Sustained Phase Of Contraction Research Articles

Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta

BackgroundElectrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments.ResultsIntracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644).ConclusionThe isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr(850) phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr(850)-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

Smooth Muscle Myosin Phosphorylated at Single Head Shows Sustained Mechanical Activity

Smooth muscle contraction is regulated by the phosphorylation of myosin. It is well known that tonic smooth muscles can maintain force with low energy consumption (latch state); however, the molecular mechanism underlying this phenomenon is unresolved. Here we show that single-head phosphorylated smooth myosin (SHPMII) exhibits fast ( approximately 24 s(-1)) and slow prolonged ( approximately 1 s(-1)) actin interactions, whereas double-head phosphorylated myosin (DHPMII) predominantly exhibits the fast ( approximately 29 s(-1)) interaction, suggesting that the phosphorylated head of SHPMII is mechanically as active as that of DHPMII. Both the fast and the slow actin interactions of SHPMII support the positive net mechanical displacement of actin. The actin translocating velocity of SHPMII was much slower than that of DHPMII, which is consistent with the slow actin interaction of SHPMII. We propose that the "latch state" can be explained by the motor characteristics of SHPMII that is present during the sustained phase of contraction.

An important role for the Na+-Ca2+ exchanger in the decrease in cytosolic Ca2+ concentration induced by isoprenaline in the porcine coronary artery.

The role of the Na+-Ca2+ exchanger (NCX) in the mechanism of the isoprenaline (Iso)-induced vasorelaxation was investigated by simultaneously monitoring the intracellular Ca2+ concentration ([Ca2+]i) and tension of fura-2-loaded medial strips of porcine coronary arteries. Normal physiological salt solution (PSS) contained 137.3 mM Na+ and 5.9 mM K+. During the sustained phase of contraction, Iso induced only a transient decrease in [Ca2+]i when contraction was induced by depolarization with 118 mM K+ solution containing 25.2 mM Na+. When contraction was induced with 30 mM K+ in PSS containing 113.2 mM Na+, Iso induced a sustained decrease in [Ca2+]i, whereas in contractions induced by 30 mM K+ in a low Na+ (25.2 mM Na+) PSS, Iso transiently decreased [Ca2+]i. Replacement of Ca2+ with Ba2+ (which cannot be extruded by the Ca2+ pumps but can be extruded through the NCX) resulted in decreased [Ba2+]i induced by Iso in normal but not in low Na+ PSS. On the other hand, Iso induced a sustained decrease in [Ca2+]i when strips were pre-contracted by U46619, a thromboxane A2 analogue, in PSS. Various types of K+ channel blockers (iberiotoxin, 4-aminopyridine, apamin or glibenclamide) or combinations of these blockers failed to completely inhibit the Iso-induced decreases in [Ca2+]i and tension. However, Iso-induced sustained decreases in [Ca2+]i during the contraction induced by U46619 were greatly inhibited in a low Na+ PSS. The Iso-induced decrease in tension during contraction by U46619 was greatly inhibited by 2',4'-dichlorobenzamil, a forward- and reverse-mode NCX inhibitor, but not by ouabain, a selective inhibitor of Na+,K+-ATPase. These results indicate that the NCX is involved in the Iso-induced reduction of [Ca2+]i and tension of the porcine coronary arterial smooth muscle.

Alpha1-Adrenoceptor-mediated phosphorylation of myosin in rat-tail arterial smooth muscle.

The mechanism of alpha1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the alpha1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 microM. The contractile response to a submaximal concentration of cirazoline (0.3 microM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 microM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22+/-0.06 mol of Pi/mol of LC20 (n=3) and reached a maximum of 0.58+/-0.08 mol of Pi/mol of LC20 (n=3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by re-addition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 microM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 microM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 microM cirazoline were partially inhibited by chloroethylclonidine (50 microM), suggesting the involvement of both alpha1A and alpha1B adrenoceptors in these contractile responses.

P-664 - α1-Adrenoceptor-mediated phosphorylation of myosin in rat tail arterial smooth muscle

The mechanism of alpha1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the alpha1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 microM. The contractile response to a submaximal concentration of cirazoline (0.3 microM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 microM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22+/-0.06 mol of Pi/mol of LC20 (n=3) and reached a maximum of 0.58+/-0.08 mol of Pi/mol of LC20 (n=3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by re-addition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 microM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 microM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 microM cirazoline were partially inhibited by chloroethylclonidine (50 microM), suggesting the involvement of both alpha1A and alpha1B adrenoceptors in these contractile responses.

