Inward remodeling of resistance arteries requires reactive oxygen species-dependent activation of matrix metalloproteinases

Inward eutrophic remodeling is the most prevalent structural change of resistance arteries in hypertension. Sympathetic and angiotensin (ANG)-induced vasoconstriction has been associated with hypertension and with the production of matrix metalloproteinases (MMPs) and ROS. Therefore, we hypothesize that prolonged exposure to norepinephrine (NE) and ANG II induces arteriolar inward remodeling dependent on the activation of MMPs and the production of ROS. This hypothesis was tested on rat cremaster arterioles that were isolated, cannulated, pressurized, and exposed to either NE (10(-5.5) mol/l) + ANG II (10(-7) mol/l) or vehicle (control) for 4 h. The prolonged exposure to NE + ANG II induced inward remodeling, as evidenced by the reduced maximal arteriolar passive diameter observed after versus before exposure to the vasoconstrictor agonists. NE + ANG II also increased the arteriolar expression and activity of MMP-2 and the production of ROS as determined, respectively, by real-time RT-PCR, gel and in situ zymography, and the use of ROS-sensitive dyes with multiphoton microscopy. Inhibition of MMP activation (with GM-6001) or ROS production (with apocynin or tempol) prevented the NE + ANG II-induced inward remodeling. Inhibition of ROS production prevented the activation of MMPs and the remodeling process, whereas inhibition of MMP activation did not affect ROS production. These results indicate that prolonged stimulation of resistance arterioles with NE + ANG II induces a ROS-dependent activation of MMPs necessary for the development of arteriolar inward remodeling. These mechanisms may contribute to the structural narrowing of resistance vessels in hypertension.     

Tyrosine phosphorylation increases Ca2+ sensitivity of vascular smooth muscle contraction

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 microM) and phosphatase (sodium o-vanadate, 5 microM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCI. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCI alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD2 values. Neither basal 45Ca2+ uptake nor verapamil-sensitive 45Ca2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca2+ entry but through increased Ca2+ sensitivity of the intracellular contractile pathway.     

Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 microM) or the beta-adrenergic blocker dl-propranolol (1 microM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.     

Fetal and adult cerebral artery K(ATP) and K(Ca) channel responses to long-term hypoxia.

High-altitude long-term hypoxia (LTH) alters cerebral vascular contractile and relaxation responses in both fetus and adult. We tested the hypotheses that LTH-mediated vascular responses were secondary to altered K+ channel function and that in the fetus these responses differ from those of the adult. In middle cerebral arteries (MCA) from both nonpregnant adult and fetal (approximately 140 days gestation) sheep, which were either acclimatized to high altitude (3,820 m) or sea-level controls, we measured norepinephrine (NE)-induced contractions and intracellular Ca2+ concentration ([Ca2+]i) simultaneously, in the presence or absence of different K+ channel openers or blockers. In adult MCA, LTH was associated with approximately 20% decrease in NE-induced tension and [Ca2+]i, with a significant increase in Ca2+ sensitivity. In contrast, in fetal MCA, LTH failed to affect significantly NE-induced contraction or [Ca2+]i but significantly decreased the ATP-sensitive K+ (K(ATP)) channel and Ca2+-activated K+ (K(Ca)) channel-mediated relaxation. The significant effect of K(ATP) and K(Ca) channel activators on the relaxation responses and the fact that K+ channels play a key role in myogenic tone support the hypotheses that K+ channels play an important role in hypoxia-mediated responses. These results also support the hypothesis of significant developmental differences with maturation from fetus to adult.     

Further investigation of a potential calcium channel modulator isolated from rat erythrocytes

