Effects of exogenously applied calponin on Ca2+-regulated force in skinned smooth muscle of the rabbit mesenteric artery

To help elucidate the physiological role of calponin (a thin-filament-linked regulatory protein) in smooth muscle contraction, the effects of its exogenous application were investigated on actin-activated MgATPase activity in crude actomyosin from chicken gizzard, and on contraction induced by Ca²⁺-dependent and -independent means in arterial smooth muscle strips skinned by saponin or β-escin. Calponin concentration dependently inhibited actin-activated MgATPase activity with a proportional increase in its binding to actomyosin and also attenuated Ca²⁺-induced contractions, in the presence or absence of calmodulin, in skinned arterial strips. Calponin, when phosphorylated by protein kinase C, reduced both its ability to bind to actomyosin and its inhibitory action on actomyosin MgATPase. The phosphorylated calponin also had no effect on the maximum Ca²⁺-induced contraction in skinned smooth muscle, suggesting that these actions of calponin are not non-specific. Calponin attenuated the Ca²⁺-independent contraction observed in myosin light chain thio-phosphorylated strips, or on application of trypsin-treated myosin light chain kinase. However, calponin had no effect on maintained rigor contraction. These results suggest that in vascular smooth muscle, calponin may play a physiological role in the inhibition of Ca²⁺-regulated force, possibly through a direct action on active actin-myosin interactions.     

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine

The effect of azelastine, an orally effective antiasthmatic/antiallergic drug, on the generation of oxygenderived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC₃₀=3.9 M) and AM (IC₃₀=9.8 M). In AM zymosan-induced CL was inhibited 21.7% by 10 M azelastine, whereas in PMNL it remained unchanged up to 10 M azelastine. Azelastine has a significantly stronger inhibitory effect (IC₃₀=4.2 M) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibitis oxygen-derived free radical generation by interaction with protein kinase C.     

Inhibition of passive cutaneous anaphylaxis (PCA) by azelastine: dissociation of its antiallergic activities from antihistaminic and antiserotonin properties

Azelastine and methysergide injected i.v. 5 min prior to antigen challenge and disodium cromoglycate (DSCG) injected i.v. immediately before antigen challenge produced dose-dependent inhibition of IgE-mediated 72 h passive cutaneous anaphylaxis (PCA) responses with ID50S of 0.3, 0.2 and 1.0 mg/kg, respectively. Thus, azelastine is about three times as effective as DSCG (a mast cell stabilizing agent) and somewhat less active than methysergide (a specific serotonin "D" receptor antagonist). Oral administration of azelastine and other drugs 2 h prior to antigen challenge produced strong inhibitory effects on PCA. The ID50S (mg/kg) were as follows: azelastine = 1.4; astemizole = 1.6; ketotifen = 2.0; aminophylline = 4.6; and diphenhydramine = 10.9. After 4 h of oral administration, azelastine and other drugs inhibited PCA responses with the following ID50S (mg/kg): azelastine = 1.8; astemizole = 2.3; ketotifen = 2.3; and aminophylline = 12.5. Azelastine administered orally 24 h before antigen challenge was still capable of exerting significant anti-PCA activity with an ID50 of 2.6 mg/kg, whereas none of the other drugs tested produced any significant inhibitory effects on PCA. In subsequent experiments, it was established that the antiallergic and antihistaminic activities of azelastine are inseparable 2 h after oral administration (ID50 of azelastine mg/kg, p.o., 2 h: PCA = 2.6 and histamine = 3.1). However, the persistence of the oral antiallergic (anti-PCA) effects of azelastine for 24 h (ID50 = 3.7 mg/kg) does not seem to be associated with its antihistaminic or antiserotonin activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependent phosphorylation and Ca2+ uptake in membrane fractions of the mesenteric artery

