Permeabilized Smooth Muscle Research Articles

Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M

We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1. This new isoform contains an insert in the 542–597 position not present in the M110 previously cloned (Chen et al. (1994) FEBS Lett. 356, 51–55) from the same species. The encoded cDNA was expressed as a soluble GST-fusion protein in E. coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle. In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 ± 3.5-fold (S.E. of the mean, n = 3). In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 μM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 μM) to relax the muscle. These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity. The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.

Role of protein kinase C in the endothelin-induced contraction in the rabbit saphenous vein

The roie of protein kinase C in the endothelin-induced contraction was examined in the isolated rabbit saphenous vein in which endothelin-1, endothelin-3, sarafotoxin S6c and IRL 1620 (succinyl-[Glu 9,Ala 11,15]endothelin-1-(8–21))-induced contraction at the threshold concentrations of 0.1–1 pM. A selective inhibitor of protein kinase C, 500 nM calphostin C (2-[12-[2-(benzyloxy)propyl]-3, 10-dihydro-4,9-dihydroxy-2,6,7,11-tetramethoxy-3,10-dioxo-1-perylenyl]-1-methylethyl carbonic acid 4-hydroxyphenyl ester), shifted the concentration-response curves for these agonists to the right 7.4- to 109-fold. In the vein in which the endothelin ET B receptor was desensitized, sarafotoxin S6c and IRL 1620 were ineffective whereas endothelin-1 and higher concentrations of endothelin-3 induced contractions by activating the endothelin ET A receptor. Caiphostin C (500 nM) shifted the concentration-response curves for endothelin-1 and endothelin-3 to the right more than 155-fold. Down-regulation of protein kinase C (by treatment with phorbol 12-myristate 13-acetate for 20 h) shifted the concentration-response curves for these agonists to the right before and after desensitization of the endothelin ET B receptor 3.7- to 59-fold. In the permeabilized smooth muscle, Ca 2+-induced contraction was enhanced by endothelin-1, endothelin-3 and sarafotoxin S6c at concentrations much higher than those needed to induce contraction (threshold concentration was 3 nM). Caiphostin C and down-regulation of protein kinase C shifted the concentration-response curves for endothelin-1 and endothelin-3 to the right and downwards without changing the effect of sarafotoxin S6c. In the permeabilized muscle in which the endothelin ET B receptor was desensitized, endothelin-1 and endothelin-3 still augmented the Ca 2+-induced contraction. Calphostin C and down-regulation of protein kinase C shifted the concentration-response curves for endothelin-1 and endothelin-3 to the right and downwards. These results suggest that protein kinase C is involved in the contraction mediated by the endothelin ET A and ET B receptors; and Ca 2+ sensitization mediated by the endothelin ET A receptor is due to activation of protein kinase C whereas Ca 2+ sensitization mediated by the endothelin ET B receptor may be due not only to the activation of protein kinase C but also to other mechanisms.

Smooth muscle contractility is modulated by myosin tail-S2-LMM hinge region interaction

The functional significance of two major smooth muscle myosin isoforms, which differ in the nonenzymic COOH-terminal tail region, is not known. We report here that a 13-amino acid peptide, which mimics a region of the tail unique to the SM1 myosin isoform, inhibits contraction velocity in permeabilized smooth muscle. This peptide is shown to bind to the S2-light meromyosin (LMM) hinge region of myosin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photoaffinity labeling, and immunoelectron microscopy. Our results suggest that novel intermolecular contacts between the tail and S2-LMM hinge regions of adjacent myosin molecules in the thick filament may modulate contractility and provide a basis for distinct isoform function.

Kinetics of Prephosphorylation Reactions and Myosin Light Chain Phosphorylation in Smooth Muscle: FLASH PHOTOLYSIS STUDIES WITH CAGED CALCIUM AND CAGED ATP

