Wall stretch and thromboxane A2 activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation.

Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 µM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli.

Modulation of L-type Ca²âº channel activity by neuronal nitric oxide synthase and myofilament Ca²âº sensitivity in cardiac myocytes from hypertensive rat.

Neuronal nitric oxide synthase (nNOS) is important in cardiac protection in diseased heart. Recently, we have reported that nNOS is associated with myofilament Ca(2+) desensitization in cardiac myocytes from hypertensive rats. So far, the effect of myofilament Ca(2+) desensitization or nNOS on L-type Ca(2+) channel activity (I(Ca)) in cardiac myocyte is unclear. Here, we examined nNOS regulation of I(Ca) in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Our results showed that basal I(Ca) was not different between sham and hypertension (from -60 to +40 mV, 0.1 Hz). S-methyl-L-thiocitrulline (SMTC), a selective nNOS inhibitor, increased peak I(Ca) similarly in both groups. However, chelation of intracellular Ca(2+) [Ca(2+)]i with BAPTA increased I(Ca) and abolished SMTC-augmentation of I(Ca) only in hypertension. Myofilament Ca(2+) desensitization with butanedione monoxime (BDM), a myosin ATPase inhibitor, decreased I(Ca) in both groups but to a greater extent in hypertension. Intracellular BAPTA or nNOS inhibition reinstated I(Ca) in the presence of BDM to the basal level, suggesting Ca(2+)-dependent inactivation of I(Ca) by nNOS and greater vulnerability in hypertension. Increasing stimulation frequencies (2, 4 and 8 Hz) attenuated myofilament Ca(2+) sensitivity in sham and reduced peak ICa in both groups. Nevertheless, SMTC or BAPTA exerted no effect on I(Ca) at high frequencies in either group. These results suggest that nNOS attenuates I(Ca) via Ca(2+)-dependent mechanism and the vulnerability is greater in hypertension subject to myofilament Ca(2+) desensitization. nNOS or [Ca(2+)]i does not affect I(Ca) at high stimulation frequencies. The results were recapitulated with computer simulation.

Low K⁺ current in arterial myocytes with impaired K⁺-vasodilation and its recovery by exercise in hypertensive rats.

K(+) channels determine the plasma membrane potential of vascular myocytes, influencing arterial tone. In many types of arteries, a moderate increase in [K(+)]e induces vasorelaxation by augmenting the inwardly rectifying K(+) channel current (I Kir). K(+)-vasodilation matches regional tissue activity and O2 supply. In chronic hypertension (HT), small arteries and arterioles undergo various changes; however, ion channel remodeling is poorly understood. Here, we investigated whether K(+) channels and K(+)-induced vasodilation are affected in deep femoral (DFA) and cerebral artery (CA) myocytes of angiotensin II-induced hypertensive rats (Ang-HT). Additionally, we tested whether regular exercise training (ET) restores HT-associated changes in K(+) channel activity. In Ang-HT, both the voltage-gated K(+) channel current (I Kv) and I Kir were decreased in DFA and CA myocytes, and were effectively restored and further increased by combined ET for 2 weeks (HT-ET). Consistently, K(+)-vasodilation of the DFA was impaired in Ang-HT, and recovered in HT-ET. Interestingly, ET did not reverse the decreased K(+)-vasodilation of CA. CA myocytes from the Ang-HT and HT-ET groups demonstrated, apart from K(+) channel changes, an increase in nonselective cationic current (I NSC). In contrast, DFA myocytes exhibited decreased I NSC in both the Ang-HT and HT-ET groups. Taken together, the decreased K(+) conductance in Ang-HT rats and its recovery by ET suggest increased peripheral arterial resistance in HT and the anti-hypertensive effects of ET, respectively. In addition, the common upregulation of I NSC in the CA in the Ang-HT and HT-ET groups might imply a protective adaptation preventing excessive cerebral blood flow under HT and strenuous exercise.

Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart.

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.

Exercise training increases inwardly rectifying K(+) current and augments K(+)-mediated vasodilatation in deep femoral artery of rats.

