Effect of Mg2+ on stress, myosin phosphorylation, and ATPase activity in detergent-skinned swine carotid media.

Smooth muscle contraction has a relatively high requirement for free magnesium (Mg2+). In this study we examined the effect of Mg2+ concentration ([Mg2+]) on Ca2+-dependent stress development and stress maintenance, myosin ATPase activity, and myosin light chain (MLC) phosphorylation levels in Triton X-100 detergent-skinned fibers of the swine carotid media. Increasing [Mg2+] in a stepwise fashion from 0.1 to 6 mM 1) decreased the magnitude and Ca2+ sensitivity of stress development but augmented the amount of stress maintained without proportional MLC phosphorylation, 2) produced a greater decrease in the Ca2+ sensitivity of MLC phosphorylation than that of stress development, and 3) decreased myosin ATPase activity. These findings demonstrate that Mg2+ differentially modulates the MLC phosphorylation-dependent development of stress and the MLC phosphorylation-independent maintenance of stress. We suggest that increases in [Mg2+] enhance stress maintenance by increasing [MgADP], thus increasing the number of cross bridges in a force-generating state, and by a direct effect on the pathway responsible for Ca2+-dependent, MLC phosphorylation-independent contractions.

Ca(2+)- and protein kinase C-dependent stimulation of mitogen-activated protein kinase in detergent-skinned vascular smooth muscle.

Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery.

Sodium hydrosulfite contractions of smooth muscle are calcium and myosin phosphorylation independent.

In an effort to further understand the processes underlying hypoxic pulmonary vasoconstriction, we examined the mechanism by which sodium hydrosulfite (Na2S2O4), a potent reducing agent and oxygen scavenger, induces smooth muscle contraction. In rat pulmonary arterial strips, sodium hydrosulfite (10 mM) induced contractions that were 65.9 +/- 12.8% of the response to 60 mM KCl (n = 9 segments). Contractions were not inhibited by nisoldipine (5 microM) or by repeated stimulation with caffeine (10 mM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (10 microM), or cyclopiazonic acid (10 microM), all of which eliminated responses to contractile agonists. Maximum force generation after exposure to sodium hydrosulfite was 0.123 +/- 0.013 mN in the presence of 1.8 mM calcium and 0.127 +/- 0.015 mN in the absence of calcium. Sodium hydrosulfite contractions in pulmonary arterial segments were not due to the generation of H2O2 and occurred in the presence of chelerythrine (10 microM), which blocked phorbol ester contractions, and solution hyperoxygenation. Similar contractile responses were obtained in rat aortic and tracheal smooth muscles. Finally, contractions occurred in the complete absence of an increase in myosin light chain phosphorylation. Therefore sodium hydrosulfite-induced smooth muscle contraction is not specific to pulmonary arterial smooth muscle, is independent of calcium and myosin light chain phosphorylation, and is not mediated by either hypoxia or protein kinase C.

Caldesmon inhibits active crossbridges in unstimulated vascular smooth muscle: an antisense oligodeoxynucleotide approach.

Caldesmon is a thin-filament-associated protein believed to be important in the regulation of smooth muscle contraction, although the precise mechanism is unknown. We used antisense oligodeoxynucleotides to produce intact swine carotid smooth muscle tissue deficient in h-caldesmon. Caldesmon content was decreased by 78% after 7 days in culture with antisense oligodeoxynucleotides but was unchanged in tissues in the presence of sense oligodeoxynucleotides or vehicle. Antisense oligodeoxynucleotides produced a significant decrease in the caldesmon/actin ratio, but no change was measured in the calponin/actin ratio, suggesting that the effect was specific to caldesmon and not other thin-filament-associated proteins. Basal and KCl-stimulated levels of myosin light chain phosphorylation were not different among tissues from all 3 groups. In contrast, h-caldesmon-deficient tissues produced 62% less KCl-induced force than controls. Unstimulated h-caldesmon-deficient smooth muscle tissues stretched and then released, redeveloped force, demonstrating active crossbridge cycling; strips containing normal h-caldesmon content did not redevelop force on release. We suggest that in resting vascular smooth muscle, active crossbridges are inhibited by caldesmon. Therefore, regulation of smooth muscle includes a thin-filament-based disinhibition component.

Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate.

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.

Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery.

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95-98). MAP kinase phosphorylation, stimulated by histamine (50 microM) or phorbol 12,13-dibutyrate (PDBu, 0.1 microM), was inhibited by PD-098059 (100 microM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of beta-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.

Mechanism of galanin-induced contraction of longitudinal smooth muscle of the rat jejunum.

Intact and alpha-toxin-permeabilized longitudinal smooth muscle were mounted for measurement of force and myosin light chain phosphorylation. Galanin contracted intact jejunum with a half-maximum effective concentration of 9.2 +/- 0.1 nM. Neither atropine, hexamethonium, guanethidine, nor tetrodotoxin affected the contraction. The contraction was also unaffected by depletion of intracellular Ca2+ or by addition of thapsigargin; removal of extracellular Ca2+ or addition of nifedipine abolished the contraction. Galanin increased myosin light chain phosphorylation levels concomitantly with force. During continued tissue stimulation, force fell to suprabasal values, whereas myosin light chain phosphorylation levels remained elevated. Galanin increased Ca2+ sensitivity of contraction in alpha-toxin-permeabilized tissues, and this was reversed by either guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. These results suggest that galanin-induced contraction of longitudinal jejunal smooth muscle is dependent on a pertussis toxin-sensitive G protein that is apparently not coupled to the release of intracellular Ca2+ but to the influx of extracellular Ca2+ and involves an initial myofilament Ca2+ sensitization followed by Ca2+ desensitization.

Cyclic AMP and cyclic GMP relax phorbol ester-induced contractions of rat aorta by different mechanisms.

This study was designed to test the hypothesis that 8-Br-cAMP and 8-Br-cGMP dependent relaxation of phorbol dibutyrate stimulated contractions of intact rat aorta are independent of changes in the level of myosin light chain phosphorylation. Phorbol dibutyrate stimulated contraction with a concomitant increase in myosin light chain phosphorylation in normal tissues and without an increase in myosin light chain phosphorylation in calcium-depleted tissues. Phorbol dibutyrate stimulated contractions in normal CaCl2-containing physiological salt solution were relaxed in a concentration-dependent manner by 8-Br-cAMP and 8-Br-cGMP. Phorbol dibutyrate-induced contractions in the absence of Ca2+ were only relaxed by 8-Br-cGMP; 8-Br-cAMP had no effect. The relaxation induced by 8-Br-cGMP was associated with a decrease in myosin light chain phosphorylation suggesting that cGMP-dependent protein kinase may alter the activity of either the myosin light chain kinase or phosphatase. The relaxation induced by 8-Br-cAMP was not associated with a decrease in phosphorylation suggesting that cAMP-dependent protein kinase may uncouple myosin light chain phosphorylation from force.

Calcium-and phorbol ester-dependent calponin phosphorylation in homogenates of swine carotid artery.

Calponin inhibits actin-activated myosin adenosinetriphosphatase (ATPase) activity, and phosphorylation reverses this inhibition. Calponin phosphorylation has been demonstrated in reconstituted contractile protein systems, but studies using intact smooth muscle have produced mixed results. The goal of this study was to determine if vascular smooth muscle contains the necessary biochemical machinery to catalyze calponin phosphorylation. We used swine carotid homogenate, which allows access to the intracellular components and contains all endogenous proteins and enzymes in physiologically relevant concentrations. We demonstrated that calponin is phosphorylated in response to Ca2+ (0.27 +/- 0.04 mol P(i)/mol calponin) and in response to phorbol 12,13-dibutyrate in the presence or absence of Ca2+ (0.48 +/- 0.09 mol P(i)/mol calponin). Calponin phosphorylation was inhibited by the protein kinase C inhibitor staurosporine but not by the Ca(2+)- and calmodulin-dependent protein kinase II inhibitor KN-62. We conclude that Ca(2+)-dependent and -independent isoforms of protein kinase C but not the Ca(2+) -and calmodulin-dependent protein kinase II catalyze calponin phosphorylation in the swine carotid artery.

Effects of hypertension on hypercholesterolemia-induced changes in contraction of rabbit aorta and carotid artery.

