Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle.

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.

Characterization of the fluorescein isothiocyanate-reactive site of gizzard myosin ATPase.

We describe the reaction of fluorescein 5'-isothiocyanate with gizzard myosin, and investigate the effect of this fluorescent modification on ATPase activities. Changes in the ATPase activities upon modification occur rapidly, paralleling the reaction of 'fast reacting lysine residues' during the fast phase of the reaction. The loss in the ATPase activity is linearly correlated with the extent of modification. About 90% of the ATPase activity is lost with the incorporation of 2.6 mol of reagent per mol of myosin. The fluorescent label is mainly incorporated into the heavy chain of the myosin molecule. Using limited tryptic digestion of labeled S1, we have shown that the fluorescent dye remains in the 18 kDa fragment. The amino acid composition and the partial sequence of the peptide from the N-terminal end is presented. The results presented here suggest the participation of the 18 kDa peptide in the nucleotide binding domain of gizzard myosin.

A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity.

Methods are described for isolating smooth muscle cells from the tracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated, Ca2+ tolerant, and contracted rapidly and substantially when exposed to cholinergic agonists, KCl, serotonin, or caffeine. Adult cells were longer and wider than preterm cells. Mean cell length in 1.6 mM CaCl2 was 194 +/- 57 (SD) microm (n = 66) for adult cells and 93 +/- 32 microm (n = 20) for preterm cells (P < 0.05). Mean cell width at the widest point of the adult cells was 8.2 +/- 1.8 microm (n = 66) and 5.2 +/- 1.5 microm (n = 20) for preterm cells (P < 0.05). Cells were loaded into a perfusion dish maintained at 35 degreesC and exposed to agonists, and contractions were videotaped. Cell lengths were measured from 30 video frames and plotted as a function of time. Nonlinear fitting of cell length to an exponential model gave shortening velocities faster than most of those reported for airway smooth muscle tissues. For a sample of 10 adult and 10 preterm cells stimulated with 100 microM carbachol, mean (+/- SD) shortening velocity of the preterm cells was not different from that of the adult cells (0.64 +/- 0.30 vs. 0.54 +/- 0.27 s-1, respectively), but preterm cells shortened more than adult cells (68 +/- 12 vs. 55 +/- 11% of starting length, respectively; P < 0.05). The preparative and analytic methods described here are widely applicable to other smooth muscles and will allow contraction to be studied quantitatively at the single-cell level.

Using light scattering to locate less than a microgram of protein per band in polyacrylamide tube gels after isoelectric focusing.

After separation by isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHGE) in 9.2 M urea polyacrylamide tube gels, proteins with Mr > 20,000 can be precipitated by shaking the gels in 25% methanol. When a fiber-optic illuminator is placed in contact with the end of the gel, the cylindrical gel transmits light by internal reflection. In regions of the gel where protein is precipitated, this light is scattered, making it visible when the gel is viewed in a darkened room against a black background. The sensitivity of the method is moderate: less than 1 microgram protein per band (in a 3-mm diameter gel) can be detected. Because the protein in the bands precipitates rapidly (30 min), the solution used (25% methanol) is fairly benign to proteins, and the apparatus is not expensive, this technique should be useful in several situations including electrophoretic purification schemes. This method is especially useful for evaluating the quality of an IEF or NEPHGE tube gel before using it for the second dimension of a two-dimensional gel.

Isolation of smooth muscle cells from swine carotid artery by digestion with papain.

A new method is described for the preparation of viable, elongated smooth muscle cells from the swine carotid artery. Cells were prepared by papain digestion of pressurized arteries in calcium-free solution. After digestion, the arteries were everted, and fine strips were teased from the intimal surface of the media in calcium-free solution, releasing single cells. Viability was assessed by exclusion of trypan blue and by appearance under phase-contrast microscopy. By these criteria, approximately 20% of the isolated cells were viable. The most distinguishing and unexpected characteristic of these cells was their length. Mean length of the relaxed viable cells was 240.4 +/- 47.4 microns (SD, n = 76), which is much longer than previously reported for arterial smooth muscle cells. Calcium (1.6 mM) caused most of the viable cells to contract slightly, and the mean cell length in calcium was 194.4 +/- 57.7 microns. Cells in 1.6 mM calcium contracted substantially in response to 10 microM histamine or the calcium ionophore A23187 (10 microM), demonstrating that histamine receptors and the contractile apparatus were still functional.

