Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.     

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.     

Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle

It is now well-established that phosphorylation of the 20,000-dalton light chain of smooth muscle myosin (LC20) is a prerequisite for muscle contraction. However, the relationship between myosin dephosphorylation and muscle relaxation remains controversial. In the present study, we utilized a highly purified catalytic subunit of a type-2, skeletal muscle phosphoprotein phosphatase (protein phosphatase 2A) and a glycerinated smooth muscle preparation to determine if myosin dephosphorylation, in the presence of saturating calcium and calmodulin, would cause relaxation of contracted uterine smooth muscle. Addition of the phosphatase catalytic subunit (0.28 microM) to the muscle bath produced complete relaxation of the muscle. The phosphatase-induced relaxation could be reversed by adding to the muscle bath either purified, thiophosphorylated, chicken gizzard 20,000-dalton myosin light chains or purified, chicken gizzard myosin light chain kinase. Incubation of skinned muscles with adenosine 5'-O-(thiotriphosphate) prior to the addition of phosphatase resulted in the incorporation of 0.93 mol of PO4/mol of LC20 and prevented phosphatase-induced relaxation. Under all of the above conditions, changes in steady-state isometric force were associated with parallel changes in myosin light chain phosphorylation over a range of phosphorylation extending from 0.01 to 0.97 mol of PO4/mol of LC20. We found no evidence that dephosphorylation of contracted uterine smooth muscles, in the presence of calcium and calmodulin, could produce a latch-state where isometric force was maintained in the absence of myosin light chain phosphorylation. These results show that phosphorylation or dephosphorylation of the 20,000-dalton myosin light chain is adequate for the regulation of contraction or relaxation, respectively, in glycerinated uterine smooth muscle.     

Carbachol-induced protein phosphorylation changes in bovine tracheal smooth muscle

The changes in protein phosphorylation associated with bovine tracheal smooth muscle contraction were studied by labeling intact muscle strips with [32P]PO4(3-) and analyzing the phosphoproteins by two-dimensional gel electrophoresis. Among 20 to 30 phosphoproteins resolvable with the two-dimensional electrophoresis system, the phosphorylation of 12 proteins was reproducibly affected by treatment with carbachol, in a time-dependent manner. Five of these proteins have been identified as 20-kDa myosin light chain, caldesmon, synemin, and two isoelectric variants of desmin. The other 7 are low molecular weight (Mr less than 40,000) cytosolic proteins. One cytosolic protein and myosin light chain are quickly but transiently phosphorylated by carbachol, the peak of myosin light chain phosphorylation being at about 1 min after agonist addition. In contrast, both variants of desmin, synemin, caldesmon, and 5 cytosolic proteins are phosphorylated at varying rates and remain phosphorylated for the duration of carbachol action. These "late" phosphorylation changes occur simultaneously with the dephosphorylation of one cytosolic protein. These carbachol-induced phosphorylation changes, like the contractile response, appear to be calcium-dependent. The addition of 12-deoxyphorbol 13-isobutyrate, a protein kinase C activator, causes a dose-dependent, sustained contraction of tracheal smooth muscle which develops more slowly than that induced by carbachol. This contractile response is associated with the same protein phosphorylation changes as those observed after prolonged carbachol treatment. In contrast, forskolin, an adenylate cyclase activator and a potent smooth muscle relaxant, induces the phosphorylation protein 3 and one variant of desmin. These observations strongly suggest that different phosphoproteins may be mediators of tension development and tension maintenance in agonist-induced contraction of tracheal smooth muscle.     

Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility

Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.     

Effects of ATP reduction on the pattern of force development and myosin light chain phosphorylation in intact arterial smooth muscle

The effect of reduction of ATP content on phosphorylation of the 20 kDa light chain of myosin (MLC) and force development in intact carotid arterial smooth muscle was investigated. With reduction of ATP to 23% of control by treatment with 2-deoxyglucose there was reduction in basal, in peak and 30 min MLC phosphorylation during contraction (P less than 0.001). The rate of force development was reduced, but maximal force was the same as control. By treatment with 50 microM iodoacetate, the resting ATP content was unchanged but fell to 22% after 30 min contraction. Basal MLC phosphorylation was the same as control, but peak (P less than 0.001) and 30 min phosphorylation were lower (P less than 0.005), even though the rate and magnitude of force development were greater. The results indicate that neither rate nor magnitude of force development correlate with MLC phosphorylation. Basal and initial MLC phosphorylation may play a cooperative role in contractile function.     

Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle

In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase C in glycerinated muscles to levels at least 40 times higher than those present during contraction of intact, phorbol ester-stimulated muscles does not activate contraction nor does it significantly modify the contraction of smooth muscle which occurs in response to the Ca2+/calmodulin-dependent phosphorylation of Ser19 by myosin light chain kinase.     

