Dependent Myosin Light Chain Phosphorylation Research Articles

Cavβ3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis.

Tight control of cytoplasmic Ca2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. We investigated the function of Cavβ3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3-/- (Cavβ3-deficient) mice as controls. We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins. Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ concentration and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

Metastatic Growth from Dormant Cells Induced by a Col-I–Enriched Fibrotic Environment

Breast cancer that recurs as metastatic disease many years after primary tumor resection and adjuvant therapy seems to arise from tumor cells that disseminated early in the course of disease but did not develop into clinically apparent lesions. These long-term surviving, disseminated tumor cells maintain a state of dormancy, but may be triggered to proliferate through largely unknown factors. We now show that the induction of fibrosis, associated with deposition of type I collagen (Col-I) in the in vivo metastatic microenvironment, induces dormant D2.0R cells to form proliferative metastatic lesions through beta1-integrin signaling. In vitro studies using a three-dimensional culture system modeling dormancy showed that Col-I induces quiescent D2.0R cells to proliferate through beta1-integrin activation of SRC and focal adhesion kinase, leading to extracellular signal-regulated kinase (ERK)-dependent myosin light chain phosphorylation by myosin light chain kinase and actin stress fiber formation. Blocking beta1-integrin, Src, ERK, or myosin light chain kinase by short hairpin RNA or pharmacologic approaches inhibited Col-I-induced activation of this signaling cascade, cytoskeletal reorganization, and proliferation. These findings show that fibrosis with Col-I enrichment at the metastatic site may be a critical determinant of cytoskeletal reorganization in dormant tumor cells, leading to their transition from dormancy to metastatic growth. Thus, inhibiting Col-I production, its interaction with beta1-integrin, and downstream signaling of beta1-integrin may be important strategies for preventing or treating recurrent metastatic disease.

Activation of Glucose-6-phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O(2) tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N(2), 5% CO(2)) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD(+) and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca(2+) sensitivity to the myofilaments and diminished Ca(2+)-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca(2+)-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD(mut(-/-)) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP(+) ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.

Activation of Glucose‐6‐Phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O2 tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose‐6‐phosphate dehydrogenase (G6PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95%N2‐5%CO2) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH and HPV, substantiating the idea that glucose metabolism and G6PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD+ and decrease in the ATP‐to‐ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated G6PD. Silencing G6PD expression in PA using a targeted siRNA abolished HPV and diminished Ca2+‐independent and Ca2+‐dependent myosin light chain phosphorylation otherwise increased by hypoxia. Similarly, G6PD expression and activity were significantly reduced in lungs from G6PDmut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating G6PD activity and the NADPH‐to‐NADP+ ratio to the HPV response clearly indicated a positive linear relationship between G6PD activity and HPV. Based on these findings, we propose that G6PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension. (Supported by NIH grant RO1HL085352)

Enhanced Contractile Response of the Basilar Artery to Platelet-Derived Growth Factor in Subarachnoid Hemorrhage

The level of platelet-derived growth factor (PDGF) in cerebrospinal fluid is elevated in subarachnoid hemorrhage (SAH). Therefore, the contractile effect of PDGF on the basilar artery was examined in SAH. A rabbit double-hemorrhage SAH model was used. In the medial layers of the control basilar artery, PDGF had no effect on contraction up to 1 nmol/L, whereas 3 nmol/L PDGF induced slight contraction. In SAH, PDGF induced an enhanced contraction with an increase in [Ca(2+)](i) at 1 nmol/L and higher concentrations. The levels of [Ca(2+)](i) and tension induced by 1 nmol/L PDGF in SAH were 17% and 20%, respectively, of those obtained with 118 mmol/L K(+) depolarization. The PDGF-induced elevation of [Ca(2+)](i) and contraction seen in SAH were abolished in the absence of extracellular Ca(2+). In alpha-toxin-permeabilized strips of SAH animals, PDGF induced no further development of tension during contraction induced by 300 nmol/L Ca(2+), suggesting no direct effect on myofilament Ca(2+) sensitivity. Genistein at 10 micromol/L completely inhibited the tension induced by 1 nmol/L PDGF. The level of myosin light-chain phosphorylation was significantly increased by 1 nmol/L PDGF. These results show that the contractile response to PDGF of the basilar artery was enhanced in SAH. The PDGF-induced contraction depended mostly on tyrosine phosphorylation and Ca(2+)-dependent myosin light-chain phosphorylation. The enhancement of the responsiveness to PDGF may therefore contribute to the development of cerebral vasospasm after SAH.