The pre-myosin light chain (MLC20) phosphorylation components of the lag phase (td) of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca2+ from 4-(2-nitrophenyl)-EGTA (NP-EGTA). Calmodulin (CaM) shortened the td (470 ms at 0 added CaM) that followed Ca2+ release, but its effect (td = approximately 200 ms) saturated at 40 microM. Photolysis of caged ATP following preequilibration with identical [Ca4CaM] shortened td to 41 ms. The rate of phosphorylation was very fast (3.5 s-1 at 22 degrees C in the presence of 5 microM exogenous CaM) following photolysis of caged ATP, and, following Ca2+ release, phosphorylation was accelerated by CaM. Simultaneous photolysis of caged ATP and NP-EGTA was followed by a td of 194 ms at 5 microM CaM and a rate of MLC20 phosphorylation intermediate between these parameters following photolysis of, respectively, NP-EGTA and caged ATP. In the presence of the normal, total endogenous CaM content (37 +/- 4 microM) of protal vein smooth muscles td was 565 ms. Steady state maximum force at pCa 5.5 was increased by much lower (100 nM) exogenous [CaM] than was required (> 2.5 microM) to shorten the td. We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 microM and probably less during a rapid Ca2+ transient. We conclude that the [CaM] dependence of the kinetics of MLC20 phosphorylation and force development (t1/2 and td) initiated by Ca2+ reflects the recruitment of a slowly diffusible component of total CaM. The relatively long duration of td (197 ms) at saturating [CaM] suggests the contribution to td of an additional component, possibly a prephosphorylation activation/isomerization of the Ca4CaM myosin light chain kinase complex (Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820). The relatively short delay (108 ms in the presence of 40 microM CaM) following simultaneous photolysis of NP-EGTA and caged ATP suggests that preincubation with ATP (prior to photolysis of NP-EGTA) may inhibit the formation of a preactive Ca2CaM myosin light chain kinase complex.

A Calponin Peptide Enhances Ca2+ Sensitivity of Smooth Muscle Contraction without Affecting Myosin Light Chain Phosphorylation

In permeabilized smooth muscle, exogenously applied calponin binds to myofibrils and reduces Ca(2+)-activated tension (Itoh, T., Suzuki, S., Suzuki, A., Nakamura, F., Naka, M., and Tanaka, T. (1994) Pflügers Arch. Eur. J. Physiol. 427, 301-308). A calponin peptide (calponin Phe173-Arg185), which inhibits the binding of calponin to actin, blocks the action of calponin and enhances the contraction induced by submaximal Ca2+ in permeabilized vascular smooth muscle. Unlike calmodulin, this peptide enhances the Ca(2+)-induced contraction without a corresponding increase in the level of myosin light chain phosphorylation. These results suggest that calponin decreases the sensitivity of smooth muscle to Ca2+ at a given level of myosin light chain phosphorylation.

Measurement of SR free Ca2+ and Mg2+ in permeabilized smooth muscle cells with use of furaptra.

The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded saponin-permeabilized cultured aortic smooth muscle (A7r5) cells. Ca(2+)-independent fluorescence emitted by furaptra trapped within organelles, excited at 346 nm (isosbestic point), decreased with a half time of 30 min. All Ca2+ measurements appeared to be from SR, because the apparent Ca2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsigargin-induced SR Ca(2+)-adenosinetriphosphatase inhibition caused near-total depletion of Ca2+, and the metabolic poisons oligomycin and rotenone had no effect. Calibration curves relating 370 nm-to-346 nm ratios to [Ca2+] and to [Mg2+] were calculated in situ; dissociation constants for Ca2+ and Mg2+ binding were 49 microM and 6.8 mM, respectively. Resting [Ca2+]SR was 75-130 microM, with a mean of 97.2 +/- 2.2 microM (n = 376), whereas [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-dependent declines in [Ca2+]SR, and pretreatment with guanosine 5'-triphosphate caused a large increase in the rate of inositol 1,4,5-trisphosphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will be a valuable tool with which to directly study [Ca2+]SR and SR function.

Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle.