Biochemical events associated with activation of smooth muscle contraction

Biochemical events associated with activation of smooth muscle contraction were studied in neurally stimulated bovine tracheal smooth muscle. A latency period of 500 ms preceded increases in isometric force and myosin light chain phosphorylation. However, stimulation resulted in the rapid hydrolysis of inositol phospholipids as demonstrated by increases in inositol phosphates by 500 ms. Inositol trisphosphate increased 2-fold with no significant change in inositol tetrakisphosphate. The apparent activation state of myosin light chain kinase was assessed indirectly through measurements of the fractional activation of a second calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. The fractional activation of cyclic nucleotide phosphodiesterase increased after neural stimulation to a maximal extent by 500 ms and remained at this level for at least 4 s. The monophosphorylation of myosin light chain increased after 500 ms and reached a maximum value by 2 s. Diphosphorylation also occurred but to a much lesser extent. Fractional activation of cyclic nucleotide phosphodiesterase and myosin light chain phosphorylation both decreased after 10 min continuous stimulation, although the force response remained at a maximal level. These observations demonstrate that inositol trisphosphate formation and activation of cyclic nucleotide phosphodiesterase (and hence most likely myosin light chain kinase) by calmodulin precede myosin light chain phosphorylation and that these events are sufficiently rapid to mediate the contractile response of neurally stimulated tracheal smooth muscle.     

Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

Effect of vanadate on force and myosin light chain phosphorylation in skinned aortic smooth muscle.

The effects of vanadate were examined on Ca2+-activated force and phosphorylation of 20-kDa myosin light chain in membrane-permeabilized rabbit aortic smooth muscle strips. Addition of vanadate during maximum contraction reduced the force in a dose-dependent manner, and inhibited it almost completely at 1 mM. Two-dimensional polyacrylamide gel electrophoretic analyses revealed that vanadate also reduced the phosphorylation of 20- kDa myosin light chain in a dose-dependent manner from approximately 50% in the absence of vanadate to approximately 20% in the presence of 1 mM vanadate. The effects of 1 mM vanadate on purified myosin light chain kinase and phosphatase were then examined using purified myosin as substrate, and it was found that vanadate neither inhibited myosin light chain kinase nor activated myosin light chain phosphatase. These results indicate that the reduction in the 20-kDa myosin light chain phosphorylation level by vanadate may be effected through its inhibition of the force generation in skinned smooth muscle strip, as evidenced by the finding that vanadate eliminated the enhancement of myosin light chain kinase activity brought about by the interaction between purified myosin and actin.     

Stretch-induced myosin light chain phosphorylation and stretch-release-induced tension development in arterial smooth muscle

Stretching arteries from resting length to 1.7 times the resting length increased myosin light chain phosphorylation from 40 to 70% in a graded fashion, reaching a plateau at 1.6 times the resting length. When the fully stretched arteries were released, active tension developed without any exogenous stimulating agent. This stretch-release-induced tension approached the same magnitude as that of the control K+-induced tension. Stretch-induced phosphorylation and the subsequent tension development upon release of stretch were prevented by incubating the arteries in physiological salt solutions containing ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or chlorpromazine. The inhibition produced by EGTA was reversible. Stretch-induced phosphorylation decreased as a function of time, regardless of whether stretch was maintained, or slackened slowly, or released quickly. While tension developed upon release of stretch, light chain phosphorylation simultaneously decreased. As tension reached and maintained its maximal value, phosphorylation continued to decrease. Thus, light chain phosphorylation is necessary for activation of arterial muscle contraction, but it need not be maintained during tension development or maintenance.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

Myosin phosphorylation, twitch potentiation, and fatigue in human skeletal muscle

Twitch tension and phosphate incorporation into the phosphorylatable light chains (P-light chains) of myosin were studied during a 10-min recovery period following a 10- or 60-s maximal voluntary isometric contraction (MVC) in 18 subjects. Analysis of muscle biopsy samples obtained before, immediately after, 1 min, and 10 min following the 10-s MVC revealed that the 10-s MVC produced a modest but transient metabolic displacement from rest, a 35% decrease in phosphocreatine, and a threefold elevation in lactate concentration. Immediately after the 60-s MVC, ATP was decreased by 20%, phosphocreatine decreased by 84%, and lactate was elevated by 15-fold. Lactate remained elevated over the 10-min recovery period. Twitch force was maximally potentiated following the 10-s MVC and declined to rest by 10 min of recovery. Twitch force was 0.66 of rest value immediately after the 60-s MVC, then increased over the next 4 min to reach a potentiated value 21% greater than rest, before declining. Significant phosphate incorporation into P-light chains was observed immediately after both contractions, but dephosphorylation to rest values at the end of recovery was only noted for the 60-s condition. These results demonstrate an inconsistent relationship between twitch tension enhancement and P-light chain phosphorylation in the in vivo human model.     