Influence of altering cellular magnesium content on vascular smooth muscle contractility.

Tissue and cellular Mg levels in porcine carotid arterial strips were varied by 4-h incubation in high-K+, Ca2+-free solutions containing variable amounts of MgCl2 (0, 0.6, 1.2, 3, 5, 10, and 15 mM). The total Mg content, designated tissue Mg, was determined immediately after the incubation and also 1 and 3 h after reexposure to a normal physiological salt solution (PSS) containing 1.2 mM Mg2+. Cellular Mg levels were calculated from this data. The tissue Mg was profoundly altered immediately following the incubation, with values ranging from 7.3 mumol/g dry wt to 71.4 mumol/g dry wt, but remained significantly elevated following reexposure to normal PSS only in those strips incubated in 15 mM Mg2+. The calculated cellular Mg levels, however, did remain significantly elevated if 1 microM ouabain was included in both the incubation and post-incubation solutions. The response to high K+ in tissues subjected to the same 4-h incubation procedure exhibited the same pattern as the cellular Mg levels. Postincubation changes in the response to norepinephrine, either in the presence or absence of external Ca2+, depended on both the dose of norepinephrine and the level of Mg2+ in the incubation medium. It appears sarcoplasmic Mg levels are capable of modulating arterial contractility through influences at both the level of contractile proteins and the delivery of activator Ca2+.

Unusual [Ca2+] dependence of vascular smooth muscle cell shortening velocity.

The relationship between unloaded shortening velocity and Ca2+ concentration was determined for hog carotid arterial smooth muscle cells, freshly isolated by digestion with papain. Cells were exposed to various [Ca2+] for 60 s at 37 degrees C and then stimulated with 10 microM histamine. Cell length was measured by a video analysis system. Shortening velocity was expressed as an exponential rate constant by fitting the cell lengths to the following equation: length = Lmin + (Lmax-Lmin)exp[-v (time-latency)], where Lmax is length before contraction, Lmin is shortest length reached, time is time elapsed after addition of agonist, latency is time from addition of agonist until contraction starts, and v is the exponential rate constant (s-1). Cells shortened substantially, usually reaching one-fourth to one-third of their initial length within 1 min. Shortening velocities of the cells were much faster than published values of maximum shortening velocity in muscle strips from this same tissue. At 1.6 mM Ca2+, v was 0.173 +/- 0.015 s-1. When Ca2+ was increased to 5 or 10 mM, v was not significantly different. However, when Ca2+ was decreased to 0.5 and 0.16 mM, v increased to 0.288 and 0.258 s-1, respectively. The difference between 0.5 and 1.6 mM was significant. The unexpected increase in shortening velocity at low Ca2+ was also seen when 10 mM caffeine was used as a stimulus: v at 1.6 mM Ca2+ was 0.156 s-1, whereas v at 0.16 mM Ca2+ was 0.272 s-1. The high shortening velocities we measured suggest that measurements made on multicellular tissues seriously underestimate the potential shortening velocity of isolated individual cells.

Estimate of cellular force generation in an arterial smooth muscle with a high actin: myosin ratio.

An in vitro preparation from the media of the pig carotid artery develops somewhat higher force/cell cross-sectional area with one-fifth the myosin content of skeletal muscle cells. The following results suggest that this performance reflects cellular properties rather than the arrangement of cells within the tissue: (1) force development at the peak of the length-force curve is independent of the length of the tissue segment in a strip of constant cross-section, and (2) average cell length is directly proportional to tissue length. We conclude that the contractile system of arterial smooth muscle cells is specialized for force generation and that the mechanical properties of the pig carotid media preparation provide valid estimates of cellular function.

Histamine-induced rhythmic contraction of hog carotid artery smooth muscle.

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 microM histamine. Strips required at least 1.45 mM 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 microM 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 H1 receptors with 0.1 microM diphenhydramine relaxed the muscle strips. The H2 receptor antagonist cimetidine (5 microM) 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 H1 receptors. These results show that this large artery, commonly considered a multi-unit smooth muscle, can sometimes exhibit single-unit behavior.

Photoaffinity labelling of gizzard myosin with 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate.