Mechanisms responsible for the in vitro relaxation of a novel dibenzothiepine derivative (NSU-242) on tracheal and vascular smooth muscles

In our previous general screening experiments, we found that NSU-242, a dibenzothiepine derivative (1–10 mg/kg), inhibited antigen-induced immediate asthmatic response in actively sensitized guinea pigs in a dose-dependent manner. The purpose of the present study was to assess the mechanism of the relaxing effect of NSU-242 on smooth muscle contractions in isolated smooth muscle tissues of the porcine trachea and rat aorta. NSU-242 administration resulted in a concentration-dependent inhibition of the tracheal-tissue contractions induced by carbachol and high K + and the aortic-tissue contractions induced by norepinephrine and high K +. The IC 50 values of these inhibitions were 1–10 μM, and there was no selectivity for the type of stimulation. In tracheal tissue, the relaxations were accompanied by neither changes in cAMP nor changes in cGMP. Carbachol (1 μM) and high K + (59.2 mM) increased myosin light chain (MLC) phosphorylation in the trachea, and NSU-242 (3–30 μM) had no effect on the level of MLC phosphorylation. Furthermore, NSU-242 (300 μM) had no effect on contractions in membrane-permeabilized tracheal tissue. FITC–phalloidin staining of the actin fiber in cultured vascular smooth muscle cells (A7r5) indicated that NSU-242 (10–100 μM) altered the configuration of actin stress fiber in the cytosol. However, unlike cytochalasin D, NSU-242 did not inhibit actin polymerization as assessed by in vitro assay. These results suggest that NSU-242 inhibits smooth muscle contractions without any effect on the Ca 2+-dependent MLC phosphorylation. NSU-242 may uncouple the force generated by the activated actomyosin interaction, possibly by modifying the actin assembly in smooth muscle cells without a direct effect on actin molecules.

Myosin Light Chain Modification Depending on Magnetic Fields: II. Experimental

Recent experiments have revealed that Ca2+ -calmodulin dependent myosin light chain phosphorylation in a cell-free preparation exhibits unexpectedly high sensitivity to weak magnetic fields. This enzyme system is a well-studied biochemical system, which appears to depend upon ion binding. A previous article in this journal discussed the theoretical background of myosin phosphorylation and the ion-dependent interactions of EMF with soft tissues. Because of the electromagnetic field (EMF) sensitivity of this cell-free system, experiments were designed to test the effect of a pulsed radio frequency (PRF) field, pulsating magnetic fields (TEMF), gradient magnetic fields (Magnabloc), and homogeneous static magnetic fields (in Helmholtz arrangement) designed for clinical application. It is concluded that these various magnetic fields affect this cell-free enzyme system by modulating ion–protein interactions.

Myosin Light Chain Modification Depending on Magnetic Fields: II. Experimental

Recent experiments have revealed that Ca2+ -calmodulin dependent myosin light chain phosphorylation in a cell-free preparation exhibits unexpectedly high sensitivity to weak magnetic fields. This enzyme system is a well-studied biochemical system, which appears to depend upon ion binding. A previous article in this journal discussed the theoretical background of myosin phosphorylation and the ion-dependent interactions of EMF with soft tissues. Because of the electromagnetic field (EMF) sensitivity of this cell-free system, experiments were designed to test the effect of a pulsed radio frequency (PRF) field, pulsating magnetic fields (TEMF), gradient magnetic fields (Magnabloc), and homogeneous static magnetic fields (in Helmholtz arrangement) designed for clinical application. It is concluded that these various magnetic fields affect this cell-free enzyme system by modulating ion–protein interactions.

Calmodulin-dependent cyclic nucleotide phosphodiesterase activity is altered by 20 microT magnetostatic fields.