Angiotensin II stimulates phosphorylation of the myosin light chain in cultured vascular smooth muscle cells

The vasoactive peptide angiotensin II stimulates phosphorylation of myosin light chain in 32P-labeled confluent cultures of vascular smooth muscle cells derived from rat mesenteric arteries. Myosin light chain was identified and its 32P-phosphorylation level quantitated following selective immunoprecipitation with an antiserum raised against purified human uterine smooth muscle myosin. Following exposure to 0.1 nM angiotensin II, phosphorylation of the light chain peaked at 4 min and then slowly decreased. The stimulation of light chain phosphorylation at 4 min is half-maximal at approximately 0.2 mM angiotensin II; the maximal response is approximately 210% of the unstimulated level. Basal myosin light chain phosphorylation was markedly reduced by incubation of cells with dibutyryl cyclic AMP or the calmodulin-inhibitor chlorpromazine. These data suggest that angiotensin II-mediated contraction in intact blood vessels involves phosphorylation of the myosin light chain, and that phosphorylation is inhibited by a cAMP-mediated process and may be calmodulin-dependent.     

Presence of specific bradykinin receptors in arterial and venous smooth muscle

The relationship between bradykinin action and its concentration was examined on isolated rings of the rabbit aorta, femoral artery, jugular vein and on isolated strips of the rat portal vein. The sensitivity of femoral artery and portal vein smooth muscles to bradykinin was disclosed. Venous smooth muscles were more sensitive to bradykinin as compared with arterial smooth muscles. Dissociation constants for the rabbit aorta, femoral artery, jugular vein and for the rat portal vein were 3.98 X 10(-6), 6.3 X 10(-6), 1.26 X 10(-7), and 7.6 X 10(-9)M, respectively. Effects of endogenous bradykinin in vivo might result from its primary action on the venous smooth muscle, action on the arterial smooth muscle and veno-arterial interactions.     

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.     

A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha -adrenoceptor stimulation

The purpose of this study was to investigate the potential roleof mitogen-activated protein (MAP) kinase in smooth muscle contractionby monitoring MAP kinase activation, caldesmon phosphorylation, andcontractile force during agonist stimulation. Isometric tension inresponse to KCl and phenylephrine (PE) was measured from strips offerret aorta. MAP kinase activation was monitored by Western blot usinga phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmonantibodies. We report here that treatment of smooth muscle strips withPD-098059, a specific inhibitor of MAP kinase kinase, did notdetectably modify the KCl-evoked contraction but significantlyinhibited the contraction to PE in the absence of extracellularCa²⁺. In this experimentalcondition, where the contraction occurs in the absence of increases in20-kDa myosin light chain phosphorylation, PD-098059 also inhibitedsignificantly MAP kinase and caldesmon phosphorylation. Collectively,these results demonstrate a direct cause-and-effect relationshipbetween MAP kinase activation and Ca²⁺-independent smooth musclecontraction and support the concept of caldesmon phosphorylation as themissing link between both events.

     

PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca(2+)](i)) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 x 10(-6) M] in the absence or presence of ERK1/2 inhibition (U-0126, 10(-5) M). After PDBu +/- ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmon(Ser789), myosin light chain(20) (MLC(20)), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca(2+)](i). PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmon(Ser789) and MLC(20) levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 x 10(-7) M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC(20) phosphorylation in the thick filament pathway and increases Ca(2+) sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmon(Ser789) was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase.     

Purified rabbit brain protein kinase C relaxes skinned vascular smooth muscle and phosphorylates myosin light chain

To clarify the role of protein kinase C in the mechanical response, the effects of exogenous protein kinase C and its cofactors were investigated on skinned smooth muscle preparations of the rabbit mesenteric artery. Addition of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine (PS) caused slow inactivation of a maximal Ca2+ contraction of the muscle fiber and correspondingly increased protein kinase C phosphorylation of myosin light chain. Neither protein kinase C nor enzyme cofactors (PS and TPA) produced relaxation of this tissue and all three components caused significant relaxation. Furthermore, when the muscle fiber was activated by Ca2+-insensitive fragment of MLC-kinase, addition of protein kinase C with PS and TPA decreased the tension and increased protein kinase C phosphorylation of myosin light chain. This evidence suggests that protein kinase C phosphorylation of myosin light chain may play an inhibitory role in the contraction of vascular smooth muscle.