Differential [Ca2+]i signaling of vasoconstriction in mesenteric microvessels of normal and reduced uterine perfusion pregnant rats

Vascular resistance and blood pressure (BP) are reduced during late normal pregnancy (Norm-Preg). In contrast, studies in human preeclampsia and in animal models of hypertension in pregnancy (HTN-Preg) have suggested that localized reduction in uterine perfusion pressure (RUPP) in late pregnancy is associated with increased systemic vascular resistance and BP; however, the vascular mechanisms involved are unclear. Because Ca2+ is a major determinant of vascular contraction, we hypothesized that the intracellular free calcium concentration ([Ca2+]i) signaling of vasoconstriction is differentially regulated in systemic microvessels during normal and RUPP in late pregnancy. Pressurized mesenteric microvessels from Norm-Preg and RUPP rats were loaded with fura 2 in preparation for simultaneous measurement of diameter and [Ca2+]i (presented as fura 2 340/380 ratio). Basal [Ca2+]i was lower in RUPP (0.73 +/- 0.03) compared with Norm-Preg rats (0.82 +/- 0.03). Membrane depolarization by 96 mM KCl, phenylephrine (Phe, 10(-5) M), angiotensin II (ANG II, 10(-7) M), or endothelin-1 (ET-1, 10(-7) M) caused an initial peak followed by maintained vasoconstriction and [Ca2+]i. KCl caused similar peak vasoconstriction and [Ca2+]i in Norm-Preg (45.5 +/- 3.3 and 0.89 +/- 0.02%) and RUPP rats (46.3 +/- 2.1 and 0.87 +/- 0.01%). Maximum vasoconstriction to Phe, ANG II, and ET-1 was not significantly different between Norm-Preg (28.6 +/- 4.8, 32.5 +/- 6.3, and 40 +/- 4.6%, respectively) and RUPP rats (27.8 +/- 5.9, 34.4 +/- 4.3, and 38.8 +/- 4.1%, respectively). In contrast, the initial Phe-, ANG II-, and ET-1-induced 340/380 ratio ([Ca2+]i) was reduced in RUPP (0.83 +/- 0.02, 0.82 +/- 0.02, and 0.83 +/- 0.03, respectively) compared with Norm-Preg rats (0.95 +/- 0.04, 0.93 +/- 0.01, and 0.92 +/- 0.02, respectively). Also, the [Ca2+]i-vasoconstriction relationship was similar in KCl-treated but shifted to the left in Phe-, ANG II-, and ET-1-treated microvessels of RUPP compared with Norm-Preg rats. The lower agonist-induced [Ca2+]i signal of vasoconstriction and the leftward shift in the [Ca2+]i-vasoconstriction relationship in microvessels of RUPP compared with Norm-Preg rats suggest activation of [Ca2+]i sensitization pathway(s). The similarity in vasoconstriction in RUPP and Norm-Preg rats suggests that such a [Ca2+]i sensitization pathway(s) may also provide a feedback effect on Ca2+ mobilization/homeostatic mechanisms to protect against excessive vasoconstriction in systemic microvessels during RUPP in late pregnancy.     

Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species

Insulin resistance (IR) and associated hyperinsulinemia are major risk factors for coronary artery disease. Mechanisms linking hyperinsulinemia to coronary vascular dysfunction in IR are unclear. We evaluated insulin-induced vasodilation in isolated small coronary arteries (SCA; approximately 225 microm) of Zucker obese (ZO) and control Zucker lean (ZL) rats. Vascular responses to insulin (0.1-100 ng/ml), ACh (10(-9)-10(-5) mol/l), and sodium nitroprusside (10(-8)-10(-4) mol/l) were assessed in SCA by measurement of intraluminal diameter using videomicroscopy. Insulin-induced dilation was decreased in ZO compared with ZL rats, whereas ACh and sodium nitroprusside elicited similar vasodilations. Pretreatment of arteries with SOD (200 U/ml), a scavenger of reactive oxygen species (ROS), restored the vasorelaxation response to insulin in ZO arteries, whereas ZL arteries were unaffected. Pretreatment of SCA with N-nitro-L-arginine methyl ester (100 micromol/l), an inhibitor of endothelial nitric oxide (NO) synthase (eNOS), elicited a vasoconstrictor response to insulin that was greater in ZO than in ZL rats. This vasoconstrictor response was reversed to vasodilation in ZO and ZL rats by cotreatment of the SCA with SOD or apocynin (10 micromol/l), a specific inhibitor of vascular NADPH oxidase. Lucigenin-enhanced chemiluminescence showed increased basal ROS levels as well as insulin (330 ng/ml)-stimulated production of ROS in ZO arteries that was sensitive to inhibition by apocynin. Western blot analysis revealed increased eNOS expression in ZO rats, whereas Mn SOD and Cu,Zn SOD expression were similar to ZL rats. Thus IR in ZO rats leads to decreased insulin-induced vasodilation, probably as a result of increased production of ROS by vascular NADPH oxidase, leading to decreased NO bioavailability, despite a compensatory increase in eNOS expression.     

Maturation depresses cGMP-mediated decreases in [Ca2+]i and Ca2+ sensitivity in ovine cranial arteries

Because cerebrovascular cGMP levels vary significantly during maturation, we examined the hypothesis that the ability of cGMP to relax cerebral arteries also changes during maturation. In concentration-response experiments, potassium-induced tone in basilar arteries was significantly more sensitive to a nonmetabolizable cell-permeant cGMP analogue 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in term fetal [-log one-half maximal concentration (EC(50)) = 4.4 +/- 0.1 M] than in adult (-log EC(50) = 4.0 +/- 0.1 M) ovine basilar arteries. Serotonin-induced tone also revealed significantly greater sensitivity to the cGMP analogue in fetal (-log EC(50) = 4.9 +/- 0.1 M) than in adult (-log EC(50) = 4.7 +/- 0.1 M) basilars. In fura 2-loaded preparations, 8-pCPT-cGMP had no significant effect on cytosolic calcium concentrations in potassium-contracted arteries but at 6 microM significantly reduced calcium only in fetal basilars (Delta = 33 +/- 8%). Higher 8-pCPT-cGMP concentrations reduced cytosolic calcium in both fetal and adult basilars. Similarly, in both potassium- and 5-hydroxytryptamine (5-HT)-contracted preparations, low concentrations of 8-pCPT-cGMP reduced myofilament calcium sensitivity only in fetal basilars (Delta = 29 +/- 6 and Delta = 42 +/- 10%, respectively), whereas higher concentrations reduced calcium sensitivity in both fetal and adult arteries. In beta-escin-permeabilized arteries, equivalent reductions in basal and agonist-enhanced myofilament calcium sensitivity were produced by much lower 8-pCPT-cGMP concentrations in fetal (172 and 61 microM, respectively) than in adult (410 and 231 microM, respectively) basilars. The mechanisms mediating cGMP-induced vasorelaxation appear similar in fetal and adult arteries, with the exception that they are much more sensitive to cGMP in fetal than adult arteries. These age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult cerebral arteries.     

Impaired overload-induced hypertrophy is associated with diminished mTOR signaling in insulin-resistant skeletal muscle of the obese Zucker rat

Recent data have suggested that insulin resistance may be associated with a diminished ability of skeletal muscle to undergo hypertrophy (Paturi S, Gutta AK, Kakarla SK, Katta A, Arnold EC, Wu M, Rice KM, Blough ER. J Appl Physiol 108: 7-13, 2010). Here we examine the effects of insulin resistance using the obese Zucker (OZ) rat with increased muscle loading on the regulation of the mammalian target of rapamycin (mTOR) and its downstream signaling intermediates 70-kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Compared with that observed in lean Zucker (LZ) rats, the degree of soleus muscle hypertrophy as assessed by changes in muscle wet weight (LZ: 35% vs. OZ: 16%) was significantly less in the OZ rats after 3 wk of muscle overload (P < 0.05). This diminished growth in the OZ rats was accompanied by significant impairments in the ability of the soleus to undergo phosphorylation of mTOR (Ser(2448)), p70S6k (Thr(389)), rpS6 (Ser(235/236)), and protein kinase B (Akt) (Ser(473) and Thr(308)) (P < 0.05). Taken together, these data suggest that impaired overload-induced hypertrophy in insulin-resistant skeletal muscle may be related to decreases in the ability of the muscle to undergo mTOR-related signaling.     