Reactivity of aortic and carotid strip from control; hypertensive; hypercholesterolemic; and hypertensive/hypercholesterolemic rabbits were studied. Maximal stress was less in strips from hypertensive/hypercholesterolemic animals. Norepinephrine sensitivity was increased in the carotid artery from hypertensive/hypercholesterolemic animals (EC50: 0.11 microM; 0.35 microM control). CaCl2 sensitivity during norepinephrine-induced contractions was enhanced by hypertension and hypercholesterolemia (carotid EC50: 0.10 mM; 0.38 mM control; aorta EC50: 0.12 mM; 0.82 mM control). Similar results were obtained during membrane depolarization. 5-hydroxytryptamine sensitivity (EC50: 0.15 microM carotid; 0.18 microM aorta) was decreased during hypertension (EC50: 0.51 microM carotid; 1.13 microM aorta) and by hypercholesterolemia (EC50: 1.76 microM carotid; 1.53 microM aorta). Our results support the hypothesis that hypertension and hypercholesterolemia increase vascular sensitivity by increasing Ca2+ permeability. Our results also suggest that hypertension and hypercholesterolemia selectively decrease 5-hydroxytryptamine-induced contractions.

Decreased PO2 and rabbit aortic smooth muscle mechanics.

Rabbit thoracic aorta was used to determine the effects of decreasing PO2 on the mechanical properties of contractions in response to norepinephrine (NE) and KCl. Aortae were aerated with 45% O2/5% CO2/50% N2 and stimulated with 10 microM NE or 50 mM KCl. At 5 min of stimulation, 5% CO2/95% N2 aeration was introduced for 15 min, defined as hypoxia. This time period was previously shown to produce similar decrease in [ATP] in either stimulation condition. Force, stiffness and isotonic shortening velocity were monitored during the initial stimulation, during hypoxia and during re-oxygenation. Hypoxia produced a substantial and rapid decrease in force and a concomitant decrease in stiffness during NE stimulation; delayed and smaller decreases in force and stiffness were observed during KCl stimulation. The force-stiffness relationship was steeper during KCl than NE stimulation, and hypoxia did not affect these relationships. Isotonic shortening velocity was significantly depressed by hypoxia during both stimulations although the decrease during KCl stimulation required a longer time. These data demonstrate that relaxation of an agonist-induced contraction in response to hypoxia results from a decrease in the number of activated crossbridges and not formation of rigor bridges.

Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery.

Caldesmon phosphorylation has been proposed to be involved in regulation of smooth muscle contraction. Mitogen-activated protein (MAP) kinase has been suggested to be the caldesmon kinase; stimulation-induced MAP kinase activation in intact vascular smooth muscle, however, has not been demonstrated. We measured temporal profiles of MAP kinase activation in response to histamine stimulation and membrane depolarization in intact swine carotid artery. Phosphotyrosine levels of 42- and 44-kDa MAP kinases were elevated during contraction in response to histamine or KCl. The temporal profile of MAP kinase activation/inactivation was similar to that for contraction/relaxation of the vascular tissue in response to KCl or histamine stimulation. MAP kinase activated during contractile stimulation phosphorylates caldesmon with a specific activity significantly greater than that for myelin basic protein-(95-98). We propose that MAP kinase is activated in response to all forms of contractile stimulation. We also suggest that activated MAP kinase phosphorylates and disinhibits the effects of caldesmon on actin-myosin interactions. This disinhibition allows an inherent level of myosin ATPase activity to be expressed.

Regulation of vascular smooth muscle contraction: myosin light chain phosphorylation dependent and independent pathways.

Ca(2+)-dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of smooth muscle contraction. However, MLC phosphorylation alone cannot account for all aspects of contractile regulation, suggesting the involvement of other elements. In this article we present evidence obtained from Triton X-100 detergent skinned and intact tissue which demonstrates that vascular smooth muscle contraction can be initiated by a Ca(2+)-dependent mechanism that does not require prior MLC phosphorylation. We show that Ca2+ can initiate contractions supported by cytidine triphosphate (CTP) and that these contractions are inhibited by calmodulin antagonists, suggesting a Ca(2+)-calmodulin dependence of force distinct from that for MLC phosphorylation. Evidence is presented to demonstrate that carotid medial fibers contain a mitogen-activated protein (MAP) kinase which is activated by Ca2+ and may catalyze caldesmon phosphorylation. Based in part on our results and those of other investigators, we propose that direct Ca(2+)-calmodulin binding to caldesmon or phosphorylation of caldesmon by a Ca(2+)-dependent MAP kinase disinhibits caldesmon. Disinhibition of caldesmon allows an inherent basal level of actin-activated myosin ATPase activity to be expressed. The result is the slow development of force.