1. Exogenous arachidonic acid (AA) inhibits the protein phosphatase that dephosphorylates smooth muscle myosin, thus sensitizing the contractile response to Ca2+; it also inhibits voltage-gated Ca2+ channels in smooth muscle. The purpose of the present study was to determine whether endogenous AA is increased by agonists in a manner consistent with its role as a messenger regulating myosin phosphatase and Ca2+ channels. Both AA and diacylglycerol (DAG) were measured in [3H]AA-labelled intact and permeabilized (with staphylococcal alpha-toxin) rabbit femoral arteries stimulated with the alpha 1-adrenergic agonist phenylephrine (PE) (intact and permeabilized smooth muscles) or by guanosine-5'-O-(3-thiotriphosphate (GTP gamma S; permeabilized smooth muscles in which the [Ca2+] was maintained constant). Arachidonic acid mass was determined with gas chromatography and mass spectrometry (GC-MS). 2. In intact smooth muscle, PE increased both AA and DAG levels significantly, to 210 and 145% of baseline values, respectively. Another Ca2+-sensitizing agent, the thromboxane analogue U46619, caused a similar increase in AA and DAG levels in rabbit pulmonary artery. 3. In permeabilized smooth muscle at constant [Ca2+](pCa 6.5) GTP gamma S-induced AA and DAG release preceded force development and GTP gamma S (50 microM, 10 min) increased AA mass to 61-88 microM. 4. Phorbol-12,13-dibutyrate (PDBu), another Ca2+-sensitizing agent, also increased both AA and DAG levels in permeabilized smooth muscle at pCa 6.5, whereas the inactive analogue, 4 alpha-phorbol, did not have a Ca2+-sensitizing effect, nor did it increase AA and DAG levels. 5. In the virtual absence of Ca2+ (pCa > 8) GTP gamma S also increased AA and DAG levels by 3.5- and 1.6-fold, respectively. The effect of free Ca2+ itself on AA and DAG release was modest in the physiological range (pCa 7.0 to pCa 6.0), but pCa 4.5 caused an approximately 3- to 4-fold increase in AA and DAG levels, compared with the levels at pCa 8. In permeabilized ileum smooth muscle maintained at constant [Ca2+] (pCa 6.0), carbachol also significantly increased AA to 1.75 times its original value within 1 min of its application. 6. Our results are consistent with, although do not prove, the roles of AA and DAG as second and/or co-messenger(s) in smooth muscle, while the increases in AA and DAG levels induced by PDBu raise the possibility that they contribute to some of the cellular effects of phorbol esters.

Effects of GTP gamma S on muscarinic receptor-stimulated inositol phospholipid hydrolysis in permeabilized smooth muscle from the small intestine.

1. Smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig were permeabilized with Staphylococcus aureus alpha toxin (alpha-toxin) and used to investigate the role of G-protein activation in the regulation of muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis. 2. The efficiency of alpha-toxin permeabilization was estimated by the release of [3H]-2-deoxyglucose ([3H]-2DG) after prior loading or lactate dehydrogenase (LDH) enzyme release from the smooth muscle fragments. 3. In alpha-toxin-permeabilized smooth muscle, but not in non-permeabilized muscle, GTP gamma S induced time- and concentration-dependent increases in labelled inositol phosphates. Carbachol (CCh) increased labelled inositol phosphates in both permeabilized and non-permeabilized muscle, although the increases were greater in non-permeabilized smooth muscle. The response to 100 microM CCh was severely reduced by 0.5 microM atropine. 4. In permeabilized muscle the effects of GTP gamma S or CCh on inositol phosphate levels were reduced by treatment with pertussis toxin (PTX) and completely inhibited by GDP beta S. 5. GTP gamma S caused a concentration-dependent inhibition of the CCh-induced increases in the levels of labelled inositol phosphates. Dibutyryl cyclic AMP or Sp-cAMPs (adenosine-3',5'-cyclic phosphorothiolate-Sp) reduced the effects of CCh on inositol phosphate levels. 6. The results suggest that muscarinic AChR activation induces inositol phospholipid hydrolysis via more than one G-protein in this smooth muscle and that several mechanisms may contribute to the modulation of both stimulatory and inhibitory responses observed.

Comparison of the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and chemical energy usage in intact and permeabilized smooth and skeletal muscles.

