Pressor effects of the vasoconstrictor hormone arginine vasopressin (AVP), observed when systemic AVP concentrations are less than 100 pM, are important for the physiological maintenance of blood pressure, and they are also the basis for therapeutic use of vasopressin to restore blood pressure in hypotensive patients. However, the mechanisms by which circulating AVP induces arterial constriction are unclear. We examined the novel hypothesis that KCNQ potassium channels mediate the physiological vasoconstrictor actions of AVP. Reverse transcriptase polymerase chain reaction revealed expression of KCNQ1, KCNQ4, and KCNQ5 in rat mesenteric artery smooth muscle cells (MASMCs). Whole-cell perforated patch recordings of voltage-sensitive K ϩ (K v ) currents in freshly isolated MASMCs revealed 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-2H-indol-2-one (linopirdine)-and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991)-sensitive KCNQ currents that were electrophysiologically and pharmacologically distinct from other K v currents. Suppression of KCNQ currents by AVP (100 pM) was associated with significant membrane depolarization, and it was abolished by the protein kinase C (PKC) inhibitor calphostin C (250 nM). The KCNQ channel blocker linopirdine (10 M) inhibited KCNQ currents in MASMCs, and it induced constriction of isolated rat mesenteric arteries. The vasoconstrictor responses were not additive when combined with 30 pM AVP, and they were prevented by the L-type Ca 2ϩ channel blocker verapamil. Ethyl-N-[2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl] carbamic acid (flupirtine) significantly enhanced KCNQ currents, and it reversed constrictor responses to 30 pM AVP. In vivo, i.v. administration of linopirdine induced a dose-dependent increase in mesenteric artery resistance and blood pressure, whereas flupirtine had the opposite effects. We conclude that physiological concentrations of AVP induce mesenteric artery constriction via PKCdependent suppression of KCNQ currents and L-type Ca 2ϩ channel activation in MASMCs.Membrane voltage (V m ) determines the open probability of L-type Ca 2ϩ channels in vascular smooth muscle cells (VSMCs), and K ϩ channels represent a primary effector for adjusting V m . To the extent that Kϩ channels are open in resting VSMCs, the outward flux of K ϩ through these channels (measured as K ϩ current) will tend to stabilize the resting V m at negative (hyperpolarized) voltages and prevent opening of voltage-sensitive Ca 2ϩ channels. In contrast, reduction of outward K ϩ currents in VSMCs results in a shift to more positive V m (membrane depolarization) leading to activation of L-type Ca 2ϩ channels and entry of Ca 2ϩ into the cell. Elevation of the cytosolic Ca 2ϩ concentration in this manner can trigger VSMC contraction and vasoconstriction.KCNQ channels (Kv7 family) are voltage-sensitiveThis work was supported by the National Heart Lung and Blood Institute Grant R01 HL070670 (to K.L.B.) and the American Heart Association Grant 0715618Z (to A.R.M.).The chemical st...

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function; however, the profile of cMyBP-C degradation after myocardial infarction (MI) is unknown. We hypothesized that cMyBP-C is sensitive to proteolysis and is specifically increased in the bloodstream post-MI in rats and humans. Under these circumstances, elevated levels of degraded cMyBP-C could be used as a diagnostic tool to confirm MI. To test this hypothesis, we first established that cMyBP-C dephosphorylation is directly associated with increased degradation of this myofilament protein, leading to its release in vitro. Using neonatal rat ventricular cardiomyocytes in vitro, we were able to correlate the induction of hypoxic stress with increased cMyBP-C dephosphorylation, degradation, and the specific release of N′-fragments. Next, to define the proteolytic pattern of cMyBP-C post-MI, the left anterior descending coronary artery was ligated in adult male rats. Degradation of cMyBP-C was confirmed by a reduction in total cMyBP-C and the presence of degradation products in the infarct tissue. Phosphorylation levels of cMyBP-C were greatly reduced in ischemic areas of the MI heart compared to non-ischemic regions and sham control hearts. Post-MI plasma samples from these rats, as well as humans, were assayed for cMyBP-C and its fragments by sandwich ELISA and immunoprecipitation analyses. Results showed significantly elevated levels of cMyBP-C in the plasma of all post-MI samples. Overall, this study suggests that cMyBP-C is an easily releasable myofilament protein that is dephosphorylated, degraded and released into the circulation post-MI. The presence of elevated levels of cMyBP-C in the blood provides a promising novel biomarker able to accurately rule in MI, thus aiding in the further assessment of ischemic heart disease.

