Aging impairs Ca2+ sensitization pathways in gallbladder smooth muscle

Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca²⁺]_i by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22–24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the “in vitro” isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction

Ca2+ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase-dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca2+-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca2+ or the addition of a dihydropyridine Ca2+ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N19RhoA suppressed Ca2+-induced Thr695 phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca2+ removal and depletion of the intracellular Ca2+ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A2 analogue-induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca2+-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.     

Ca2(+)-induced contraction and hyperreactivity of pulmonary arterial smooth muscle in monocrotaline-treated rats

The mechanism of progressive pulmonary hypertension induced by monocrotaline (MCT) remains controversial. To determine whether or not functional changes in pulmonary arterial smooth muscle contribute to the development of pulmonary hypertension, we examined the reactivity of isolated pulmonary artery segments 7, 14 and 21 days after a single subcutaneous injection of MCT. In Ca2(+)-free buffer, pulmonary arteries from MCT-treated rats contracted when CaCl2 was added without any other stimulation. The pulmonary artery exposed to MCT also exhibited hyperreactivity to KCl and 5-hydroxytryptamine. These functional changes in the pulmonary artery preceded the elevation of right ventricular systolic pressure and right ventricular hypertrophy. The contraction in response to Ca2+ suggests that the pulmonary artery of rats given MCT may be contracted in situ. Vasoconstriction due to these alterations may play an important role in the development of pulmonary hypertension according to this model.     

Nicotinic acid adenine dinucleotide phosphate mediates Ca2+ signals and contraction in arterial smooth muscle via a two-pool mechanism

Previous studies of arterial smooth muscle have shown that inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose mobilize Ca2+ from the sarcoplasmic reticulum. In contrast, little is known about Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate, a pyridine nucleotide derived from beta-NADP+. We show here that intracellular dialysis of nicotinic acid adenine dinucleotide phosphate (NAADP) induces spatially restricted "bursts" of Ca2+ release that initiate a global Ca2+ wave and contraction in pulmonary artery smooth muscle cells. Depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin and inhibition of ryanodine receptors with ryanodine, respectively, block the global Ca2+ waves by NAADP. Under these conditions, however, localized Ca2+ bursts are still observed. In contrast, xestospongin C, an IP3 receptor antagonist, had no effect on Ca2+ signals by NAADP. We propose that NAADP mobilizes Ca2+ via a 2-pool mechanism, and that initial Ca2+ bursts are amplified by subsequent sarcoplasmic reticulum Ca2+ release via ryanodine receptors but not via IP3 receptors.     

Cyclic ADP-ribose contributes to contraction and Ca2+ release by M1 muscarinic receptor activation in coronary arterial smooth muscle

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca(2+) release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 micromol/L) and oxotremorine (OXO; 2.5-80 micromol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca(2+) ([Ca(2+)](i)) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca(2+)](i) in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca(2+)-free Hanks' solution. This OXO-induced rise in [Ca(2+)](i) was significantly reduced by pirenzepine (PIR; an M(1) receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from beta-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca(2+) release induced by activation of M(1) muscarinic receptors.     

Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells

Angiostensin II (Ang II) regulates the migration and proliferation of vascular smooth muscle cells. Recent studies indicate that intermediate-conductance Ca2+ -activated K+ (IKca) channels have an important role in cell migration and proliferation. It is not known, however, whether the action of Ang II is linked to IKca channel regulation. Here, we investigated the modulation of IKca channels by Ang II in artery smooth muscle cells. Functional IKca channel expression in cultured embryonic rat aorta smooth muscle (A10) cells was studied using the patch-clamp technique. These cells predominantly express IKca channels. In contrast, large-conductance Ca2+ -activated K+ (BKca) currents were rarely observed in excised patches. Ang II increased the IKca current in a contration-dependent manner. Losartan (1.0 microM), an AT1 selective antagonist, abolished the activation of IKca channels by Ang II. Pretreatment with 100 microM myristoylated protein kinase C inhibitor peptide 20-28 or 10 microM GF109203X completely abolished the AngII-induced activation of IKca currents, whereas the action of Ang II was not prevented in the presence of 100 microM Rp-cyclic 3', 5'-hydrogen phosphotiate adenosine triethylammonium, a protein kinase A inhibitor, or 1.0 microM KT-5823, a protein kinase G inhibitor. A membrane permeant analogue of diacylglycerol 1, 2-dioctanoyl-sn-glycerol (10 microM) induced the activation of IKca currents. These data suggest that Ang II activates IKca channels through the activation of protein kinase C, and the AT1 receptor is involved in the regulation of these channels.     