Summary Ca²⁺-dependent phosphorylation of endogenous substrate proteins (mol. wt 30 800, 35 500, 38 600 and 53 200) is found in a membrane subcellular fraction from rabbit mesenteric arteries. Characteristics of³²P incorporation are suggestive of a phosphoester-type phosphorylation produced by a Ca²⁺-dependent protein kinase.Ca²⁺-dependent phosphorylation and Ca²⁺ uptake rate show comparable affinities for Ca²⁺ of 3.5 × 10^–7 m and 2.4 × 10^–7 m, respectively. The dependence of both phenomena on the MgATP concentration is also similar. Ca²⁺-dependent phosphorylation and Ca²⁺ uptake are inhibited by trifluoperazine with an IC₅₀ of 3 × 10^–5 m and 5 × 10^–5 m, respectively. These results suggest that Ca²⁺ uptake might be modulated by a Ca²⁺-dependent protein kinase, which is possibly regulated by membrane-bound calmodulin.Endogenous Ca²⁺-dependent phosphorylation is stimulated up to 300% by the addition of boiled cytosol. This stimulation is due to phosphorylation of proteins of molecular weight 21 000 and 81 500 and is reversed by trifluoperazine. Since this stimulation cannot be mimicked by addition of calmodulin or phosphatidylserine, and since boiled cytosol does not stimulate Ca²⁺ uptake, it is proposed that an unknown cytosolic factor stimulates a second Ca²⁺-dependent protein kinase resulting in phosphorylation of membrane substrates unrelated to the Ca²⁺ pump.Since cAMP-dependent protein kinase is shown to cause little phosphorylation and has no effect on Ca²⁺ uptake, it is concluded that a Ca²⁺-dependent rather than a cAMP-dependent protein kinase might modulate Ca²⁺ transport in vascular smooth muscle.     

A monoclonal antibody that recognizes different conformational states of skeletal muscle troponin C and other calcium binding proteins

Summary Skeletal muscle troponin C contains four Ca²⁺-binding sites, two with a high affinity for Ca²⁺ that also bind Mg²⁺ competitively (Ca²⁺/Mg²⁺ sites) and two sites of lower affinity that are specific for Ca²⁺. We have characterized a monoclonal antibody (B₉D₉) that was produced against rabbit skeletal troponin C. The binding of this antibody to rabbit skeletal troponin C is sensitive to the binding of Ca²⁺. Increasing the Ca²⁺ concentration produces a decrease in the amount of antibody bound with a pK of approximately 6.9 which correlates with Ca²⁺ binding to the Ca²⁺/Mg²⁺ sites. Magnesium binding to rabbit skeletal troponin C had no effect on antibody binding. Thus the conformation of rabbit skeletal troponin C brought about by Ca²⁺ binding to these sites affects the antibody binding to its epitope. This epitope was unavailable for antibody binding in whole troponin. The antibody-binding site was localized in cyanogen bromide fragment CB9 of rabbit skeletal troponin C (residues 84–135). This antibody was also shown to cross-react with bovine cardiac troponin C., barnacle (Balanus nubilus) troponin C, bovine testis calmodulin and carp parvalbumin. In addition, the effect of Ca²⁺ on antibody binding seen with rabbit skeletal troponin C was also seen with bovine cardiac troponin C, and calmodulin. Thus these proteins appear to share a similar epitope and undergo similar structural changes. Wang and colleagues (1987) have presented evidence that rabbit skeletal troponin C at low pH has an elongated structure similar to that seen in the crystal structure and that at neutral pH its structure is more compact. We have found that in the absence of Ca²⁺ this antibody binds best to rabbit skeletal troponin C at low pH and its binding is reduced with increasingly alkaline pH. It is possible that the structural alterations brought about by changes in pH may also be responsible for the reduction in antibody binding. Since pH and Ca²⁺ have the same effect on antibody binding, this may mean that Ca²⁺ binding to the Ca²⁺/Mg²⁺ sites may also make rabbit skeletal troponin C more compact.     

Myosin light chain kinase: functional domains and structural motifs

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca²⁺/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca²⁺/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca²⁺/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca²⁺/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca²⁺/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.     