Absorbance measurements at 660 nm of calmodulin (CaM) dependent cyclic nucleotide phosphodiesterase activity under cell free conditions indicate that 30-min exposures to weak magnetostatic field intensities alters this activity, compared to zero magnetic field exposures. This effect depends nonlinearly on the concentration of free calcium, with maximum magnetic interaction apparently occurring at an optimal Ca(2+) concentration corresponding to 50% activation (EC(50)). If one regards Ca(2+)/CaM activation as a switching process, then increasing the magnetic field at Ca(2+) levels in excess of optimal acts to bias this switch towards lower calcium concentrations. A magnetic dependence has been previously reported by others in an homologous system, CaM dependent myosin light chain phosphorylation, implying that there may be an underlying magnetic interaction that involves the initial Ca(2+)/CaM binding process common to both enzymatic pathways. The level of magnetostatic intensity at which this effect is observed ( approximately 20 microT) implies that CaM activation may be functionally sensitive to the geomagnetic field.

The paradox of smooth muscle physiology

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (contraction) and the release of force (relaxation). The signaling events that activate contraction include Ca 2+-dependent myosin light chain phosphorylation. The signaling events that mediate relaxation include the removal of a contractile agonist (passive relaxation) and activation of cyclic nucleotide-dependent signaling pathways in the continued presence of a contractile agonist (active relaxation). The major questions that remain in contractile physiology include (1) how is tonic force maintained when intracellular Ca 2+ levels and myosin light chain phosphorylation have returned to basal levels; and (2) what is the mechanism of cyclic nucleotide-dependent relaxation? This review focuses on these specific controversies surrounding the molecular mechanisms of contraction and relaxation of vascular smooth muscle.

Mildly Oxidized Low Density Lipoprotein Rapidly Stimulates via Activation of the Lysophosphatidic Acid Receptor Src Family and Syk Tyrosine Kinases and Ca2+ Influx in Human Platelets

In contrast to native low density lipoprotein (LDL), mildly oxidized LDL (mox-LDL) induced platelet shape change and stimulated during shape change the tyrosine phosphorylation of specific proteins including Syk; the translocation of Src, Fyn, and Syk to the cytoskeleton; and the increase of cytosolic Ca(2+) due to mainly Ca(2+) entry. The stimulation of these early signal pathways by mox-LDL was inhibited by desensitization of the lysophosphatidic acid (LPA) receptor and specific LPA receptor antagonists, was independent of the alpha(IIb)beta(3)-integrin, and was mimicked by LPA. Stimulation of tyrosine phosphorylation and Syk activation were independent of the increase of cytosolic Ca(2+) and were suppressed by genistein and two specific inhibitors of the Src family tyrosine kinases, PP1 and PD173956. In contrast to PP1 and PD 173956, genistein prevented shape change by mox-LDL. The results indicate that mox-LDL, through activation of the LPA receptor, stimulates two separate early signal pathways, (a) Src family and Syk tyrosine kinases, and (b) Ca(2+) entry. The activation of these early signaling pathways by mox-LDL probably plays a role in platelet responses subsequent to shape change. The inhibition of mox-LDL-induced platelet activation by LPA receptor antagonists or dietary isoflavonoids such as genistein could have implications in the prevention and therapy of cardiovascular diseases.

Regulation of the Ca2+ dependence of smooth muscle contraction.

Cellular mechanisms for the regulation of Ca(2+)-dependent myosin light chain phosphorylation were investigated in bovine tracheal smooth muscle. Increases in the free intracellular Ca2+ concentration ([Ca2+]i), light chain phosphorylation, and force were proportional to carbachol concentration. KCaM, the concentration of Ca2+/calmodulin required for half-maximal activation of myosin light chain kinase, also increased proportionally, presumably due to Ca(2+)-dependent phosphorylation of the kinase. Isoproterenol treatment inhibited agonist-induced contraction by decreasing [Ca2+]i and thereby light chain phosphorylation. Depolarization by increasing concentrations of KCl also resulted in proportional increases in [Ca2+]i, KCaM, light chain phosphorylation, and force. However, the [Ca2+]i required to obtain a given value of either light chain phosphorylation or KCaM was greater in KCl-depolarized tissues compared to carbachol-treated tissues. In muscles contracted with KCl, isoproterenol treatment resulted in diminished light chain phosphorylation and force without alterations in [Ca2+]i or KCaM. Thus, isoproterenol inhibition of KCl-induced contraction results from a cellular mechanism different from that found in agonist-induced contraction. In neither case does isoproterenol produce relaxation by altering the calmodulin activation properties of myosin light chain kinase.

