest that NO modulates retinal ganglion cell N-type Ca channels by facilitating their voltage-dependent activation via a mechanism involving guanylyl cyclase/PKG-dependent phosphorylation. This effect could fine-tune neural integration in ganglion cells or play a role in ganglion cell disease by modulating intracellular calcium signaling.