Recepto-secretory mechanism in histamine-stimulated amylase release from rat parotid gland

The recepto-secretory mechanism in histamine-stimulated amylase release from rat parotid slices was studied using blockers of receptors and inhibitors of the intracellular messenger systems. Amylase release stimulated by histamine was inhibited by pyrilamine, an H₁-receptor blocker, but not by cimetidine, an H₂-receptor blocker. Atropine, prazosin and yohimbine had no effect on the release. Histamine-stimulated amylase release was inhibited by W-7, ML-9 and H-7, inhibitors of a calmodulin, a myosin light chain kinase (MLCK) and protein kinase C, respectively, while H-8, an inhibitor of protein kinase A, did not inhibit the release. These results suggest that histamine stimulation evokes amylase release via H₁-receptors, followed by the Ca²⁺-dependent systems involving calmodulin, MLCK and protein kinase C.     

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca²⁺-binding protein. Thin filament activation of myosin MgATPase is Ca²⁺-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca²⁺-regulated at 25°C/50 mM KCl. We isolated the Ca²⁺-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca²⁺]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.     

Tetrahexylammonium ions increase Ca2+ sensitivity of contraction of guinea-pig ileal smooth muscle

Effects of tetraalkylammonium ions, having tetraalkyl chains of increasing length from ethyl to octyl, on inositol-trisphosphate (InsP ₃)-induced Ca²⁺ release and contractile mechanics were examined in guinea-pig skinned ileal smooth muscle longitudinal strips. Although tetrahexylammonium ions (THexA) appeared to be the most potent inhibitor of Ca²⁺ release among the tetraalkylammonium ions examined, an additional and more prominent effect was found, i.e., the contraction induced by Ca²⁺ release showed a large sustained component in the presence of THexA. Potentiation of the contraction by THexA (above 30 M) was also observed in skinned fibers in which the sarcoplasmic reticulum function was destroyed by treatment with A23187. The potentiating effect of THexA was the most potent by far among the tetraalkylammonium ions examined and was elicited by Ca²⁺-dependent and GTP-binding-protein-independent mechanisms. The potentiation was not due to activation of myosin light-chain kinase. The selective inhibitors of myosin light-chain kinase, protein kinase C and calmodulin reduced THexA-induced potentiation of contraction only at concentrations above 30 M, at which non-specific effects are likely. Furthermore, relaxation induced by changing pCa from 4.5 to 8.5 was not affected by 1 mM THexA, suggesting that the potentiating effect is not mainly due to inhibition of myosin light-chain phosphatase. In conclusion, THexA sensitizes guinea-pig skinned ileal smooth muscle to Ca²⁺ in a structure-selective manner. This sensitization appears not to be mediated mainly by a GTP-binding protein, by activation of myosin light-chain kinase or protein kinase C, by enhanced Ca²⁺ binding to calmodulin, or by inhibition of myosin light-chain phosphatase.     

Pharmacodynamic and pharmacokinetic studies with azelastine in the guinea pig: evidence for preferential distribution into the lung

The correlation between the antiasthma activity of azelastine and the concentrations of azelastine and its major metabolite, desmethylazelastine, in the blood and lung were investigated in guinea pigs. Blood and lung tissue samples collected at 15 min after aeroallergen (ovalbumin, 0.5 mg/ml, 30 s, 15 psi) challenge, i.e., 2–1/4 h after oral administration of the drug, were analyzed for azelastine and its desmethyl metabolite by specific HPLC assay. Azelastine afforded protection against immediate allergic responses (IAR, characterized by gasping and bronchospasmic convulsions). Based on the reduction of the severity of IAR, the ID₅₀ was 0.04 mg/kg. It also delayed the onset of IAR and prevented mortalities. A positive correlation (r²= 0.944) between the antiallergic activity and the sum concentrations of azelastine and desmethylazelastine in the lung was observed. Preferential uptake of azelastine and desmethylazelastine by the lung was demonstrated by the mean lung/blood ratio of about 22 and 146, respectively. The selective uptake of azelastine and its active metabolite by the lungs could contribute to its antiasthmatic/antiallergic activity in guinea pigs.     