Ca2+]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction.

Phorbol diesters, potent activators of protein kinase C, can produce a slow contraction in arterial smooth muscle. Such observations have prompted proposals that protein kinase C may have direct regulatory functions in contraction. In this paper, we present evidence that [Ca2+]-dependent myosin light chain phosphorylation is responsible for the contraction induced by low-dose phorbol diester and during force development in response to high-dose phorbol diester stimulation. The relationships between myoplasmic [Ca2+], myosin phosphorylation, and steady-state stress induced by low-dose phorbol dibutyrate were similar to those observed with contractile agonists. However, prolonged exposure to high-dose phorbol dibutyrate induced high stress with elevated phosphorylation that was not associated with elevations in aequorin-estimated [Ca2+]. Our results suggest that phorbol diesters can increase myoplasmic [Ca2+], and the resulting increase in myosin phosphorylation quantitatively explains the contraction.

Myoplasmic [Ca2+] Determines Myosin Phosphorylation and Isometric Stress in Agonist-Stimulated Swine Arterial Smooth Muscle

Smooth muscle cells can regulate both their rate of stress development and the level of maintained stress. Agonist-induced steady-state stress was dependent on changes in aequorin-estimated myoplasmic [Ca2+] in the range of 120-190 nM. Higher levels of [Ca2+] were observed only transiently after stimulation and correlated with higher levels of myosin phosphorylation and faster stress development. A single regulatory system (Ca2+-dependent myosin light-chain phosphorylation) appears to control both mean crossbridge cycling rates (rate of contraction or shortening velocity) and the number of attached crossbridges (stress).

N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, a novel activator of protein kinase C

Naphthalenesulfonamide derivatives were used to study the mechanism of regulation of Ca2+-dependent smooth muscle myosin light chain phosphorylation catalyzed by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and myosin light chain kinase. Derivatives such as N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), with a hydrophobic residue at the end of a hydrocarbon chain, stimulated Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation in a Ca2+-dependent fashion. There was no significant effect of these compounds on Ca2+-calmodulin (CaM) dependent myosin light chain phosphorylation. On the other hand, derivatives with the guanidino or amino residue at the same position had an inhibitory effect on both Ca2+-phospholipid- and Ca2+-CaM-dependent myosin light chain phosphorylation. These observations suggest that activation of Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation by naphthalenesulfonamide derivatives depends on the chemical structure at the end of hydrocarbon chain of each compound. SC-9 was similar to phosphatidylserine with regard to activation, and the apparent Km values for Ca2+ of the enzyme with this compound and phosphatidylserine were 40 microM and 80 microM, respectively. Kinetic analysis indicated that 12-O-tetradecanoylphorbol 13-acetate increased the affinity of the enzyme with SC-9 for calcium ion. However, kinetic constants revealed that the Km value of protein kinase C activated by SC-9 for substrate myosin light chain was 5.8 microM, that is, about 10 times lower than that of the enzyme with phosphatidylserine, and that the Vmax value with SC-9 was 0.13 nmol X min-1, that is, 3-fold smaller than that seen with phosphatidylserine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of felodipine, nitrendipine and W-7 on arterial myosin phosphorylation, actin-myosin interactions and contraction