Magnesium and contraction of arterial smooth muscle

The present in vitro experiments demonstrate that reduction in [Mg²⁺]₀ induces contractile responses in rat aortic strips; the greater the reduction in [Mg²⁺]₀, the greater the magnitude of the mechanical response. These contractile responses are dependent upon the [Ca²⁺]₀ and the polarity of the vascular smooth muscle cell membrane. Adrenergic, cholinergic, serotonergic, or histaminergic antagonists do not interfere with these Mg²⁺-dependent contractile responses. Increases in [Mg²⁺]₀ above 1.2 mM (e.g., 2.4 and 6.0 mM), on the other hand, inhibits spontaneous contractile activity and lowers basal tension; these latter findings are not due to hyperosmolarity. Exposure of rat aorta to Mg²⁺-free Krebs-Ringer for 1 hr differentially affects the contractile responses induced by catecholamines, serotonin, angiotensin, barium, and neurophypophyseal hormones. In addition, Mg²⁺-free solution results in a lowering of the threshold and maximal force development of Ca²⁺-induced contractions of depolarized rat aorta. These data support the suggestions that (1) certain divalent cation sites in vascular smooth muscle may be nonspecific in regard to Mg and Ca, and (2) Mg ions may be important in regulating permeability, translocation and/or binding of Ca ions in vascular muscle.     

Protein kinase C mediates insulin-inhibited Ca2+ transport and contraction of vascular smooth muscle

Insulin acutely inhibits contraction of primary cultured vascular smooth muscle (VSM) cells from canine femoral artery by inhibiting contractile agonist-induced Ca²⁺ influx. Insulin also inhibits contraction at step(s) distal to intracellular Ca²⁺ concentration (Ca²⁺_i) by stimulating cyclic guanosine monophosphate (GMP) production. We wished to see whether these effects of insulin are mediated by protein kinase C (PKC). Ca²⁺ influx was assessed by measuring the rate of fluorescence quenching of intracellular fura 2 by extracellular Mn²⁺. We found that 10 μmol/L serotonin (5-HT) stimulated Mn²⁺ influx 3-fold, and 1 nmol/L insulin inhibited the 5-HT–stimulated component of Mn²⁺ influx by 63% (P < .05), but insulin had no effect in the presence of 1 μmol/L staurosporine, an inhibitor of PKC. In the absence of insulin, preincubating cells with 0.1 μmol/L phorbol 12-myristate 13-acetate (PMA) for 5 min inhibited the 5-HT–stimulated component of Mn²⁺ influx by 69% (P < .05). Insulin inhibited cell contraction induced by raising Ca²⁺_i to supraphysiologic levels with ionomycin by 75% (P < .05). We also noted that 10⁻⁶ mol/L calphostin C, another PKC inhibitor, or 16-h preincubation with PMA completely blocked this effect of insulin. Finally, 10-min exposure to insulin or PMA increased cyclic GMP production in ionomycin-treated cells by 50% and 64%, respectively (both P < .05). We conclude that insulin inhibits VSM cell contraction by inhibiting 5-HT–stimulated Ca²⁺ influx and also at step(s) distal to Ca²⁺_i by a PKC-dependent mechanism.     

Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells

The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.     

Hypersensitivity of BKCa to Ca2+ sparks underlies hyporeactivity of arterial smooth muscle in shock

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) play a critical role in blood pressure regulation by tuning the vascular smooth muscle tone, and hyposensitivity of BK(Ca) to Ca(2+) sparks resulting from its altered beta1 subunit stoichiometry underlies vasoconstriction in animal models of hypertension. Here we demonstrate hypersensitivity of BK(Ca) to Ca(2+) sparks that contributes to hypotension and blunted vasoreactivity in acute hemorrhagic shock. In arterial smooth muscle cells under voltage-clamp conditions (0 mV), the amplitude and duration, but not the frequency, of spontaneous transient outward currents of BK(Ca) origin were markedly enhanced in hemorrhagic shock, resulting in a 265% greater hyperpolarizing current. Concomitantly, subsurface Ca(2+) spark frequency was either unaltered (at 0 mV) or decreased in hyperpolarized resting cells. Examining the relationship between spark and spontaneous transient outward current amplitudes revealed a hypersensitive BK(Ca) activity to Ca(2+) spark in hemorrhagic shock, whereas the spark-spontaneous transient outward current coupling fidelity was near unity in both groups. Importantly, we found an acute upregulation of the beta1 subunit of the channel, and single-channel recording substantiated BK(Ca) hypersensitivity at micromolar Ca(2+), which promotes the alpha and beta1 subunit interaction. Treatment of shock animals with the BK(Ca) inhibitors iberiotoxin and charybdotoxin partially restored vascular membrane potential and vasoreactivity to norepinephrine and blood reinfusion. Thus, the results underscore a dynamic regulation of the BK(Ca)-Ca(2+) spark coupling and its therapeutic potential in hemorrhagic shock-associated vascular disorders.     