Tyrosine Phosphorylation and Regulation of Swine Carotid Artery Contraction

Regulation of [Ca2+]i and modulation of the [Ca2+]i sensitivity of myosin phosphorylation in smooth muscles may involve phosphorylation of one or more proteins on tyrosine residues. We tested this hypothesis by measuring tyrosine phosphorylation of proteins extracted from swine carotid artery and separated by SDS gel electrophoresis. Tyrosine phosphorylation was estimated by the binding of antiphosphotyrosine antibodies to proteins in SDS gels. We found four bands with approximate molecular weights of 120, 110, 85 and 75 kD in which tyrosine phosphorylation increased 1 min after histamine stimulation. After washout of histamine, dephosphorylation of the proteins in these four bands occurred at a slower rate than relaxation. Tyrosine phosphorylation of these four protein bands did not correlate with the [Ca2+]i sensitivity of myosin phosphorylation following agonist or high [K+]o stimulation. Phorbol dibutyrate stimulation also induced tyrosine phosphorylation of these four protein bands. These data suggest that tyrosine phosphorylation of the proteins in these four bands may be involved in the initial phase of swine carotid artery contraction. However, there was, at most, only a minor involvement of tyrosine phosphorylation of these four protein bands in the sustained phase of contraction or in the regulation of [Ca2+]i sensitivity of myosin phosphorylation in the swine carotid artery. These data do not rule out a role for other, less abundant tyrosine phosphoproteins in the regulation of sustained contraction or the [Ca2+]i sensitivity of myosin phosphorylation.

Agonist-induced production of 1,2-diacylglycerol and phosphatidic acid in intact resistance arteries. Evidence that accumulation of diacylglycerol is not a prerequisite for contraction.

The production of total amounts of 1,2-diacylglycerol as well as those specifically derived from inositol lipid hydrolysis was studied in intact rat resistance arteries stimulated with either noradrenaline, vasopressin, or angiotensin II at 20 s when the onset of contraction would be nearing its maximum, and at 5 min during the sustained phase of contraction. Total amounts of 1,2-diacylglycerol were not altered by any agonist at 20 s, or at 5 min. However, arachidonate-containing species of 1,2-diacylglycerol were differentially influenced being increased at 5 min by noradrenaline, and decreased at 20 s and 5 min by vasopressin. Only angiotensin II produced substantial increases in this class of 1,2-diacylglycerol at both time points. In order to investigate the fate of this second messenger total and inositol lipid derived phosphatidic acids were then measured at both 20 s and 5 min. Noradrenaline induced a rise in both total and arachidonate-containing phosphatidic acid at both times as did vasopressin. Only small increases were induced by angiotensin II at 20 s. These data demonstrate that the accumulation of 1,2-diacylglycerol generated from inositol lipid breakdown is only observed with activation by angiotensin II. Other agonists produced phosphatidic acids with time and the rate of generation of these lipids is agonist-specific. Thus phosphatidic acid may play a more prominent role during the sustained phase of contraction than previously anticipated.

Tachykinins: local gastric effects and brain stem responses

The gastric motor or mechanical effects of a group of peptides, the tachykinins, were evaluated in anesthetized cats to determine the relationship between local motor events and brain stem neurons that regulate gastric activity. The peptides evaluated were substance P, physalaemin, and eledoisin. The tachykinin-induced gastric changes were dose related and were characterized by initial distention-sustained contraction-late distention phases. At lower doses distention was the dominant effect with a sustained contraction-late distention response appearing as the dose increased. The sustained contraction-late distention phases were frequently accompanied by phasic contractions with a frequency of 2-4/min. Atropine had a significant effect on the sustained contraction phase but no effect on the phasic contractions or distention phases. Bilateral cervical vagotomy had a significant effect on the early distention phase, suggesting a link with brain stem mechanisms. The activity of brain stem units that responded to phasic distention of the stomach reflected the tachykinin-induced changes in gastric distention. Although the gastric effects of these tachykinins shared distinct similarities, certain differences in the time sequence of the distention-contraction interactions suggests the possibility that dissimilar receptor types may be involved in the mechanisms of action. Their mechanisms of action may also involve a direct effect on the effector organ.

Lactate oxidation by skeletal muscle during sustained contraction in vivo.

Estimation of lactate oxidation in vivo was carried out by a tracer technique in the muscles of the lower hindleg of the dog during tetani of different duration. The fractional (%) rate of lactate oxidation increased markedly, compared with that of the resting muscle, after the first 2 min of stimulation. Lactate oxidation afforded a large contribution to the total oxygen consumption. Fatigue appeared to play a role in limiting lactate oxidation in the later phase of sustained contraction.