Vascular smooth muscle. A review of the molecular basis of contractility.
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Actins↗
Biomedical subjects
L P Adam
Publications and source records attributed to L P Adam.
Calcium channel effectors are potent non-competitive blockers of acetylcholine receptors.
Nicardipine and other calcium channel effectors (CCEs) were studied for their effects on nicotinic acetylcholine receptor (nAChR) activity. While CCEs had no effect on frog (Rana pipiens) skeletal muscle contractions resulting from nerve stimulation or direct stimulation of the muscle, nicotinic agonist-induced contractures were blocked. Nicardipine did not affect nAChR single-channel open time or amplitude, corroborating data from endplate currents (EPCs); EPC amplitudes and decays were unaffected. All the CCEs tested, however, non-competitively blocked nAChRs. The block of nAChRs resulted in a shortened agonist-induced depolarization and thus a diminished contracture response. An increase in cultured mouse skeletal muscle (C-2) cell single-channel closed times was observed with the intracellular addition of nicardipine, verifying a direct block of nAChRs. Using single-channel analysis, nicardipine's site of action, or at least access to its site of action, was determined to be at the intracellular side of the receptor. A direct action of the CCEs on the nAChR was also shown by their ability to block phencyclidine (PCP) binding to Torpedo nobiliana membranes. All the CCEs blocked specific binding of [3H]-PCP to its binding site on the nAChR-channel complex, with bepridil and nicardipine being the most potent. These data are compatible with a model in which nicardipine and other CCEs, at concentrations which do not alter nAChR channel open time or conductance, block the effects of superfused nicotinic agonist on nAChRs either by stabilizing the formation of the nAChR desensitized state or by effecting a slow channel block.
Animals↗
Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothelin-1.
Endothelin-1 contracts porcine carotid arterial smooth muscle with an ED50 of 10 nM. Contraction is associated with phosphorylation of the 20,000 dalton-regulatory light chain subunits of vascular myosin. Phosphopeptide mapping of light chains isolated from 32PO4-loaded muscle strips stimulated by endothelin-1 (5 x 10(-8) M) and comparison with maps generated from light chains phosphorylated in vitro or muscles stimulated with KCl (110 mM) or angiotensin-II (5 x 10(-8) M) indicates that Ca2(+)-calmodulin activation of myosin light chain kinase is a biochemical pathway stimulated by all three agonists. However, a small amount of phosphate (17%) was detected in a light chain peptide phosphorylated by protein kinase C. Endothelin-1 also stimulated phosphorylation of the thin filament protein, caldesmon, (from 0.35 mol PO4/mol caldesmon to 0.52 mol PO4/mol). Collectively, these results provide evidence that the effects of endothelin-1 on force generation and maintenance in vascular muscle may be dependent upon myosin light chain phosphorylation by Ca2+ calmodulin--requiring myosin light chain kinase and upon a thin filament mechanism that is modulated by phosphorylation of caldesmon.
Angiotensin II↗
Phosphorylation of caldesmon in arterial smooth muscle.
We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.
Animals↗
Myosin light chain and heavy chain phosphorylation in smooth muscle: potential regulatory roles for calcium, phospholipids and cyclic nucleotides.
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Animals↗
Augmentation of succinylcholine-induced neuromuscular blockade by calcium channel antagonists.
The block of endplate currents (EPCs) by succinylcholine, in frog cutaneous pectoris muscles, is significantly potentiated by certain organic calcium channel antagonists, at concentrations at which the calcium channel antagonists have no effect on EPCs themselves. Succinylcholine alone blocked the current in a dose-dependent manner with an IC50 = 2.7 microM in voltage-clamped muscle fibers. The ability of succinylcholine to block endplate currents was potentiated by nicardipine (3.5- and 31-fold by 1 and 10 microM, respectively), by bepridil (4.3-fold by 1 microM), and by verapamil (7.7-fold by 10 microM). The dihydropyridine nifedipine (10 microM), did not potentiate the succinylcholine effect. Since the calcium channel antagonists do not affect endplate currents directly at these concentrations, a channel blocking or receptor antagonistic effect of these drugs is not indicated. We therefore suggest that the enhancement of block by succinylcholine may be due to the increased desensitization of the receptor. Furthermore, this enhancement of desensitization may explain the effects of these calcium channel antagonists seen in whole animal studies, where the contractures caused by acetylcholine and succinylcholine, applied systemically, are blocked.
Animals↗
Comparison of muscarinic receptor properties in hatched chick heart atrium and ventricle.
We have reported previously that chick myocardium responds to muscarinic agonists with a decrease in slow inward current in both atrial and ventricular muscle. A second ionic current, the background potassium current, is increased in the atrium but not in the ventricle. A possible explanation for the modulation of potassium current in atrium only is the existence of a unique muscarinic receptor population in atrium responsible for potassium conductance changes. We looked for differences in atrial and ventricular muscarinic receptors by pharmacological and biochemical techniques. The dissociation constants for binding of l-[3H]quinuclidinyl benzilate were 46 pM in both tissues. Estimates for binding of atropine in competitive binding experiments gave dissociation constants of 1.8 nM in atrium and 2.0 nM in ventricle. Pharmacologic evaluation of atropine occupancy of muscarinic receptor by Schild analysis showed no difference in the dissociation constants in atrium (1.7 nM) and ventricle (1.1 nM). Displacement of 0.1 nM [3H]quinuclidinyl benzilate with carbachol showed the atrium to have a higher apparent affinity for agonist than the ventricle (atrium IC50 = 8.2 X 10(-6) M, ventricle IC50 = 2.1 X 10(-5) M). Computerized curve fitting analysis detected three binding states (super high, high and low affinity) for carbachol in the atrium and ventricle in the absence and presence of 5'-guanylylimidodiphosphate (10(-4) M). We did not detect a qualitative difference between atrial and ventricular muscarinic receptors. Muscarinic-induced potassium conductance changes which occur in the atria do not appear to be due to a unique muscarinic receptor in atria.
Animals↗