Ca2+-transport ATPases of vascular smooth muscle

To characterize the Ca2+-transport properties of the plasma membrane and of the endoplasmic reticulum of bovine pulmonary artery, membrane vesicles are subfractionated by a procedure of density-gradient centrifugation that takes advantage of the selective effect of digitonin on the density of plasma-membrane vesicles. The obtained endoplasmic-reticulum fraction contains hardly any plasma-membrane vesicles, whereas the plasma-membrane fraction is still contaminated by a substantial amount of endoplasmic-reticulum vesicles. An adenosine 5'-triphosphate (ATP) energized Ca2+-transport system and a Ca2+-stimulated ATPase activity are present in both subcellular fractions. The Ca2+ transport by the plasma membrane is catalyzed by a (Ca2+,Mg2+)-ATPase of Mr 130,000. It binds calmodulin and it has a low steady-state phosphoprotein intermediate level. The endoplasmic-reticulum vesicles contain a Ca2+-transport ATPase of Mr 100,000 that is characterized by a high steady-state phosphointermediate level. It is antigenically related to the Ca2+-pump protein of cardiac sarcoplasmic reticulum. Phospholamban, the regulatory protein of the Ca2+-transport enzyme of cardiac sarcoplasmic reticulum, is also present in the endoplasmic reticulum of the pulmonary artery. A comparison of these fractions with the previously characterized fractions from porcine gastric smooth muscle reveals important differences in the basal Mg2-ATPase activity, in the ratio of the (Ca2+,Mg2+)-ATPase of the plasmalemma to that of the endoplasmic reticulum, and in the ratio of the (Na+,K+)-ATPase activity to the plasmalemmal (Ca2+,Mg2+)-ATPase activity. These differences can be ascribed in part to the species and in part to the tissue. These data suggest that in the bovine pulmonary artery the Ca2+ extrusion via the ATP-dependent Ca2+ pump may have a less predominant role, and that the Ca2+ uptake by the endoplasmic reticulum, and possibly also the Ca2+ extrusion via the Na+-Ca2+ exchanger could be more important in this tissue than in the porcine stomach.     

槟榔碱促结肠平滑肌细胞收缩及对胞内钙离子浓度的影响

目的:观察氢溴酸槟榔碱(Ah)促进培养的结肠平滑肌细胞收缩作用及对胞内游离Ca2 浓度的影响．方法:培养大鼠结肠平滑肌细胞,分为4组:正常对照组、Ah刺激组、乙酰胆碱(Ach)刺激组、阿托品预处理组．应用特异性Ca2 荧光批示剂Fluo-3／AM负载细胞,激光共聚焦显微镜检测游离Ca2 浓度和细胞收缩率．结果:正常对照组细胞未发生自主性收缩,因指示剂的衰减,荧光强度(FI)有递减的趋势; Ah刺激组在加药后短时间内胞内钙离子浓度迅速升高,出现一个波峰,FI平均基础值与峰值有差异(P<0．05),而后胞内钙离子浓度再缓慢攀升,在484．0±47．6 s达到高峰,而后有一个较长时间的平台期,在1 400 s左右恢复至静息水平,FI基础值与峰值有显著差异 (P<0．01),细胞收缩百分数为20．70％±0．07％; Ach刺激组在加药后胞内钙离子浓度升高,形成一个小波峰,FI基础值和峰值差异有显著性(P<0．01)．随后胞内钙离子浓度缓慢攀升, 在600 s左右达到高峰,而后有一个较长时间的平台期．FI基础值与峰值差异有显著性 (P<0．001);经阿托品预孵育处理后的细胞,加入Ah后,其收缩效应被完全抑制．结论:Ah可引起结肠平滑肌细胞收缩,升高细胞内游离Ca2 浓度．其收缩效应可以被M受体阻断剂阿托品所抑制．     

Endothelin-1 promotes Ca2+ antagonist-insensitive coronary smooth muscle contraction via activation of epsilon-protein kinase C

Certain forms of coronary artery disease do not respond to treatment with Ca2+ channel blockers, and a role for endothelin-1 (ET-1) in Ca2+ antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1-induced coronary smooth muscle contraction is Ca2+ antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]i was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank's solution (1 mmol/L Ca2+), ET-1 (10(-7) mol/L) caused a transient increase in [Ca2+]i (236+/-14 nmol/L) followed by a maintained increase in [Ca2+]i (184+/-8 nmol/L) and 35% cell contraction. The Ca2+ channel blockers verapamil and diltiazem (10(-6) mol/L) abolished the maintained ET-1-induced [Ca2+]i, but only partially inhibited ET-1-induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and epsilon-PKCV1-2. ET-1 caused translocation of Ca2+-dependent alpha-PKC and Ca2+-independent epsilon-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1-induced translocation of alpha-PKC, but not that of epsilon-PKC. Phorbol 12-myristate 13-acetate (10(-6) mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+ influx, caused 35% cell contraction and increase in [Ca2+]i (291+/-11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of alpha- or epsilon-PKC. In Ca2+-free (2 mmol/L EGTA) Hank's solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of epsilon-PKC that were inhibited by epsilon-PKC V1-2 inhibitory peptide. Thus, a significant component of ET-1-induced contraction of coronary smooth muscle is Ca2+ antagonist-insensitive and involves activation and translocation of Ca2+-independent epsilon-PKC, and may represent a signaling mechanism of Ca2+ antagonist-resistant forms of coronary vasospasm.     