The contractile effects of a peptide isolated from rat erythrocytes were further studied in rat aortic rings. Previous data showed that preincubation of aortic tissue with the peptide had no effect on resting tension, but significantly enhanced K+ and norepinephrine (NE) induced contraction. The calcium channel antagonist verapamil noncompetitively blocked the effect of the peptide, whereas nifedipine blockage appeared to be competitive. In the present study the peptide enhanced K+, NE, and phenylephrine (PE) induced contraction in a concentration-dependent manner, with a maximum enhancement at peptide concentrations of 10(-7)-10(-6) M. At a concentration as low as 10(-9) M, the peptide significantly enhanced K(+)-induced, but not NE- or PE-induced, contraction. The magnitude of maximal enhancement was greater for K(+)-induced contraction than that for NE- or PE-induced contraction. Preincubation of the tissues with the peptide caused a leftward shift of cumulative concentration-response curves to K+ and NE. The peptide enhancement of contraction increased with increasing K+ and NE concentration. The peptide potentiated the contractile response to Ca2+ in K(+)-depolarizing medium. It also enhanced the contractile response to NE in intracellular Ca2(+)-pool-depleted tissue following the replenishment of extracellular Ca2+, but had no apparent effect on the mobilization of intracellular calcium. Addition of nifedipine caused a rightward shift of both the peptide and Bay K 8644 concentration-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraluminal pressure stimulates MAPK phosphorylation in arterioles: temporal dissociation from myogenic contractile response

Members of the MAPK family of enzymes, p42/44 and p38, have been implicated in both the regulation of contractile function and growth responses in vascular smooth muscle. We determined whether such kinases are activated during the arteriolar myogenic response after increases in intraluminal pressure. Particular emphasis was placed on temporal aspects of activation to determine whether such phosphorylation events parallel the known time course for myogenic contraction. Experiments used single cannulated arterioles isolated from the cremaster muscle of rats with some vessels loaded with the fluorescent Ca2+-sensitive dye fura 2 (2 microM). The p42/44 inhibitor PD-98059 (50 microM) caused vasodilation but did not prevent pressure-induced myogenic constriction. The vasodilator response was accompanied by decreased smooth muscle intracellular Ca2+. Western blotting revealed a significant increase in the level of phosphorylation of p42/44 15 min after the application of a 30- to 100-mmHg pressure step. Phosphorylation of p42/44 was a late event that appeared to be temporally dissociated from contraction, which was complete within 1-5 min. EGF (80 nM) caused marked phosphorylation of p42/44 but only acted as a weak vasoconstrictor. The p38 inhibitor SB-203580 (10 microM) did not alter baseline diameter, nor did it prevent myogenic vasoconstriction. Consistent with these observations, SB-203580 did not cause a measurable change in intracellular Ca2+. The results demonstrate activation of the p42/44 class of MAPK resulting from increased transmural pressure. Such activation is, however, dissociated from the acute pressure-induced vasoconstrictor response in terms of time course and may represent the activation of compensatory, but parallel, pathways, including those related to growth and remodeling.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

Regulation of metabolism and contraction by cytoplasmic calcium in the intestinal smooth muscle

Reduced pyridine nucleotides (PNred) and oxidized flavoproteins (FPox) were measured fluorometrically in the intestinal smooth muscle strip of guinea pig taenia caeci simultaneously with contractile tension. Cytoplasmic free Ca2+ levels ([Ca2+]cyt) were also measured by a fura-2-Ca2+ fluorescence technique. PNred, FPox, and [Ca2+]cyt increased during spontaneous contraction or upon the addition of high K+ or carbachol and decreased upon the removal of these stimulants. [Ca2+]cyt increased before the increase in muscle tension. PNred increased almost simultaneously with or immediately after the onset of contraction, while FPox increased before the initiation of contraction. Both PNred and FPox decreased a few seconds after the initiation of relaxation. In the K+-depolarized, Ca2+-depleted muscle, graded elevation of external Ca2+ increased PNred, FPox, and muscle tension. The sensitivity to Ca2+ was in the order of FPox greater than PNred greater than muscle tension. Changes in PNred were inhibited when glycolysis was inhibited by substitution of external glucose with oxaloacetate, pyruvate, or beta-hydroxybutylate, but not when oxidative phosphorylation was inhibited by N2 bubbling or by NaCN. In contrast to this, changes in the FPox were inhibited by N2 bubbling or NaCN, but not by the inhibition of glycolysis. These results suggest that an elevation of intracellular Ca2+ activates carbohydrate metabolism and contractile elements independently, resulting in the reduction of cytoplasmic pyridine nucleotides, oxidation of mitochondrial flavoproteins, and development of tension in the intestinal smooth muscle.     

Effects of impaired Ca2+ homeostasis on contraction in postinfarction myocytes.