Stimulation of hypothalamic histidine decarboxylase by calcium-calmodulin and protein kinase (cAMP-dependent) inhibitor

Stimulatory effects of Ca²⁺–CaM^* and PKI on partially purified hypothalamic HD (10 fold purification) have been shown under conditions involving inhibition of the enzyme by cAMP-induced phosphorylation and under control conditions. A 11 (v/v) mixture of 0.1 mM CaCl₂ and 10 units of CaM from human red blood cells reversed the inhibition of HD induced by cAMP-dependent protein phosphorylation activity to the control level. Verapamil (0.01 mM) could partially block the former effect without affecting the control level of enzyme activity. 0.01 mM TPA did not further increase the effect of Ca²⁺–CaM on HD, in the presence of 0.01 mM ATP, indicating that this stimulation does not require the action of Ca²⁺-dependent protein kinase. The control level of HD is not influenced by 0.1 mM CaCl₂ or 0.02 mM EGTA but is raised by CaM in the presence of CaCl₂ (0.1 mM). A highly purified protein kinase (cAMP-dependent) inhibitor (PKI) from bovine heart and a crude inhibitor from rat cerebellum could also reverse the inhibitory effect of cAMP-dependent protein kinase under phosphorylating conditions and enhanced HD activity above control levels. PKI and Ca²⁺–CaM, added together, produced single, not additive effects.We conclude that cAMP-induced phosphorylation is probable the main regulatory mechanism of histamine formation and this could be influenced by both Ca²⁺–CaM and PKI. Inhibition of cAMP-dependent protein kinase as well as stimulation of phosphoprotein phosphatase and Ca²⁺–CaM-dependent phosphodiesterase might be involved in the above actions.     

Modulation of ligand-gated dopamine channels in Helix neurones

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca²⁺] by various procedures (external zero Ca²⁺ or 1 mM Co²⁺, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.     

Inhibition of Ca2+-activated tension and myosin light chain phosphorylation in skinned smooth muscle strips by the phenothiazines

The antipsychotic phenothiazine drugs trifluoperazine, chlorpromazine, and promethazine inhibited Ca²⁺-activated tension in functionally skinned rabbit ileum and rabbit pulmonary artery strips. Exogenous calmodulin rapidly reversed the inhibition of tension. Inhibition of the endogenous myosin light chain kinase by these drugs resulted with ATP or its analog, ATPS, as a substrate. The evidence presented suggests the inhibition of Ca²⁺ activation of tension in skinned smooth muscle preparations by phenothiazines is due to the inhibition of Ca²⁺-activated phosphorylation of the 20,000 dalton myosin light chain by the endogenous myosin light chain kinase.     

Visualizing CaMKII and CaM activity: a paradigm of compartmentalized signaling

Calcium (Ca²⁺) has long been recognized as a crucial intracellular messenger attaining stimuli-specific cellular outcomes via localized signaling. Ca²⁺-binding proteins, such as calmodulin (CaM), and its target proteins are key to the segregation and refinement of these Ca²⁺-dependent signaling events. This review not only summarizes the recent technological advances enabling the study of subcellular Ca²⁺-CaM and Ca²⁺-CaM-dependent protein kinase (CaMKII) signaling events but also highlights the outstanding challenges in the field.     

Antagonism of histamine and leukotrienes by azelastine in isolated guinea pig ileum

The effects of azelastine on histamine- and leukotriene C₄ and D₄ (LTC₄, LTD₄)-induced contractile responses in isolated guinea pig ileum were investigated. Following a 2-min contact with the ileum, azelastine produced competitive antagonism of histamine (pA₂=8.24). Following a 15-min contact, azelastine at 2.5×10^–9 M exerted competitive antagonism, but at higher concentrations (10, 40 and 160×10^–9M) it not only shifted histamine concentration-effect curves to the right but also suppressed its maximum. Thus, azelastine exerts a dual (competitive/noncompetitive) antagonism of histamine depending upon the concentration and duration of contact. Azelastine and FPL 55712 (a known LT receptor antagonist) produced concentration-dependent antagonism of LTC₄ and LTD₄. Azelastine and compound FPL 55712 also exerted concentration-dependent reversal (relaxation) of pre-existing LTC₄-induced contractions. In conclusion, the potent H₁-histamine and leukotriene receptor blocking activities of azelastine may contribute to its antiasthmatic/antiallergic activities.     