A hypothesis for the mechanism of receptor and G-protein-dependent enhancement of vascular smooth muscle myofilament Ca2+ sensitivity.

Agonist activation enhances smooth muscle myofilament Ca2+ sensitivity. The increased force accompanying receptor stimulation (over Ca2+ alone) requires GTP and is reversed by GDP beta S, demonstrating a G-protein dependence. Protein kinase C (PKC) activators, such as phorbol esters, mimic and PKC inhibitors block the agonist-induced increase in Ca2+ sensitivity, suggesting a role for PKC in the regulation of Ca2+ sensitivity. Myosin light chain (MLC) phosphorylation levels are transiently increased by agonist stimulation, but steady-state levels of MLC phosphorylation are similar to those in response to Ca2+ alone. Thus, G-protein-mediated inhibition of MLC phosphatase may account for the initial increase in force development but not the increase in steady-state force. In contrast to MLC, calponin phosphorylation levels are maintained during agonist stimulation of intact vascular smooth muscle. We propose that stimulation of smooth muscle by membrane depolarization increases MLC phosphorylation, but as a result of inhibition by unphosphorylated calponin only a portion of the phosphorylated cross bridges attach to actin. Agonist stimulation produces the same steady-state level of MLC phosphorylation but also leads to calponin phosphorylation via a PKC-dependent pathway. Thus, during agonist stimulation, all phosphorylated cross bridges can interact with actin, thereby generating significantly greater levels of force.

Regulation of Ca(2+)-dependent ATPase activity in detergent-skinned vascular smooth muscle.

We measured force, actin-activated myosin adenosinetriphosphatase (ATPase) activity, and myosin light-chain (MLC) phosphorylation levels in Triton X-100 detergent-skinned media of swine carotid arteries. Pseudo-ATPase activity composed of MLC kinase and phosphatase activities contributed maximally 12% to steady-state tissue ATPase activity. An increase in the Ca2+ concentration ([Ca2+]) induced an increase in force, MLC phosphorylation, and actin-activated myosin ATPase activity; this protocol was defined as the force development phase of contraction. Force maintenance was defined as the state induced by decreasing the [Ca2+] after a maximal contraction. Lowering the [Ca2+] decreased MLC phosphorylation to levels similar to those measured during force development at each [Ca2+]. In contrast, force remained at elevated levels while actin-activated myosin ATPase activity fell to significantly lower levels than those measured during the development phase for each [Ca2+]. We suggest that the significantly lower actin-activated myosin ATPase activity observed during a state of elevated force, compared with the development phase of a contraction, is evidence of slowly cycling latch bridges.

Phorbol ester-induced contractions of swine carotid artery are supported by slowly cycling crossbridges which are not dependent on calcium or myosin light chain phosphorylation.

This study determined if phorbol ester-induced contraction of vascular smooth muscle requires calcium-dependent myosin light chain (MLC) phosphorylation and, if not, whether the mechanical characteristics of the contraction in terms of stiffness and crossbridge cycling are similar to those during a calcium- and MLC phosphorylation-dependent contraction. Carotid arterial strips were exposed to 1.0 microM phorbol 12,13-dibutyrate (PDBu) in the presence of normal physiological salt solution (PSS) or after calcium depletion in calcium-free PSS and compared with contraction elicited by calcium-containing 110 mM KCl-PSS. PDBu induced maximal stress in both the presence and absence of calcium. While there was a temporal correlation between MLC phosphorylation and shortening velocity during KCl depolarization, shortening velocity was dissociated from MLC phosphorylation during PDBu stimulation. The stress-stiffness relationship was not different during KCl and PDBu stimulation, suggesting similar crossbridge interactions even though MLC phosphorylation levels were significantly different. These results demonstrate that PDBu-induced contraction of the swine carotid artery is not dependent on calcium or MLC phosphorylation. We suggest the possibility that activation of a calcium-independent PKC isoform may result in the expression of an inherent level of actin-activated myosin ATPase activity resulting in the slow development of stress.