The primary goal of this study was to determine the utility of 2,3-butanedione monoxime as a tool for determining and separating the chemical energy usage associated with force production from that of force-independent, or 'activation' processes in smooth and skeletal muscles. We determined the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and high energy phosphate usage in intact and permeabilized smooth (rabbit taenia coli) and skeletal (mouse extensor digitorum longus) muscles. In the intact taenia coli, 2,3-butanedione monoxime depressed the tonic phase of the tetanus, contractures evoked by high potassium (90 mM) and by carbachol (10(-5) M) and the small force response evoked by these agonists after treatment with D-600 (10(-5) M). In the electrically stimulated intact taenia coli 2,3-butanedione monoxime (0-20 mM) caused a proportional inhibition of tetanic force output, myosin light chain phosphorylation and high energy phosphate usage (ED50 approximately 7 mM for all these parameters). At 20 mM 2,3-butanedione monoxime, force and energy usage fell to near zero and the degree of myosin light chain phosphorylation decreased to resting values, indicating a shut-down of both force-dependent and force-independent energy usage at high concentrations of 2,3-butanedione monoxime. In permeabilized taenia coli, 2,3-butanedione monoxime had little or no depressant effects on force production, ATPase activity or calcium sensitivity. 2,3-butanedione monoxime had a very modest inhibitory effect on the in vitro motility of unregulated actin filaments interacting with thiophosphorylated myosin. In solution, 2,3-butanedione monoxime inhibited myosin light chain kinase, but not the phosphatase (SMP-IV). These results suggest that the major effect of 2,3-butanedione monoxime is not on the contractile proteins themselves, but rather on calcium delivery during excitation, thereby reducing the degree of activation of myosin light chain kinase and subsequent activation of myosin by light chain phosphorylation. Thus, 2,3-butanedione monoxime is not useful for the determination of the energetics of activation processes in smooth muscle because of its inhibition of both force-dependent and force-independent processes. In contrast, in the intact mouse extensor digitorum longus, 2,3-butanedione monoxime inhibits tetanic force production (ED50 approximately 2 mM) to a much greater extent than myosin light chain phosphorylation. When 2,3-butanedione monoxime was used to manipulate force production in muscles at L(o), it was found that approximately 60% of the total energy usage was force-independent and the remainder was force-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyamines increase Ca2+ sensitivity in permeabilized smooth muscle of guinea pig ileum

The effects of polyamines were investigated in strips of smooth muscle from guinea pig ileum permeabilized with beta-escin (0.005%). Spermine (1 mM) inhibited transient contractions induced in Ca(2+)-free medium by carbachol (0.1 mM) and GTP gamma S (0.1 mM) but potentiated responses to caffeine (20 mM) and D-myo-inositol 1,4,5-trisphosphate (40 microM). At high ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid concentration (10 mM) and in the presence of A-23187 (10 microM), force at optimal and suboptimal Ca2+ concentrations was increased both by spermine and by carbachol. Spermine did not potentiate contraction in Ca(2+)-free medium or after full thiophosphorylation of the regulatory 20-kDa myosin light chains but slightly potentiated contractions produced by partial thiophosphorylation. Also, spermidine and putrescine, as well as the aminoglycoside antibiotic neomycin, increased sensitivity to Ca2+, with potency correlating with number of positive charges. After permeabilization by a high concentration (0.1%) of beta-escin, the sensitivity to Ca2+ was increased by spermine but not by GTP gamma S. In preparations permeabilized by Triton X-100, spermine slightly increased Ca2+ sensitivity but not maximal force. Tissue contents of putrescine, spermidine, and spermine in intact ileum muscle were 8, 98, and 184 nmol/g, respectively. Permeabilization by 0.005 and 0.1% beta-escin reduced spermine contents by 40 and 53%, respectively. Effects of added polyamines in permeabilized preparations may thus reflect physiological effects of endogenous polyamines modulating contraction in the intact tissue.

PH dependence of inositol 1,4,5‐trisphosphate‐induced Ca2+ release in permeabilized smooth muscle cells of the guinea‐pig.

1. The dependence on pH of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied in saponin-skinned smooth muscle cells from guinea-pig portal vein, using the indicator fura-2 to monitor Ca2+ release. 2. Increasing pH between 6.7 and 7.3 enhanced the rate of IP3-induced Ca2+ release at all the Ca2+ concentrations above 30 nM without changing the bell-shaped dependence of the Ca2+ release rate on Ca2+ concentration with a peak near 300 nM. 3. The ascending limb of the biphasic Ca2+ dependence was shifted slightly toward the lower Ca2+ concentration at pH 7.3, suggesting an increase in the Ca2+ sensitivity of IP3-induced Ca2+ release at the higher pH. 4. With the elevation in pH from 6.7 to 7.3 at 100 nM Ca2+, about 7-fold higher IP3 concentration was required to release half of the Ca2+ in the store within 15 s. This pH-dependent change in the IP3 sensitivity was smaller at 1 microM Ca2+ and was indiscernible in the absence of Ca2+. 5. These results suggest that H+ may inhibit binding of IP3 and Ca2+ to the modulator sites of the Ca2+ release mechanism. However, these effects on the binding sites may not fully explain the complex effect of pH, and there may be pH-dependent step(s) involved in the gating mechanism of IP3 channels. The present study demonstrates the importance of pH as a modulator of IP3-induced Ca2+ release.