Motor neuron targeting of IGF-1 attenuates age-related external Ca2+-dependent skeletal muscle contraction in senescent mice

A population of fast muscle fibers from aging mice is dependent on external Ca(2+) to maintain tetanic force during repeated contractions. We hypothesized that age-related denervation in muscle fibers plays a role in initiating this contractile deficit, and that prevention of denervation by IGF-1 overexpression would prevent external Ca(2+)-dependent contraction in aging mice. IGF-1 overexpression in skeletal muscle prevents age-related denervation, and prevented external Ca(2+)-dependent contraction in this work. To determine if the effects of IGF-1 overexpression are on muscle or nerve, aging mice were injected with a tetanus toxin fragment-C (TTC) fusion protein that targets IGF-1 to spinal cord motor neurons. This treatment prevented external Ca(2+)-dependent contraction. We also show evidence that injections of the IGF-1-TTC fusion protein prevent age-related alterations to the nerve terminals at the neuromuscular junctions. We conclude that the slow age-related denervation of fast muscle fibers underlies dependence on external Ca(2+) to maintain tetanic force in a population of muscle fibers from senescent mice.     

The control of Ca2+ influx and NFATc3 signaling in arterial smooth muscle during hypertension

Many excitable cells express L-type Ca(2+) channels (LTCCs), which participate in physiological and pathophysiological processes ranging from memory, secretion, and contraction to epilepsy, heart failure, and hypertension. Clusters of LTCCs can operate in a PKCalpha-dependent, high open probability mode that generates sites of sustained Ca(2+) influx called "persistent Ca(2+) sparklets." Although increased LTCC activity is necessary for the development of vascular dysfunction during hypertension, the mechanisms leading to increased LTCC function are unclear. Here, we tested the hypothesis that increased PKCalpha and persistent Ca(2+) sparklet activity contributes to arterial dysfunction during hypertension. We found that PKCalpha and persistent Ca(2+) sparklet activity is indeed increased in arterial myocytes during hypertension. Furthermore, in human arterial myocytes, PKCalpha-dependent persistent Ca(2+) sparklets activated the prohypertensive calcineurin/NFATc3 signaling cascade. These events culminated in three hallmark signs of hypertension-associated vascular dysfunction: increased Ca(2+) entry, elevated arterial [Ca(2+)](i), and enhanced myogenic tone. Consistent with these observations, we show that PKCalpha ablation is protective against the development of angiotensin II-induced hypertension. These data support a model in which persistent Ca(2+) sparklets, PKCalpha, and calcineurin form a subcellular signaling triad controlling NFATc3-dependent gene expression, arterial function, and blood pressure. Because of the ubiquity of these proteins, this model may represent a general signaling pathway controlling gene expression and cellular function.     

Regulation of contraction and relaxation in arterial smooth muscle.

Intracellular calcium concentration ([Ca2+]i)-dependent activation of myosin light chain kinase and its phosphorylation of the 20-kd light chain of myosin is generally considered the primary mechanism responsible for regulation of contractile force in arterial smooth muscle. However, recent data suggest that the relation between [Ca2+]i and myosin light chain phosphorylation is variable and depends on the form of stimulation. The dependence of myosin phosphorylation on [Ca2+]i has been termed the "[Ca2+]i sensitivity of phosphorylation." The [Ca2+]i sensitivity of phosphorylation is "high" when relatively small increases in [Ca2+]i induce a large increase in myosin phosphorylation. Conversely, the [Ca2+]i sensitivity of phosphorylation is "low" when relatively large increases in [Ca2+]i are required to induce a small increase in myosin phosphorylation. There are two proposed mechanisms for changes in the [Ca2+]i sensitivity of phosphorylation: Ca(2+)-dependent decreases in the [Ca2+]i sensitivity of phosphorylation induced by phosphorylation of myosin light chain kinase by Ca(2+)-calmodulin protein kinase II and agonist-dependent increases in the [Ca2+]i sensitivity of phosphorylation by inhibition of a myosin light chain phosphatase. I will review the proposed mechanisms responsible for the regulation of [Ca2+]i and the [Ca2+]i sensitivity of phosphorylation in arterial smooth muscle.     

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.