The relative effects of felodipine, a dihydropyridine with purported calmodulin antagonistic properties, have been compared with the calmodulin inhibitor, W-7, for inhibition of Ca 2+-dependent force development and direct inhibition of Ca 2+-calmodulin mediated arterial myosin light chain phosphorylation and actin-myosin interactions. Felodipine (IC 50 3 × 10 −9 M) was approximately 30 000 × more potent than W-7 (IC 50 10 −4 M) and equipotent with another dihydropyridine, nitrendipine, in inhibiting isometric force development in K +-depolarized aortic smooth muscle strips. In contrast, W-7 (IC 50 4 × 10 −5 M) was approximately 5 × more potent than felodipine (IC 50 2 × 10 −4 M) in inhibiting Ca 2+-dependent myosin light chain phosphorylation or superprecipitation of arterial actomyosin. Concentration-related inhibition of both parameters by W-7 was tightly coupled to concomitant inhibition of force development in intact smooth muscle. In contrast, inhibition of myosin light chain phosphorylation and superprecipitation by felodipine was only apparent at concentrations ⩾ 10 −5 M while maximal inhibition of force development occurred at a concentration as low as 10 −7 M. Inhibition of contractility by W-7 was minimal in paced rabbit atria, whereas inhibition by felodipine was similar to that seen with nitrendipine. These results suggest that the pharmacologically-relevant mechanism of Ca 2+ antagonism in smooth muscle by felodipine is similar to nitrendipine (blockade of the Ca 2+ entry channel) and does not involve direct inhibition of Ca 2+-calmodulin stimulated myosin light chain phosphorylation and subsequent actin-myosin interactions.

The contractile activity of leukocyte contractile protein

Contractile activity of myosin-B from porcine leukocytes was regulated by Ca2+, which was associated with the level of phosphorylation of its myosin light chain. In another series of experiment, it was found that the myosin-B lost its contractility by washing with 1 mM NaHCO3. However, the washed myosin-B restored its Ca2+-sensitive contractility by the addition of not only a fraction prepared from the NaHCO3-wash of the myosin-B, but also “native tropomyosin (NT) ” which was the regulatory protein fraction from arterial smooth muscle. From the above results, its was considered that the mechanism of Ca2+-sensitive contractility of leukocyte-myosin-B was analogous to that of arterial contractile protein. Some preparations of leukocyte-myosin-B showed the same properties as the washed myosin-B in the Ca2+-sensitive contractile activity. In the myosin B, the Mg2+-ATPase activity was regulated by Ca2+ through “NT” fraction from arterial smooth muscle. On the other hand, leukocyte-myosin and arterial tropomyosin (TM) were purified from leukocyte-myosin B and arterial “NT”, respectively. In a synthetic actomyosin consisting of skeletal actin and leukocyte-myosin, arterial NT enhanced the actinactivated Mg2+-ATPase activity of leukocyte-myosin. The enhancement was much higher in the presence of Ca2+ and associated with an increase in the level of phosphorylation of myosin light chain. The arterial TM enhanced also the Mg2+-ATPase activity of the synthetic actomyosin, but its enhancement was independent of Ca2+. The enhancement was lower than that of NT and was not associated with an increase in the phosphorylation of myosin light chain. These results indicate that leukocyte contractile protein is essentially regulated by the level of Ca2+-dependent myosin light chain phosphorylation, but its contractility is augumented by TM associated with actin, independent of Ca2+

Ca2+-calmodulin-dependent phosphorylation and platelet secretion.

Protein phosphorylation may play a critical role in stimulus-coupled secretion of platelets. Some platelet proteins become phosphorylated on exposure to agents such as thrombin and collagen, and the smallest of these phosphoproteins (molecular weight 20,000), has been identified as a light chain of myosin. Phosphorylation of myosin light chain increases the activity of actin-activated myosin ATPase and the resultant contraction of the actomyosin presumably mediates the release reaction. Platelet myosin light chain kinase has been identified as a calcium-dependent enzyme requiring calmodulin for its activity. Calmodulin is a Ca2+-binding protein with a molecular weight of approximately 18,000 which seems to be involved in a wide variety of cellular processes. Although a growing body of evidence suggests that non-muscle actomyosin, such as that isolated from platelets, is regulated by Ca2+-calmodulin-dependent light chain phosphorylation, the precise relationship between the phosphorylation and the function of platelets is not clearly established. We now present pharmacological evidence that a calmodulin-mediated system, such as Ca2+-dependent myosin light chain phosphorylation, also plays an important role in the phenomenon of the release reaction. N-(6-aminohexyl)-5-chloro-1-napthalene-sulphonamide (W-7) (refs 13-15) is shown to bind selectively to calmodulin in vitro and inhibit its biological activity.