卵细胞受精相关胞质钙离子波、钙离子震荡信号特征及其形成机制

胞质[Ca2 ]i震荡的动力学变化在哺乳动物早期胚胎发育中发挥重要作用。卵母细胞的成熟伴随间断的、快速的[Ca2 ]i震荡的时空表达;在受精过程中精子因子诱导的反复[Ca2 ]i震荡的振幅和持续时间是卵细胞最有效的激活信号,这种信号形成自然连续的受精[Ca2 ]i波,并以长时持续[Ca2 ]i震荡形式在受精卵空间传递并持续数小时,直至受精完成;受精卵内源性的Ca2 释放所引起的[Ca2 ]i震荡形成第一次卵裂信号,启动早期胚胎的发育。精子PLCζ和cPKCs是形成受精卵[Ca2 ]波、[Ca2 ]震荡的重要因素。     

Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats

Obesity, insulin resistance, dyslipidemia, and hypertension are components of the pathophysiological state known as metabolic syndrome. Adrenergic vasoconstriction is mediated through increases in cytosolic Ca2+ and the myofilaments' sensitivity to Ca2+. In many pathophysiological states, there is an enhanced role for Rho kinase (ROK)-mediated increases in Ca2+ sensitivity of the contractile apparatus. Thus we hypothesized that there is a greater role for ROK-mediated increases in Ca2+ sensitivity in alpha1-adrenergic vasoconstriction in arteries from obese Zucker (OZ) rats. Therefore, small gracilis muscle arteries from 11- to 12-wk-old and 16- to 18-wk-old lean and OZ rats were isolated, cannulated, and pressurized to 75 mmHg. For some experiments, vessels were loaded with fura 2-AM. Changes in luminal diameter and vessel wall Ca2+ concentration ([Ca2+]) were measured in response to phenylephrine (PE), the thromboxane mimetic U-46619, and KCl. alpha1-Adrenergic vasoconstriction was similar between 11- to 12-wk-old lean and obese animals and greater in older obese animals compared with controls. PE-induced increases in vascular smooth muscle cell [Ca2+] were blunted in OZ animals compared with lean controls in both age groups of animals. KCl and U-46619 elicited similar vasoconstriction and vascular smooth muscle cell [Ca2+] in both groups. ROK inhibition attenuated PE vasoconstriction to a greater degree in arteries from 11- to 12-wk-old OZ rats compared with lean animals; ROK inhibition in arteries from older rats right shifted both concentration-response curves to the same point. Total RhoA and ROKalpha protein expressions were similar between groups. These results suggest an enhanced role for the ROK pathway in alpha1-adrenergic vasoconstriction in metabolic syndrome.     

Mechanisms of [Ca2+]i elevation by H2O2 in islets of rats

Activity of reactive oxygen species is elevated in diabetes mellitus and has been implicated in the destruction of cellular components. The toxic effect of reactive oxygen species was investigated by testing the effect of H2O2 on [Ca2+]i in isolated islets of Langehans. H2O2 increased [Ca2+]i in a dose-dependent manner, which was irreversible at high concentrations. The maximum effect of H2O2 on [Ca2+]i was larger than those of KCl, glucose, ATP, carbachol and endothelin-1. The effect of H2O2 was only partially attenuated by removal of external Ca2+ and by the in-organic Ca2+ channel blocker nickel, but was not blocked by voltage-dependent or -independent Ca2+ channel blockers nimodipine, nicardipine, SK&F 96365, econazole and lanthanum. H2O2, disrupted [Ca2+]i homeostasis in islets by affecting both release and influx of Ca2+ and causing dysfunction of Ca2+ clearance systems and may contribute to the pathological process of diabetes.     

