Phosphorylation of the 20,000-dalton light chain of myosin is closely correlated with cross-bridge cycling in arterial smooth muscle. Evidence is presented that dephosphorylation can produce an attached, noncycling cross-bridge (latch-bridge) which is responsible for the high economy of force maintenance in this tissue.
The hypothesis that Ca2+ initiates contraction in smooth muscle by activating an endogenous myosin light chain kinase (MLCK) that phosphorylates the 20,000 dalton light chain (LC 20) of myosin was tested in tissues prepared from the media of swine carotid arteries. Unstimulated tissues with low levels of tone exhibited low levels of phosphorylated LC 20. On stimulation with a high-K+ physiological salt solution containing 1.6 mM CaCl2, LC 20 phosphorylation increased to 0.6 mol P/mol LC 20 within 30 s. This increase preceded force development, which required 2-4 min to attain a maximum steady-state value of 3.34 +/- 0.15 (SE) X 10(5) N/m2. These results support the hypothesis, as the stimulus was submaximal for the preparation. However, LC 20 phosphorylation declined significantly from its peak value before steady-state force was attained, reaching near control levels after 10 min of stimulation. The results suggest that Ca2+-stimulated LC 20 phosphorylation is an important physiological control mechanism but that additional factors are involved in the maintenance of tonic isometric force.
Electrical activity of enzymatically isolated, smooth muscle cells from hog carotid arteries was recorded under current clamp and voltage clamp. Under the experimental conditions, membrane potential usually was not stable, and spontaneous hyperpolarizing transients of approximately 100-msec duration were recorded. The amplitude of the transients was markedly voltage dependent and ranged from about 20 mV at a membrane potential of 0 mV to undetectable at membrane potentials negative to -60 mV. Under voltage clamp, transient outward currents displayed a similar voltage dependency. These fluctuations reflect a K+ current; they were abolished by 10 mM tetraethylammonium chloride, a K+ channel blocker, and the current fluctuations reversed direction in high extracellular K+ concentration. Modulators of intracellular Ca2+ concentration also affected electrical activity. Lowering intracellular Ca2+ concentration by addition of 10 mM EGTA to the pipette solution or suppressing sarcoplasmic reticulum function by superfusion with caffeine (10 mM), ryanodine (1 microM), or histamine (3-10 microM) blocked the rapid voltage and current spikes. However, caffeine and histamine induced a much slower hump of outward current before blocking the rapid spikes. This slower transient outward current could be elicited only once after external Ca2+ was removed and is consistent with an activation of K+ channels by Ca2+ released from internal stores. In contrast, removal of external Ca2+ alone failed to abolish the rapid spikes. These results suggest that 1) a Ca2+-dependent K+ conductance can markedly affect the electrical behavior of arterial smooth muscle cells and 2) internal Ca2+ stores, probably the sarcoplasmic reticulum, can support rapid and frequent releases of Ca2+. Exposure to a low concentration of histamine (3 microM) caused synchronization of the irregular, rapid fluctuations giving rise to slow, periodic oscillations of Ca2+-activated K+ conductance with a frequency of 0.1-0.3 Hz. These regular oscillations are reminiscent of periodic Ca2+-induced Ca2+ release, were inhibited by 10 mM caffeine, and point to a modulation of sarcoplasmic reticulum Ca2+ release by histamine.
SUMMARY. Smooth muscle strips isolated from the hog common carotid artery can contract rhythmically, exhibiting low frequency, large amplitude oscillations in tension when stimulated with 10 IIM histamine. Strips required at least 1.45 ITIM calcium and 2.5 mM potassium to exhibit this rhythmic activity. Rhythmic contractions could be converted to tonic contractions by removal of potassium or ouabain treatment. Relaxation by 2 mM lanthanum, 1 mM manganese, or 1 fiM verapamil implies that the external medium is the source of calcium mediating the contractions. The involvement of adrenergic nerve terminals in this response was ruled out, since propranolol, phentolamine, tetrodotoxin, bretylium, or 6-hydroxydopamine treatment did not alter the oscillations. Blockade of H! receptors with 0.1 HM diphenhydramine relaxed the muscle strips. The H 2 receptor antagonist cimetidine (5 fiM) had no effects. Attempts to obtain rhythmic contractions by stimulating with other vasoactive agents (norepinephrine, acetylcholine, 5-hydroxytryptamine, angiotensin II, and elevated potassium concentrations) were unsuccessful, suggesting that this is a specific histamine response mediated solely by Hi receptors. These results show that this large artery, commonly considered a multi-unit smooth muscle, can sometimes exhibit single-unit behavior. (CircRes 55: 480-485, 1984)
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