A calmodulin-binding peptide relaxes skinned muscle from guinea-pig taenia coli

During smooth muscle activation the calcium calmodulin complex interacts with myosin light chain kinase (MLCK) whereby activating it. A synthetic peptide analogue (RS20) corresponding to the calmodulin recognition sequence of MLCK has been synthesized and previously found to inhibit the calmodulin stimulated light chain kinase activity. Here we studied the effect of this peptide on skinned fibers from guinea pig taenia coli. Maximal contractions induced by 30 M Ca²⁺ at 0.1 M calmodulin could be completely relaxed by the peptide at 1 M. The inhibitory effect was accompanied by partial dephosphorylation only of the regulatory myosin light chain. Relaxation could be reversed by addition of calmodulin which also increased the extent of light chain phosphorylation.The calmodulin concentration required for reversing the inhibition depended on the concentration of the inhibitory peptide suggesting that the peptide competed with MLCK for the calmodulin binding site. As the calcium-calmodulin-peptide mixture constitutes a calmodulin buffer, our results suggest, that the peptide is a calmodulin antagonist unique in terms of its potency and that less than nanomolar concentrations of free calmodulin may be required for inducing smooth muscle contractions.     

Ca2+ sensitizing effects of EMD 53998 after troponin replacement in skinned fibres from porcine atria and ventricles

Skinned fibres from porcine ventricles exhibited a higher Ca²⁺ sensitivity (pCa₅₀, i.e. -log₁₀ Ca²⁺ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca²⁺ sensitivity and Ca²⁺ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca²⁺ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa₅₀=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca²⁺ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca²⁺ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca²⁺-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 M). The Ca²⁺-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.     

Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction

An increase in the cytosolic Ca²⁺ concentration is a prerequisite in activation of contractile activity of smooth muscle. The shape of the Ca²⁺-signal is determined by spatial distribution and kinetics of Ca²⁺-binding sites in the cell. The increase in cytosolic Ca²⁺ activates myosin light chain kinase (MLCK) which in turn phosphorylates the regulatory light chains of myosin II. This Ca²⁺-dependent MLC₂₀ phosphorylation is modulated in a Ca²⁺-independent manner by inhibiting the constitutive active myosin light chain phosphatase mediated by the monomeric GTPase Rho and the Rho-associated kinase as well as protein kinase C or by increasing its activity through cGMP. Furthermore, the activity of MLCK may be decreased due to phosphorylation by CaM kinase II and perhaps p21 activated protein kinase. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades which not only show a temporal but also a spatial activation pattern.     

Cyclic-AMP mediated relaxation of chemically skinned fibres of smooth muscle

Smooth muscle from guinea pig taenia coli was chemically skinned with Triton X-100 and stored in ATP-salt solution containing 50% glycerol at –20°C. Fibre bundles were relaxed at Ca²⁺-concentrations below 10^–7 M, but contracted at 10^–6 M Ca²⁺. The isometric tension developed could be partly relaxed by the addition of c-AMP (in the presence of NaF), and it could also be inhibited following preincubation with the catalytic subunit of c-AMP dependent protein kinase. The inhibitory effect was much more pronounced at intermediate Ca²⁺-concentrations (e.g. 10^–6) than at concentrations producing a maximum contraction, suggesting that Ca-sensitivity had been lowered. Sodium fluoride which was required to potentiate the c-AMP effects was found to have a slight relaxing effect per se. The c-AMP effect may be mediated through activation of cyclic AMP-dependent kinase, producing phosphorylation of the myosin light chain kinase which, according to adelstein et al. (1978), may result in a net dephosphorylation of the myosin light chains and a concomittant inhibition of the contractile response.