AIMS: A moderate increase in extracellular [K(+)] ([K(+)](e)) induces relaxation of small arteries by activating inwardly rectifying K(+) current (I(Kir)). The K(+)-induced vasodilatation is an important mechanism for exercise-induced hyperaemia in skeletal muscle. We investigated whether I(Kir) and K(+)-induced vasodilatation are enhanced in deep femoral arteries (DFAs) from exercise-trained rats (ET rats; treadmill running for 20 min at 20 m/min, 3 days/week for 2 weeks). The effects of exercise training on K(+)-induced vasodilatation and I(Kir) were also investigated in cerebral (CA) and mesenteric arteries. METHODS AND RESULTS: The K(+)-induced vasodilatation of DFAs and the density of I(Kir) and voltage-gated K(+) current (I(Kv)) were increased in DFA myocytes of ET rats. The myogenic tone of the DFA was unchanged by exercise. Although similar functional up-regulations of I(Kir) and I(Kv) were observed in CA myocytes, the K(+)-induced vasodilatation was not increased in the CA of ET rats. Interestingly, concomitant to the increases in I(Kir) and I(Kv), background Na(+) conductance was also increased in the CA myocytes. However, such an effect was not observed in DFA myocytes from ET rats. Neither I(Kir) nor K(+)-induced vasodilatation was observed in mesenteric arteries of ET rats. CONCLUSION: The present study provides evidence that regular exercise up-regulates I(Kir) in DFA and CA myocytes. Although the increase in I(Kir) was observed in two types of arteries, augmentation of K(+)-induced relaxation was observed only in the DFA of ET rats, possibly due to the increased Na(+) conductance in CA myocytes. The increases in I(Kir) and K(+)-induced vasodilatation of the arteries of skeletal muscle suggest novel mechanisms of improved exercise hyperaemia with physical training.

Inhibition of Ca2+-release-activated Ca2+ channel (CRAC) and K+ channels by curcumin in Jurkat-T cells.

The increase in cytoplasmic Ca(2+) concentration (Δ[Ca(2+)](c)) mediated by the Ca(2+)-release-activated Ca(2+) channel (CRAC) is a critical signal for the activation of lymphocytes. Also, the voltage-gated K(+) channel (K(v)) and intermediate-conductance Ca(2+)-activated K(+) channel (IKCa1/SK4) have drawn attention as pharmacological targets for regulating immune responses. Since polyphenolic agents have various immunomodulatory effects, here we compared the effects of curcumin, rosmarinic acid, resveratrol, and epigallocatechin gallate on the ionic currents through CRAC (I(CRAC)), K(v) (I(Kv)), SK4 (I(SK4)) and on the Δ[Ca(2+)](c) of Jurkat-T cells using the patch clamp technique and fura-2 spectrofluorimetry. Curcumin (10 µM) inhibited store-operated Ca(2+) entry (SOCE). Consistently, dose-dependent inhibition of I(CRAC) by curcumin was confirmed in Jurkat-T (IC(50), 5.9 µM) and the HEK293 cells overexpressing Orai1 and STIM1 (IC(50), 0.6 µM). Also, curcumin inhibited both I(Kv) (IC(50), 11.9 µM) and I(SK4) (IC(50), 4.2 µM). The other polyphenols (rosmarinic acid, resveratrol, and epigallocatechin gallate at 10 - 30 µM) had no effect on SOCE and showed only a partial inhibition of the K(+) currents. In summary, among the tested polyphenolic agents, curcumin showed prominent inhibition of major ion channels in lymphocytes, which might contribute to the anti-inflammatory effects of curcumin. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10209FP].

Inhibition of Ca2+-Release-Activated Ca2+ Channel (CRAC) and K+ Channels by Curcumin in Jurkat-T Cells.

The increase in cytoplasmic Ca2+ concentration (Δ[Ca2+]c) mediated by the Ca2+-release-activated Ca2+ channel (CRAC) is a critical signal for the activation of lymphocytes. Also, the voltage-gated K+ channel (Kv) and intermediate-conductance Ca2+-activated K+ channel (IKCa1/SK4) have drawn attention as pharmacological targets for regulating immune responses. Since polyphenolic agents have various immunomodulatory effects, here we compared the effects of curcumin, rosmarinic acid, resveratrol, and epigallocatechin gallate on the ionic currents through CRAC (ICRAC), Kv (IKv), SK4 (ISK4) and on the Δ[Ca2+]c of Jurkat-T cells using the patch clamp technique and fura-2 spectrofluorimetry. Curcumin (10 µM) inhibited store-operated Ca2+ entry (SOCE). Consistently, dose-dependent inhibition of ICRAC by curcumin was confirmed in Jurkat-T (IC50, 5.9 µM) and the HEK293 cells overexpressing Orai1 and STIM1 (IC50, 0.6 µM). Also, curcumin inhibited both IKv (IC50, 11.9 µM) and ISK4 (IC50, 4.2 µM). The other polyphenols (rosmarinic acid, resveratrol, and epigallocatechin gallate at 10 - 30 µM) had no effect on SOCE and showed only a partial inhibition of the K+ currents. In summary, among the tested polyphenolic agents, curcumin showed prominent inhibition of major ion channels in lymphocytes, which might contribute to the anti-inflammatory effects of curcumin. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10209FP].