The significance of altered Ca2+ influx and efflux pathways on contractile abnormalities of myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) was investigated by varying extracellular Ca2+ concentration ([Ca2+]o, 0.6-5.0 mM) and pacing frequency (0.1-5.0 Hz). Myocytes isolated from 3-wk MI hearts were significantly longer than those from sham-treated (Sham) hearts (125 +/- 1 vs. 114 +/- 1 micrometer, P < 0.0001). At high [Ca2+]o and low pacing frequency, conditions that preferentially favored Ca2+ influx over efflux, Sham myocytes shortened to a greater extent than 3-wk MI myocytes. Conversely, under conditions that favored Ca2+ efflux (low [Ca2+]o and high pacing frequency), MI myocytes shortened more than Sham myocytes. At intermediate [Ca2+]o and pacing frequencies, differences in steady-state contraction amplitudes between Sham and MI myocytes were no longer significant. Collectively, the interpretation of these data was that Ca2+ influx and efflux pathways were subnormal in MI myocytes and that they contributed to abnormal cellular contractile behavior. Because Na+/Ca2+ exchange activity, but not whole cell Ca2+ current, was depressed in 3-wk MI rat myocytes, our results on steady-state contraction are consistent with, but not proof of, the hypothesis that depressed Na+/Ca2+ exchange accounted for abnormal contractility in MI myocytes. The effects of depressed Na+/Ca2+ exchange on MI myocyte mechanical activity were further evaluated in relaxation from caffeine-induced contractures. Because Ca2+ uptake by sarcoplasmic reticulum was inhibited by caffeine and with the assumption that intracellular Na+ and membrane potential were similar between Sham and MI myocytes, myocyte relaxation from caffeine-induced contracture can be taken as an estimate of Ca2+ extrusion by Na+/Ca2+ exchange. In MI myocytes, in which Na+/Ca2+ exchange activity was depressed, the half time of relaxation (1.54 +/- 0.14 s) was significantly (P < 0.02) prolonged compared with that measured in Sham myocytes (1.10 +/- 0.10 s).     

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.     

The effect of lanthanum on the mechanical function of the isolated perfused rat heart at different extracellular calcium concentrations.

The effects of 50 microM lanthanum (La3+) on the contractile force, rate and coronary flow of rat hearts perfused with solutions containing 2.5, 5, 7.5 mM calcium (Ca2+) have been investigated. La3+ produced a rapid and marked decrease in contractile force within 1-3 min ("early La(3+)-effect"). The inhibition of contractility by La3+ was reduced progressively when the Ca2+ ion concentration in the perfusion fluid was raised from 2.5 to 7.5 mM. However, after 10-80 min of La3+ perfusion the contractile force was increased significantly ("late La(3+)-effect"). Elevation of Ca2+ during exposure to La3+ increased its effect. During the late La(3+)-effect, a marked decrease in heart rate and a significant increase in time to reach peak tension, time for half relaxation and twitch duration was observed. High concentrations of perfusate Ca2+ decreased the chronotropic response to La3+, in contrast, elevated Ca2+ potentiated La(3+)-induced increase in time to reach peak tension, time for half relaxation and twitch duration. La3+ produced a significant decrease in coronary flow. High Ca2+ augmented the decrease coronary flow. The findings indicate that La3+ may produce marked effects on myocardial function. High extracellular Ca2+ reduces the La(3+)-induced initial decrease in force of contraction, but potentiates the late increase in contractile force by La3+. Elevated external Ca2+ also increases the effects of La3+ on twitch parameters, heart rate and coronary flow.     

Hypoxic contractile response in isolated human pulmonary artery: role of calcium ion

