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Intracellular Ca2+ concentrations ([Ca2+]i) are regulated in a spatiotemporal manner via both entry of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Metabotropic glutamate receptor subtype 1 (mGluR1) is a G protein-coupled receptor that stimulates the inositol 1,4,5-trisphosphate-Ca2+ signaling cascade, whereas Cav2.1 is a pore-forming channel protein of P/Q-type voltage-sensitive Ca2+ channels. In this investigation, we showed that mGluR1 and Cav2.1 are colocalized at dendrites of cerebellar Purkinje neurons and form the heteromeric assembly in both the brain and heterologously expressing COS-7 cells. This assembly occurs through the direct interaction between their carboxyl-terminal intracellular domains. Calcium imaging and whole-cell recording showed that mGluR1 inhibits Cav2.1-mediated [Ca2+]i increases and Ba2+ currents in HEK 293 cells expressing Cav2.1 with auxiliary alpha2/delta and beta1 subunits, respectively. This inhibition occurred in a ligand-independent manner and was enhanced by pre-activation of mGluR1 in a ligand-dependent manner. In contrast, simultaneous stimulation of mGluR1 and Cav2.1 induced large [Ca2+]i increases. Furthermore, the temporally regulated inhibition and stimulation of [Ca2+]i increases by mGluR1 and Cav2.1 were observed at dendrites but not soma of cultured Purkinje neurons. These data suggest that the assembly of mGluR1 and Cav2.1 provides the mechanism that ensures spatiotemporal regulation of [Ca2+]i in glutamatergic neurotransmission.

In the present study, we examined the roles of G(12), G(13), G(q), and G(i) in endothelin-1-induced hypertrophic responses. Endothelin-1 stimulation activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) in cultured rat neonatal myocytes. The activation of JNK, but not ERK, was inhibited by the expression of carboxyl terminal regions of G alpha(12) and G alpha(13). JNK activation was also inhibited by expression of the G alpha(12)/G alpha(13)-specific inhibitor regulator of G protein signaling (RGS) domain of p115RhoGEF and the G alpha(q)-specific inhibitor RGS domain of the G protein-coupled receptor kinase 2 (GRK2-RGS). JNK activation was not, however, inhibited by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2 (GRK2-ct), which is a G beta gamma-sequestering polypeptide. Additionally, JNK activation but not ERK activation was inhibited by the expression of C3 exoenzyme that inactivates small GTPase Rho. These results suggest that JNK activation by G alpha(12), G alpha(13), and G alpha(q) is involved in Rho. On the other hand, ERK activation was inhibited by pertussis toxin treatment, the receptor-G(i) uncoupler, and GRK2-ct. Thus, ERK was activated by G alpha(i)- and G beta gamma-dependent pathways. These results clearly demonstrate that differential pathways activate JNK and ERK.

N-type voltage-dependent Ca(2+) channels (VDCCs), predominantly localized in the nervous system, have been proposed to play vital roles in a variety of neuronal functions such as neurotransmitter release at sympathetic nerve terminals. To directly approach the elucidation of the physiological significance of N-type VDCCs in the autonomic nervous system, alpha(1B) subunit (Ca(V) 2.2)-deficient mice were generated, in which peripheral neurons showed a complete and selective elimination of N-type channel currents sensitive to omega-conotoxin GVIA (the peptide toxin from the fish-hunting cone snail Conus geographus), without a significant effect on the activity of other VDCC types. In isolated left atria prepared from N-type-deficient mice, the positive inotropic response mediated by the sympathetic nervous system was dramatically decreased, whereas the negative inotropic response mediated by parasympathetic neurons was nearly intact compared with those of normal mice. The baroreflex response to bilateral carotid occlusion was markedly reduced in the mutant mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct in vivo evidence for an essential role of N-type VDCCs in maintaining the normal function of the sympathetic nervous system in circulatory regulation, demonstrating a potential of N-type VDCC-deficient mice as a useful model for studying disorders attributable to sympathetic nerve dysfunction.

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.