The reaction of a photoaffinity analog, 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate (BZ2ATP) with gizzard myosin is described. The incorporation of BZ2ATP into myosin is both specific and stoichiometric. About 2.2 mol BZ2ATP are incorporated/mol myosin resulting in the significant loss of EDTA(K+) ATPase activity. The Mg2+ and actin-activated ATPase activities are slightly inhibited. Addition of ATP (millimolar) during the photolysis reaction significantly inhibits incorporation of BZ2ATP into myosin. Our data show that the label is mainly incorporated into the heavy chain of myosin with some label in the 20-kDa light chain. Limited proteolysis of radioactively labeled myosin subfragment 1 with trypsin reveals the presence of radioactivity mainly in the 50-kDa fragment and some in the 29-kDa and 25-kDa fragments. However, our data on the ATP-sensitive incorporation of BZ2ATP into the tryptic fragments suggest that the 50-kDa peptide, not the 29-kDa peptide, may be located at or around the active site.

A simple method of preparing myosin from porcine aortae.

A simple method for preparing myosin from porcine aortae is described. The procedure yields a highly pure myosin devoid of any obvious contaminants, such as tropomyosin and actin. The proteolysis of the heavy chain, frequently observed in the earlier procedures, is greatly minimized by the use of several protease inhibitors and by reducing the purification time. The procedure therefore can be used to isolate and purify myosin from porcine aortae for studies where the purity and integrity of the heavy chain are highly desirable.

Myosin dephosphorylation during rapid relaxation of hog carotid artery smooth muscle.

Changes in myosin light chain phosphorylation were measured during histamine-induced rhythmic contractions of hog carotid artery smooth muscle strips. Histamine made the muscle strips contract spontaneously every 1-5 min, and this allowed measurement of the time course of phosphorylation in relation to force development under conditions where diffusion of the agonist through tissue would not complicate the interpretation of the data. In the absence of histamine, phosphorylation was low [0.12 +/- 0.04 mol P/mol of the 20,000-Da light chain (LC 20)]. Phosphorylation was slightly (but not significantly) higher in the presence of 10 microM histamine in the relaxed state between contractions (0.20 +/- 0.03 mol P/mol LC 20). In muscle strips frozen during force development, when force had reached half of its peak value, phosphorylation was 0.38 +/- 0.06 mol P/mol LC 20. The highest levels of phosphorylation (0.49 +/- 0.04 mol P/mol LC 20) were found in strips frozen at the peak of the rhythmic contractions. Strips frozen when force had declined to half of the peak force showed low levels of phosphorylation (0.17 +/- 0.07 mol P/mol LC 20), indicating that the myosin light chain phosphatase activity was quite high. Mathematical modeling of the kinase and phosphatase reactions suggested that the apparent first-order phosphatase rate constant was at least 0.08 s-1 under these conditions. To obtain a better estimate of this rate constant, a second series of phosphorylation measurements were made early in the relaxation phase of the rhythmic contractions. The highest phosphatase rate constant obtained from these measurements was 0.23 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Role of the sarcoplasmic reticulum.

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.

Myosin light chain phosphorylation associated with contraction in arterial smooth muscle.

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.

Estimates of cellular mechanics in an arterial smooth muscle.

Estimates of force generation or shortening obtained from smooth muscle tissues are valid for individual cells only if each cell is contracting homogeneously and if cells anatomically arranged in series are mechanically coupled. These two assumptions were tested and shown to be valid for the pig carotid media under certain conditions. Homogeneity of cellular responses in carotid strips was estimated from the motion of markers on the tissue during K+ -induced isometric contractions. When tissues were stretched to L0 (the optimum length for force generation), there was little marker movement on stimulation. However, considerable marker movement was observed on stimulation at shorter muscle lengths, reflecting localized shortening or stretching. The mechanical coupling of the very small cells in the media was determined by measuring the dependence of cell length on tissue length. Tissues were fixed with glutaraldehyde during isometric contractions at various tissue lengths (0.4--1.1 x L0). The fixed tissues were macerated with acid and the lengths of the dispersed cells were measured. Cell lengths were broadly distributed at all muscle lengths. However, the direct proportionality between mean cell length and muscle length (as a fraction of L0) indicated that cells which are anatomically in series are coupled force-transmitting structures. We conclude that valid estimates of cellular mechanical function in this preparation can be obtained from tissue measurements at lengths greater than about 0.9L0.