Endothelin differentially affects rat gastric longitudinal and circular smooth muscle.

We have determined the response of two types of rat gastric smooth muscle to the cumulative addition of endothelin-1 (ET-1). Both the longitudinal smooth muscle (tonic) from the forestomach and the circular smooth muscle (phasic) from the fundus were used in this study. Longitudinal smooth muscle contracted in a concentration-dependent manner in response to ET-1. The ET-1-induced contractions were abolished by the use of either nifedipine or calcium-free solutions. The maximal stress developed was 1.13 +/- 0.12 x 10(5) N/m2, or about 61% of the maximal carbachol response in this tissue. The EC50 for the ET-1-induced contraction was 11.4 +/- 2.3 nM. In contrast to the contractile effect on longitudinal smooth muscle, ET-1 produced a potent concentration-dependent inhibition of both the spontaneous phasic activity and carbachol-stimulated activity of the circular smooth muscle. The maximal inhibitory effect in response to ET-1 occurred at about 10 nM. The abolition of phasic activity lasted about 15 minutes before phasic activity returned. ET-1 also inhibited carbachol-induced increases in the phasic activity of circular smooth muscle with a similar potency. Inhibitors of arachidonic acid products, indomethacin or nordihydroguaiaretic acid, did not affect the response to ET-1 of either longitudinal or circular smooth muscle. Similarly, inhibition of nitric oxide, vasoactive intestinal peptide (VIP) calcitonin-related peptide (CGRP) and inhibition of endogenous neural pathways by tetrodotoxin (TTX), atropine, hexamethonium, cold storage or phentolamine did not reverse the inhibitory response of the circular smooth muscle to ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery.

This study was designed to investigate the mechanism of endothelin-1 (ET-1) contractions in Staphylococcus alpha-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with alpha-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 microM GTP significantly enhanced myofilament Ca2+ sensitivity as compared with the addition of Ca2+ alone (EC50, 0.47 microM Ca2+ for Ca2+ alone and 0.13 microM Ca2+ for ET-1 plus (GTP). This enhanced sensitivity was reversed by GDP beta S. ET-1-induced contractions were relaxed at a constant [Ca2+] by the addition of 30 microM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 microM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 microM Ca2+, steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca2+ dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2+ sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide analogs of the pseudosubstrate domain of smooth muscle myosin light chain kinase inhibit actomyosin ATPase activity at concentrations that do not inhibit superprecipitation.

The inhibitory effect of calmodulin antagonists, synthetic peptide analogs of the pseudosubstrate domain of smooth muscle MLC kinase, and an inhibitor based on the sequence of MLC were examined using bovine aortic actomyosin and isolated chicken gizzard MLC. Much lower concentrations of the peptides were necessary to inhibit actomyosin ATPase activity than to inhibit superprecipitation. In contrast, calmodulin antagonists inhibited both ATPase activity and superprecipitation at similar concentrations. The peptide analogs were competitive with isolated MLC, but not calmodulin, for inhibition of MLC kinase. These results suggest that in addition to the calmodulin dependence of MLC phosphorylation, a second calmodulin-like protein may be important in actin-myosin interactions. The data also suggest that the pseudosubstrate hypothesis may not completely account for regulation of MLC kinase activity.

Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries.

Norepinephrine (NE) plus guanosine triphosphate (GTP) increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized smooth muscle. We used alpha-toxin-permeabilized rabbit mesenteric arteries to determine the temporal relationships among force, myosin light chain (MLC) phosphorylation, stiffness, and shortening velocity during contractions in response to Ca2+ alone and to the same [Ca2+] in the presence of NE plus GTP. The addition of NE plus GTP caused a marked increase in the tonic contraction but only transiently elevated the level of MLC phosphorylation over that observed in the presence of Ca2+ alone. NE plus GTP induced similar increases in force and stiffness, but shortening velocity depended solely on the [Ca2+]. A regulated MLC phosphatase could explain the initial increase in force and MLC phosphorylation, but not the maintenance of enhanced force while MLC phosphorylation levels fell to values similar to those in response to Ca2+ alone. Therefore, additional elements must be involved in the maintenance of the receptor and G protein-dependent increase in myofilament Ca2+ sensitivity.