We have previously demonstrated that treatment with l - cis diltiazem reduced cardiac infarct size in vivo. To examine the effect of l - cis diltiazem on Ca 2+ overload induced by ischemia/reperfusion, we used a model for Ca 2+ overload produced by metabolic inhibition in isolated guinea pig myocytes. Intracellular Ca 2+ concentration ([Ca 2+ ] i ) was quantified by fura-2 fluorescence microscopy and Ca 2+ overload was induced by inclusion of 1 μM of carbonyl cyanide m -chrolophenylhydrazone (CCCP) for 40 min treatment followed by washout for 30 min. This treatment caused a large [Ca 2+ ] i elevation as well as a sustained contracture of the cardiomyocytes. The increase was suppressed by 10 μM of 2-[2-[4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea methanesulphonate (KB-R7943), a specific inhibitor of the Na + /Ca 2+ exchanger, but not by nitrendipine (10 μM). l - cis Diltiazem (10 μM) attenuated the [Ca 2+ ] i increase, suggesting that l - cis diltiazem elicits a cardioprotective effect via attenuation of the [Ca 2+ ] i increase induced by metabolic inhibition and energy repletion.

Cardiac natriuretic peptides, atrial and brain natriureticpeptides (ANP and BNP, respectively) are known to haveanti-cardiac hypertrophy effects. ANP and BNP bind totheir common receptor, guanylyl cyclase-A, which subse-quently activates cGMP-protein kinase G (PKG) pathway.Precise molecular mechanisms by which cardiac natriu-retic peptides protect hearts against pathological cardiachypertrophy still remain unclear, however. Transientreceptor potential (TRP) C6, an ion channel responsiblefor the receptor-activated Ca2+ entry, has been shown tobe a positive regulator of calcineurin-NFAT signalingpathway that drives pathologic cardiac remodeling [1]. Inthis study to elucidate the molecular pathways, by whichcardiac natriuretic peptides negatively regulate pro-hyper-trophic signaling, we investigated effects of ANP onTRPC6-calcineurin-NFAT signaling.

Atrial natriuretic peptide (ANP), via its cGMP-forming guanylyl cyclase-A (GC-A) receptor, is critically involved in the regulation of arterial blood pressure and intravascular volume. To elucidate the role of the endothelial effects of ANP, we generated mice with conditional, endothelium (EC)-restricted ablation of the GC-A gene. Our observations in these and control mice demonstrated that ANP, via GC-A, mildly stimulates systemic transendothelial albumin transport in the microvasculature of skeletal muscle and skin. These and other studies indicate that concerted renal diuretic/natriuretic and mild endothelial hyperpermeability actions of ANP are essential to adjust intravascular fluid volume. However, this notion apparently contradicts published in vitro and in vivo studies showing that ANP can strenghen the pulmonary barrier under inflammatory conditions, suggesting that the hormone either acts differently on pulmonary vs systemic endothelium, or it exerts opposite effects on quiescent endothelia (enhanced permeability) and an inflammation-activated endothelium (barrier stabilization). Endothelial hyperpermeability is characteristic of many systemic diseases, such as allergic responses, edema, and sepsis. In particular, histamine markedly and acutely enhances endothelial permeability. Activation of endothelial Gq-coupled H1 receptors activates phospholipase C and elevates intracellular [Ca2+], ultimately leading to actin-myosin contraction and paracellular leakiness. Here we combined studies in microvascular endothelial cells and intravital microscopy of vascular permeability in the m. cremaster microcirculation to study whether ANP counteracts not only pulmonary but also systemic inflammation, i.e. histamine-induced hyperpermeability. Our observations reveal that transient receptor potential canonical (TRPC) 6 channels mediate the inflammatory effects of histamine. Most importantly, they characterize a regulatory pathway by which histamine-induced activation of TRPC6 channels and subsequent calcium-dependent acute endothelial hyperpermeability are prevented by ANP/GC-A-induced, cGMP-dependent protein kinase I (cGKI) - mediated inhibitory phosphorylation of these channels.