The mechanism for hypoxic pulmonary vasoconstriction (HPVC) was investigated in human pulmonary arterial strips. Hypoxia in the presence of histamine (10(-6) M) caused marked pulmonary arterial contraction, which was reversed by O2. The hypoxic contraction in the presence of histamine was inhibited by diphenhydramine, but not by cimetidine. The hypoxic histamine-mediated contraction was attenuated but still present in the absence of extracellular Ca2+, or by the inhibitors of voltage-dependent Ca2+ influx. However, it was inhibited significantly by a further depletion of intracellular Ca2+, or by HA 1004, an intracellular calcium antagonist. A low concentration (10(-7) M) of a calcium ionophore, A23187, enhanced the hypoxic contraction in the presence of histamine, whereas procaine completely inhibited it. W-7, a calmodulin inhibitor, significantly decreased the hypoxic histamine-mediated contraction, but 12-O-tetradecanoylphorbol-13-acetate (TPA), a C-kinase promotor, had no effect. The hypoxic contractile response was also observed in the presence of both A23187 and KCl instead of histamine, but the hypoxia-induced contraction with KCl alone was much smaller than that. These results indicate that hypoxia in the presence of certain other vasoactive agents has a potent contractile effect on the human pulmonary artery and that the response is dependent on Ca2+. Enhancement of both Ca2+ influx and Ca2+ release from intracellular storage sites by hypoxia, which interacts with calmodulin, were suggested to be involved in the mechanism of HPVC.     

Use of acute phenolic denervation to show the neuronal dependence of Ca2+-induced relaxation of isolated arteries

We recently showed that perivascular sensory nerves of mesenteric resistance arteries (MRA) express a receptor for extracellular Ca2+ (CaR) and proposed that activation of the CaR by Ca2+ causes nerve-dependent vascular relaxation. We now describe a novel procedure for acutely denervating isolated arteries and have used this method to test the hypothesis that Ca2+-induced relaxation of MRA is nerve dependent. MRA were studied using a wire myograph equipped with electrodes for electrical field stimulation (EFS) which caused sympathetic nerve-mediated contraction, and when applied in the presence of guanethidine, induced nerve-mediated relaxation. Ca2+-induced relaxation was produced by the cumulative addition of Ca2+ to MRA precontracted with norepinephrine. Exposure of MRA to 6.5% phenol in ethanol for 20 sec significantly attenuated EFS-induced contraction and relaxation, and Ca2+-induced relaxation. The magnitude of the relaxation response to EFS correlated significantly with the decrease in Ca2+-induced relaxation. In contrast, endothelium-dependent relaxation induced by acetylcholine was slightly, but nonsignificantly decreased by phenol treatment and did not correlate with Ca2+-induced relaxation. These data indicate that brief exposure of isolated MRA to phenol significantly impairs perivascular nerve function and support the hypothesis that Ca2+-induced relaxation is neurally mediated.     

Three different vasoactive responses of rat tail artery to nicotine

The vasoactive effects of nicotine on isolated rat tail artery tissues were studied. Nicotine transiently contracted rat tail artery tissues (EC50, 55.6 +/- 2 microM) in an extracellular Ca2+ dependent and endothelium-independent fashion. The blockade of alpha1-adrenoceptors, but not alpha2-adrenoceptors or P2X purinoceptors, inhibited the nicotine-induced contraction by 38 +/- 7% (p < 0.05). Nicotine (1 mM) depolarized membrane by 13 +/- 3 mV, but did not affect L-type Ca2+ channel currents, of the isolated rat tail artery smooth muscle cells. The phenylephrine-precontracted tail artery tissues were relaxed by nicotine (EC50, 0.90 +/- 0.31 mM), which was significantly inhibited after the blockade of nicotinic receptors. Simultaneous removal of phenylephrine and nicotine, after a complete relaxation of the phenylephrine-precontracted tail artery strips was achieved by nicotine at accumulated concentrations (> or =10 mM), triggered a Ca2+-dependent rebound long-lasting vasoconstriction (n = 20). This rebound contraction was abolished in the absence of calcium or in the presence of tetracaine in the bath solution. Pretreatment of vascular tissues with a nicotinic receptor antagonist did not affect the nicotine-induced vasoconstriction or nicotine withdrawal induced rebound contraction. The elucidation of the triphasic vascular effects of nicotine and the underlying mechanisms is important for a better understanding of the complex vascular actions of nicotine.     