Impaired Ca2+ handling in penile arteries from prediabetic Zucker rats: involvement of Rho kinase

Diabetes is associated with an increased vascular tone usually involved in the pathogenesis of diabetic cardiovascular complications such as hypertension, stroke, coronary artery disease, or erectile dysfunction (ED). Enhanced contractility of penile erectile tissue has been associated with augmented activity of the RhoA/Rho kinase (RhoK) pathway in models of diabetes-associated ED. The present study assessed whether abnormal vasoconstriction in penile arteries from prediabetic obese Zucker rats (OZRs) is due to changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and/or in myofilament Ca(2+) sensitivity. Penile arteries from OZRs and lean Zucker rats (LZRs) were mounted on microvascular myographs for simultaneous measurements of [Ca(2+)](i) and tension. The relationships between [Ca(2+)](i) and contraction for the α(1)-adrenergic vasoconstrictor phenylephrine (PE) were left shifted and steeper in OZRs compared with LZRs, although the magnitude of the contraction was similar in both groups. In contrast, the vasoconstriction induced by the thromboxane A(2) receptor agonist U-46619 was augmented in arteries from OZRs, and this increase was associated with an increase in both the sensitivity and maximum responses to Ca(2+). The RhoK inhibitor Y-27632 (10 μM) reduced the vasoconstriction induced by PE to a greater extent in OZRs than in LZRs, without altering Ca(2+). Y-27632 inhibited with a greater potency the contraction elicited by high KCl in arteries from OZRs compared with LZRs without changing [Ca(2+)](i). RhoK-II expression was augmented in arteries from OZRs. These results suggest receptor-specific changes in the Ca(2+) handling of penile arteries under conditions of metabolic syndrome. Whereas augmented vasoconstriction upon activation of the thromboxane A(2) receptor is coupled to enhanced Ca(2+) entry, a RhoK-mediated enhancement of myofilament Ca(2+) sensitivity is coupled with the α(1)-adrenergic vasoconstriction in penile arteries from OZRs.     

谷氨酸促进大鼠海马神经元的内钙升高

谷氨酸能影响大鼠海马神经元胞内钙信号的变化,进而影响海马神经元神经冲动的发放和学习记忆过程。运用荧光测钙技术实时监测了大鼠海马神经元内钙信号的动态变化,同时分析了谷氨酸对其胞内钙信号的影响。试验表明：谷氨酸能够显著提高胞内游离钙离子的浓度;细胞外钙离子的存在、谷氨酸刺激时间及刺激频率的增加都能引起胞内钙信号不同程度的升高;但谷氨酸的过度刺激会引起钙离子浓度的超负荷,从而导致神经元结构和功能的损坏。     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.     

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).     

Apical P2XR contribute to [Ca2+]i signaling and Isc in mouse renal MCD

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.     

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.     

Antiphase oscillations of endothelium and smooth muscle [Ca2+]i in vasomotion of rat mesenteric small arteries

The mechanisms leading to vasomotion in the presence of noradrenaline and inhibitors of the sarcoplasmic/endoplasmic reticulum calcium ATPase were investigated in isolated rat mesenteric small arteries. Isobaric diameter and isometric force were measured together with membrane potential in endothelial cells and smooth muscle cells (SMC). Calcium in the endothelial cells and SMC was imaged with confocal microscopy. In the presence of noradrenaline and cyclopiazonic acid, ryanodine-insensitive oscillations in tone were produced. The frequency was about 1 min(-1) and amplitude about 70% of the maximal tone. The amplitude was reduced by indomethacin and increased with L-NAME. Vasomotion was inhibited by nifedipine and by 40 mM potassium. The frequency was increased and amplitude decreased by removal of the endothelium and by application of charybdotoxin and apamin. The vasomotion was associated with in-phase oscillations of membrane potential in endothelial cells and SMC and oscillations of [Ca2+]i that were in near anti-phase. We suggest a working model for the generation of oscillation based on a membrane oscillator where ion channels in both endothelial cells and SMC interact via a current running between the two cell types through myoendothelial gap junctions, which sets up a near anti-phase oscillation of [Ca2+]i in the two cell types.