Cross-bridge cycling at rest and during activation. Turnover of myosin-bound ADP in permeabilized smooth muscle.

Single turnover experiments were performed on myosin-bound ADP by measuring the time course of incorporation of [3H]ADP following rapid formation of [3H]ATP by photolysis of caged [3H]ATP. Permeabilized rabbit portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and containing [14C]ATP and high specific activity caged [3H]ATP. At variable times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio 3H:14C in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0.004 s-1. When the myosin light chain was about 80% thiophosphorylated, and the muscle was generating maximum isometric force, there appeared a fast phase of ADP exchange (44% of the total) which had a rate constant of 0.2 s-1. The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon thiophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric conditions. The fraction of ADP exchanged at 10 s following photolytic release of [3H]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transition of myosin from the resting (slow ATPase) cycle into the activated (fast ATPase) cycle, and that the fraction of myosin in the fast cycle is directly determined by the degree of light chain phosphorylation. The data are also consistent with the cooperativity model described previously by Vyas et al.

P-95 - Mechanisms of manganese-dependent contractions induced by acetylcholine in β-escin permeabilized smooth muscle.

P-95 - Mechanisms of manganese-dependent contractions induced by acetylcholine in β-escin permeabilized smooth muscle.

O-316 - Manganese-dependent norepinephrine-induced contractions in β-escin permeabilized smooth muscle of guinea-pig vas deferens.

O-316 - Manganese-dependent norepinephrine-induced contractions in β-escin permeabilized smooth muscle of guinea-pig vas deferens.

A mechanism underlying histamine-induced contraction in isolated rabbit lingual artery

To understand the contractile response to histamine, drug-induced tension developments were measured in intact and staphylococcal alpha-toxin-treated permeabilized smooth muscle preparations of rabbit lingual artery. Histamine produced an endothelium-independent contraction; the observed effect was antagonized by diphenhydramine and was attenuated by nifedipine. Histamine produced only transient contraction in the Ca(2+)-free bathing media. In permeabilized preparations, histamine-induced contraction was abolished by ryanodine (in the presence of caffeine) or prior repeated application of caffeine; however, contraction produced by IP3 was still observed under the same experimental condition as that of the histamine study. Histamine- or caffeine-induced contraction was abolished in the permeabilized preparations by prior repeated application of IP3; caffeine or IP3 produced contraction after repeated application of histamine. Ryanodine in the presence of histamine was ineffective on caffeine-induced contraction, suggesting that histamine by itself may not have the ability to act on caffeine-sensitive Ca2+ release channels. Neomycin and H-7 completely abolished the histamine-induced contraction. Hence, it is suggested that histamine can contract the lingual artery via H1-receptor-coupled phospholipase C activation, and the contraction consists of a phasic response evoked by Ca2+ release from the IP3- and caffeine-sensitive Ca2+ storage site and the tonic response generated by the voltage-dependent influx of Ca2+. It is also postulated that IP3 produced by histamine may be able to produce Ca2+ release from only a part of the intracellular Ca2+ stores in the smooth muscle of rabbit lingual artery.

Mycalolide-B, a novel and specific inhibitor of actomyosin ATPase isolated from marine sponge

A toxin isolated from marine sponge, mycalolide-B, inhibited smooth muscle contractions without changing cytosolic Ca 2+ levels. It also inhibited Ca 2+-induced contraction in permeabilized smooth muscles. In native actomyosin prepared from chicken gizzard, mycalolide-B inhibited superprecipitation and Mg 2+-ATPase activity stimulated by Ca 2+ without changing myosin light chain phosphorylation. In the permeabilized muscle and native actomyosin preparation thiophosphorylated with ATPγS, mycalolide-B inhibited ATP-induced contraction and Mg 2+-ATPase activity, respectively, in the absence of Ca 2+. Mycalolide-B also inhibited Mg 2+-ATPase activity of skeletal muscle native actomyosin. Mycalolide-B had no effect on calmodulin-stimulated (Ca 2+Mg 2+)-ATPase activity of erythrocyte membranes. These results suggest that mycalolide-B selectively inhibits actin—myosin interaction.