Reactivation of Ca2+-dependent cytoplasmic contraction in permeabilized cell models of the heliozoon Echinosphaerium akamae

Permeabilized cell models of the large heliozoon Echinosphaerium akamae were prepared by treatment with 100 mM EGTA or 1% Triton X-100. When > 10(-6) M Ca(2+) was added to the EGTA-permeabilized cells, axopodial cytoplasm became contracted and several swellings were formed along the axopodial length. Axonemal microtubules remained intact, while higher concentration of Ca(2+) (> 10(-4) M) induced microtubule disassembly and complete breakdown of the axopodia. In Triton-permeabilized cells, cytoplasmic contraction and relaxation of the cell body were induced repeatedly by successive addition and removal of Ca(2+). The contraction did not require ATP, and was not inhibited by cytochalasin B. Electron microscopy showed, in EGTA-permeabilized axopodia, contractile tubules became granulated by the addition of Ca(2+). From these observations, it is strongly suggested that Ca(2+)-dependent granulation of the contractile tubules is responsible for the axopodial contraction.     

Activation of nitric oxide synthase (NOS3) by mechanical activity alters contractile activity in a Ca2+-independent manner in cardiac myocytes: role of troponin I phosphorylation

Cardiac myocytes express the calcium-responsive nitric oxide synthase (eNOS or NOS3). Activation of NOS3 by increased intracellular Ca2+ concentration, [Ca2+]i, has been demonstrated to decrease myocyte contractile responsiveness, although this appears to occur in a Ca2+-independent manner. Therefore, the aim of this study was to examine the possibility that contractile activity could be modulated by an NO-mediated alteration in the phosphorylation status of troponin I, which is known to alter myofilament sensitivity to Ca2+. During pacing at 3 Hz, 32P-labeled myocytes exhibited a 59 +/- 9% increase in TnI phosphorylation compared to quiescent cells (p < 0.05), an effect that was significantly attenuated by either methylene blue or l-nitroarginine (l-NA). While exposure to methylene blue significantly increased the contractile amplitude of paced myocytes, this was not accompanied by an alteration in intracellular Ca2+. These data indicate that the NO-mediated effects on myocyte contraction may be elicited through an alteration in myofilament Ca2+ sensitivity that results from an alteration in the phosphorylation status of troponin I.     

ACE inhibition prevents diastolic Ca2+ overload and loss of myofilament Ca2+ sensitivity after myocardial infarction

Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16- PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function.     

Prolonged relaxation of detergent-skinned smooth muscle involves decreased endogenous phosphatase activity

Since contraction of smooth muscle involves Ca2+-dependent phosphorylation of the 20 Kd myosin light chains, changes in endogenous phosphatase activity may participate in regulating smooth muscle contractility. We found that detergent-skinned fibers from 7 of 10 chicken gizzards studied were characterized by relatively high endogenous light chain phosphatase activity (23 mU/mg protein) and rapid relaxation (t1/2 = 1-3 min) in the absence of Ca2+ (less than 10(-8) M). In contrast, skinned fibers from 3 of the gizzards exhibited very low phosphatase activity (3 mU/mg protein) and markedly prolonged relaxation (t1/2 = 50-200 min). However, such slow relaxing fibers were converted to a form resembling rapidly relaxing fibers (t1/2 = 4-10 min) when an aortic polycation-modulable phosphatase was included in the incubation medium. Moreover this phosphatase-enhanced relaxation was associated with dephosphorylation of the light chains. Maximal isometric force (1 mN) and light chain phosphorylation (0.8 mol PO4/mol light chain) were similar in slowly and rapidly relaxing fibers. Thus, the two populations of skinned fibers, though dramatically different with respect to phosphatase activity and relaxation time, appeared to be very similar in terms of Ca2+-dependent contraction. These findings strongly suggest that prolonged relaxation of smooth muscle of the kind noted in this study, and perhaps in hypertensive or aging vascular smooth muscle, may reflect decreased endogenous phosphatase activity.     

Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle

A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.     

Molecular aspects of arterial smooth muscle contraction: focus on Rho

The vascular smooth muscle cell is a highly specialized cell whose primary function is contraction and relaxation. It expresses a variety of contractile proteins, ion channels, and signalling molecules that regulate contraction. Upon contraction, vascular smooth muscle cells shorten, thereby decreasing the diameter of a blood vessel to regulate the blood flow and pressure. Contractile activity in vascular smooth muscle cells is initiated by a Ca(2+)-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. Ca(2+)-sensitization of the contractile proteins is signaled by the RhoA/Rho-kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase, thereby maintaining force. Removal of Ca(2+) from the cytosol and stimulation of myoson phosphatase initiate the relaxation of vascular smooth muscle.