O(2) transport during maximal exercise was studied in rats bred for extremes of exercise endurance, to determine whether maximal O(2) uptake (VO(2 max)) was different in high- (HCR) and low-capacity runners (LCR) and, if so, which were the phenotypes responsible for the difference. VO(2 max) was determined in five HCR and six LCR female rats by use of a progressive treadmill exercise protocol at inspired PO(2) of approximately 145 (normoxia) and approximately 70 Torr (hypoxia). Normoxic VO(2 max) (in ml. min(-1). kg(-1)) was 64.4 +/- 0.4 and 57.6 +/- 1.5 (P < 0.05), whereas VO(2 max) in hypoxia was 42.7 +/- 0.8 and 35.3 +/- 1.5 (P < 0.05) in HCR and LCR, respectively. Lack of significant differences between HCR and LCR in alveolar ventilation, alveolar-to-arterial PO(2) difference, or lung O(2) diffusing capacity indicated that neither ventilation nor efficacy of gas exchange contributed to the difference in VO(2 max) between groups. Maximal rate of blood O(2) convection (cardiac output times arterial blood O(2) content) was also similar in both groups. The major difference observed was in capillary-to-tissue O(2) transfer: both the O(2) extraction ratio (0.81 +/- 0.002 in HCR, 0.74 +/- 0.009 in LCR, P < 0.001) and the tissue diffusion capacity (1.18 +/- 0.09 in HCR and 0.92 +/- 0.05 ml. min(-1). kg(-1). Torr(-1) in LCR, P < 0.01) were significantly higher in HCR. The data indicate that selective breeding for exercise endurance resulted in higher VO(2 max) mostly associated with a higher transfer of O(2) at the tissue level.

We tested the hypothesis that exercise training (Ex) attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in male porcine coronary arteries [left anterior descending coronary arteries (LAD)] by increasing nitric oxide (NO) release [due to increased endothelial NO synthase (NOS) expression] and/or increased bioactivity of NO. Adult male pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20-24 wk. Pigs were Ex or remained sedentary (Sed) for 16-20 wk, beginning after 4 wk on diet. Four groups of pigs were used: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. HF enhanced LAD contractions induced by KCl, aggregating platelets (AP), and serotonin (5-HT). AP and 5-HT produced EDR after blockade of cyclooxygenase with indomethacin (Indo) and smooth-muscle 5-HT(2) receptors with ketanserin. HF impaired EDR induced by AP, 5-HT, and bradykinin. Results indicate a decreased contribution of NO to EDR in HF-Sed LADs, because the percentage of bradykinin-induced EDR inhibited by N(G)-nitro-L-arginine methyl ester was 27% in NF-Sed and 34% in NF-Ex but only 17% in HF-Sed. Also, N(G)-nitro-L-arginine methyl ester + Indo results indicate that release of an Indo-sensitive vasoconstrictor contributes to blunted EDR in HF-Sed LAD. Immunoblot and immunohistochemistry results indicate the following: 1) LAD endothelial NOS protein content was similar among groups; 2) HF decreased LAD superoxide dismutase (SOD) but increased caveolin-1 content; and 3) Ex increased SOD content of HF LADs. We conclude that HF impairs EDR by impairing the contribution of NO released from NOS (due to decreased SOD and increased caveolin-1 protein content) and by production of an Indo-sensitive vasoconstrictor. Ex preserves EDR in HF LADs by decreasing the production of the constrictor and increasing NO-release by NOS and/or NO bioactivity and bioavailability.