Effects of felodipine, nifedipine and verapamil on cytosolic Ca 2+ and contraction in vascular smooth muscle

The inhibitory effects of felodipine, nifedipine and verapamil were compared in vascular smooth muscle. In rat aorta, these inhibitors attenuated the high K +-induced contraction with a parallel decrease in the cytosolic Ca 2+ level ([Ca 2+] i). Maximal inhibition was obtained with 10 nM felodipine, 100 nM nifedipine and 10 μM verapamil. The inhibitory effects were antagonized by an increase in external Ca 2+ concentration to 6.5 mM and the addition of a Ca 2+ channel activator, 100 nM Bay k 8644. These inhibitors also attenuated the contraction induced by norepinephrine although these effects were weaker than those on high K +-induced contraction. Furthermore, these inhibitors attenuated the norepinephrine-stimulated [Ca 2+] i more strongly than contraction. In contrast, none of these inhibitors inhibited the transient increase in [Ca 2+] i and muscle tension induced by norepinephrine in Ca 2+-free solution and the Ca 2+-induced contraction in permeabilized smooth muscle. These results suggest that felodipine, nifedipine and verapamil inhibit smooth muscle contraction by inhibiting Ca 2+ channels at concentrations which do not change Ca 2+ release or Ca 2+ sensitivity of contractile elements.

Kinetics of Ca2+ release and contraction induced by photolysis of caged D-myo-inositol 1,4,5-trisphosphate in smooth muscle. The effects of heparin, procaine, and adenine nucleotides.

The kinetics of Ca2+ release and contraction induced by photolytic release of inositol 1,4,5-trisphosphate (InsP3) were determined in permeabilized smooth muscle. The rate of Ca2+ release was half-maximal at 1 microM InsP3. The concentration-dependent delay of Ca2+ release at saturating InsP3 concentration was approximately 10 ms and within the uncertainty of the measurements. The relationship between the delay and InsP3 concentration showed no evidence of a high level (n = 4 or higher) of cooperativity but could not distinguish between no cooperativity (n = 1) or a low level (n = 2) of cooperativity. Submaximal [InsP3] caused only partial Ca2+ release from the InsP3-sensitive stores. InsP3-induced Ca2+ release was markedly potentiated by ATP or by adenosine 5'-(beta,gamma-methylene-triphosphate), but neither the rate nor the amplitude of release was significantly affected by procaine (2-5 mM). Heparin increased the delay between photolysis and Ca2+ release, indicating that the off rate of inert ligand(s) bound to InsP3 receptors may contribute to the physiological delay in Ca2+ release. There was a much longer (370 ms +/- 45 S.E.) delay between the rise of Ca2+ and force development, presumably reflecting events preceding and associated with myosin light chain phosphorylation.

Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium.

Arachidonic acid (AA) increased, at constant Ca2+, the levels of force and 20-kDa myosin light chain (MLC20) phosphorylation in permeabilized smooth muscle, and slowed relaxation and MLC20 dephosphorylation. The Ca(2+)-sensitizing effect of AA was not inhibited by inhibitors of AA metabolism (indomethacin, nordihydroguaiaretic acid, or propyl gallate), of protein kinase C (pseudopeptide) or by guanosine-5'-O-(beta-thiodiphosphate) and was abolished by oxidation of AA in air. A non-metabolizable AA analog, 5,8,11,14-eicosatetraynoic acid) also had Ca(2+)-sensitizing effects. Extensive treatment with saponin abolished the Ca(2+)-sensitizing effects of phorbol 12,13-dibutyrate and guanosine-5'-O-(gamma-thiotriphosphate), but not that of AA. A purified, oligomeric MLC20 phosphatase isolated from gizzard smooth muscle was dissociated into subunits by AA, and its activity was inhibited toward heavy meromyosin but not phosphorylase. We conclude that AA may act as a messenger-promoting protein phosphorylation through direct inhibition of the form of protein phosphatase(s) that dephosphorylate MLC20 in vivo.

Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle.

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.