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

Beta adrenergic agonists such as clenbuterol, salbutamol, and albuterol are sympathomimetic substances capable of activating beta receptors in vivo. One important function of these substances is to produce effects resembling those of the impulses transmitted by postganglionic fibers of the central nervous system, thereby mimicking normal innervation control in tissues such as skeletal muscle. A dietary administration of these beta adrenoceptor agonists in rat produce growth-promoting protein anabolic effects in skeletal muscle [1,15,21,23,28]. The chronic administration of these agonists causes a hypertrophy of skeletal muscle in mice [17] and in the diaphragm of hamster [31]. The stimulation of the growth of skeletal muscle as a result of an application of beta adrenergic agonists is believed to be largely because of an accelerated protein turnover rate [10] coupled with a decrease degradation rate [27], or a combination of both these processes.The growth-promoting effects of beta adrenergic agonists are not only restricted to normal innervated muscle, but also have been equally confirmed under conditions characterized by the atrophy of constituent muscle fibers such as aging, denervation atrophy, and/or dystrophic conditions. An administration of these agonists to denervated rats not only limited, but also even reversed the process of denervation atrophy [13,19,20,35]. Similarly, an examination of the effects of clenbuterol on dystrophic mice has established that the drug could serve as a valuable adjunct Key words: beta adrenergic agonists, isoproterenol, clenbuterol, gastrocnemius muscle, denervation atrophy, 3-methylhistidine, myofibrillar degeneration. Abstract:The effects of beta adrenergic agonists, clenbuterol (2 mg/kg body weight/d) and isoproterenol (12 mg/kg body weight/d), in normal innervated and denervated rat gastrocnemius muscle were investigated. The daily administration of beta adrenergic agonists to normal innervated rats for a short period (7 d) resulted in the hypertrophy of gastrocnemius as confirmed from the measurement of total tissue protein contents. The development of denervation atrophy witnessed a stimulation in the expression of acid and alkaline phosphatases, pointing to an enhanced myofibrillar degeneration. An administration of beta adrenergic agonists inhibited the expression of raised levels of these enzymes in denervated muscle. A measurement of 3-methylhistidine in muscle revealed a loss of amino acid with the progress in the development of denervation atrophy. Serum and urine samples from denervated rats showed a progressive accumulation of 3-methylhistidine. Clenbuterol and isoproterenol treatment to these rats resulted in an inhibition of 3-methylhistidine accumulation. When 3-methylhistidine was used as a marker of myofibrillar degeneration, the results seemed to suggest that the degeneration of cyto-contractile apparatus accompanying denervation atrophy is attenuated in the presence of beta adrenergic agonists, implying that these sympathomimetic drugs are capable of re...

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

An oral administration of a single dose of beta-adrenoceptor agonist clenbuterol (15 mg/kg body weight) to mice resulted in an increased collagen distribution in the subendocardium and myocardium of the left ventricle. Abundant collagen accumulation is characteristic in myonecrotic regions and around blood vessels. Hydroxyproline assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of pepsin insoluble collagen confirmed this stimulated collagen proliferation. An MMP-activity assay of tissue extract by gelatin in gel zymography demonstrated a significant inhibition of MMP-9 activity in the beta-agonist-treated group. The results suggest that clenbuterol treatment is capable of inducing structural and functional remodeling of the extracellular matrix by down-regulating MMP-9 activity and thereby causing an impairment of collagen turnover. This may lead to changes in the different hemodynamic properties of the tissue, including ventricular compliance.

Smooth muscle contraction and relaxation are generally considered to be associated with phosphorylation and dephosphorylation of the 20-kDa regulatory myosin light chain (LC20). Thus, contractions of lamb tracheal smooth muscle induced by Bay K 8644 and relaxed by calcium channel blockers (verapamil, D-600 and nitrendipine) are accompanied by an increase and decrease, respectively, of LC20 phosphorylation. Similarly, endothelin-1 (ET-1) induces a sustained contraction, which is coupled with elevated LC20 phosphorylation and reversed by LC20 dephosphorylation after application of a potassium channel agonist (EMD 52692). In contrast, calcium channel blockers relax ET-1-induced contraction without any dephosphorylation of myosin light chains (MLC), suggesting that MLC phosphatase is inhibited in this case. Obviously, MLC dephosphorylation is not a prerequisite for smooth muscle relaxation. The variable relationship between MLC phosphorylation and force during relaxation suggests that there are mechanisms other than MLC phosphorylation that are important for regulation of contraction and relaxation in smooth muscle.