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R A Murphy

Publications and source records attributed to R A Murphy.

At least 145 records · Page 8Linked to original sources
PubMed1983

Myosin phosphorylation and regulation of cross-bridge cycle in tracheal smooth muscle.

We have tested the hypothesis that phosphorylation of the 20,000-dalton myosin light chains (LC 20) in rabbit tracheal smooth muscle modulates cross-bridge kinetics and isotonic shortening velocity. The thin muscle [190 +/- 10 (SE) microns] allowed detection of rapid changes in carbachol-induced active stress development, LC 20 phosphorylation, and isotonic shortening velocities. Phosphorylation of the LC 20 in resting muscle was 0.12 +/- 0.04 mol Pi/mol LC 20. Carbachol (10(-5) M) increased the level of phosphorylation to 0.46 +/- 0.03 mol Pi/mol LC 20 within 30 s. Phosphorylation then declined significantly as steady-state active stress was reached. A positive correlation was always found between LC 20 phosphorylation and shortening velocity. This result supports the hypothesis that the level of myosin phosphorylation was related to the mean cross-bridge cycling rate rather than the number of cross bridges contributing to the developed stress. Dephosphorylation of LC 20 occurred at about the same rate as the decline in shortening velocity and stress upon stimulus washout.

Animals↗
PubMed1983

Ca2+, cAMP, and changes in myosin phosphorylation during contraction of smooth muscle.

Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. However, peak values are not maintained and phosphorylation declines, while active stress increases monotonically to a sustained steady state. The aim of this study was to determine the reason(s) for the transient change in myosin phosphorylation. Four hypotheses were considered: 1) reduced substrate, i.e., ATP depletion, 2) altered access of either the myosin kinase or phosphatase to the cross bridge, 3) reduced myosin kinase activity secondary to its phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase, and 4) reduced myoplasmic [Ca2+] during the contraction. Our results suggest that the most likely explanation is that there are two Ca2+-dependent regulatory processes: 1) myosin phosphorylation and 2) a second, unidentified site allowing stress maintenance with reduced cross-bridge cycling rates. A higher cell Ca2+ concentration appears to be necessary to activate myosin kinase and produce myosin phosphorylation than is needed for force maintenance. We suggest that agonist-induced Ca2+ transients, coupled with the differential Ca2+ sensitivity of the two regulatory systems, may explain the observed transient in myosin phosphorylation during a maintained contraction in smooth muscle.

Animals↗
PubMed1983

Ca2+, myosin phosphorylation, and relaxation of arterial smooth muscle.

Relaxation of tissues prepared from the swine carotid media following agonist (110 mM K+) washout was analyzed as a dual-exponential decay. The time course of the initial rapid phase (about 2 min) corresponded to myosin dephosphorylation and to the decay of the capacity to shorten isotonically. Because myosin was dephosphorylated to basal levels within 2 min, we hypothesize that the later, slow phase of relaxation (lasting up to 45 min) was due to a slow inactivation of nonphosphorylated cross bridges. Removing extracellular Ca2+ (0 mM CaCl2, 0.1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid) greatly enhanced the rate of the slow phase of relaxation, and raising extracellular CaCl2 to 5 mM slowed relaxation significantly. A slow rate of Ca2+ removal to a final concentration that maintains resting tone appears to produce the slow phase of relaxation. These results support hypotheses based on other studies of contracting muscles. There appear to be two populations of cross bridges interacting with the thin filament: 1) phosphorylated and capable of rapid cycling, and 2) dephosphorylated cross bridges that can maintain stress. The latter reflect an unidentified regulatory mechanism, which appears to have a high sensitivity for Ca2+.

Animals↗
PubMed1983

Control of tone in vascular smooth muscle.

Total peripheral resistance and regional blood flow are determined by the contractile activity of vascular smooth muscle cells. The control systems that determine the activity of arterial smooth muscle are described. The interactions among control systems that underlie the remarkable diversity in the responses of blood vessels are emphasized to illustrate how various classes of drugs mediate clinically important effects.

Action Potentials↗
PubMed1983

The role of myosin light chain phosphorylation in regulation of the cross-bridge cycle.

Ca2+ binding to myofibrillar regulatory sites can produce conformational changes allowing cross-bridge attachment and cycling. Measurements of smooth muscle actomyosin ATPase activity suggested that Ca2+ might act indirectly to mediate cross-bridge attachment by stimulating myosin light chain phosphorylation. However, the predicted obligatory relationship between developed force and myosin phosphorylation was not always observed in living smooth muscle. The observation that myosin phosphorylation was always tightly correlated with average cross-bridge cycling rates estimated from isotonic shortening velocities suggested that Ca2+ has two regulatory roles. One action is exerted via a Ca2+-binding protein whose identity is unknown in smooth muscle. This regulatory site acts like other Ca2+-binding regulatory proteins in muscle to permit cross-bridge interaction and to determine active stress. The second regulatory role involves stimulation of myosin light chain kinase and light chain phosphorylation. Increasing the level of phosphorylated cross-bridges increases shortening velocities or rate of force development. We suggest that the dephosphorylated cross-bridges are noncycling or slowly cycling in activated smooth muscle. Smooth muscle may be a particularly favorable experimental preparation for demonstrating a general regulatory role of myosin phosphorylation in modulating the kinetics and energetics of muscle contraction.

Actins↗
PubMed1982

High affinity of various smooth muscle myosins for skeletal F-actin demonstrated by enzyme kinetics and electron microscopy.

Myosins isolated from mixed rabbit skeletal muscle and several smooth muscles differ with respect to the rate of ATP hydrolysis and the degree of inhibition of the potassium-activated ATPase activity at saturating actin concentrations. Kinetic studies indicate that smooth muscle myosins are tightly bound to skeletal F-actin in the presence of ATp. This observation led to the hypothesis that ATP is a poor dissociating agent for smooth muscle myosin and skeletal F-actin. The kinetic interpretation was verified by electron microscopic observations of myosin binding to F-actin filaments in the presence of ATP.

Actins↗
PubMed1982

Relationship among fibre type, myosin ATPase activity and contractile properties.

At least two types of skeletal muscle myosin have been described which differ in ATPase activity and stability in alkaline or acidic media. Differences in ATPase characteristics distinguish Type I and Type II fibres histochemically. In this study, ATPase activity of myosin from muscles of several species with known histochemical and contractile properties has been determined to test the hypothesis that (1) myosin ATPase activity, (2) histochemical determination of fibre types and (3) maximum shortening velocity, all provide equivalent estimates of contractile properties in muscles of mixed fibre types. Maximum shortening velocity appears to be proportional to ATPase activity as expected from previous reports by Barany. However, both myosin ATPase and the maximum shortening velocity exhibit curvilinear relationships to the fraction of cross-sectional area occupied by Type II fibres. Therefore, we reject the hypothesis and conclude that histochemically determined myofibrillar ATPase does not accurately reflect the intrinsic ATPase activity or shortening velocity in muscles of mixed fibre types. Our data are consistent with the presence of more than two myosin isozymes or with a mixture of isozymes within single muscle fibres.

Adenosine Triphosphatases↗
PubMed1982

Suppression of maternal lymphocyte mitogenic responses by supernatants from short-term placental cell cultures.

Human placental cells were freed from lymphocyte contamination on discontinuous BSA gradients. Supernatants from short-term cultures of these purified placental cell suspensions were predominantly suppressive for maternal lymphocyte blast transformation to phytohemagglutinin and in the mixed lymphocyte culture. The suppressor factor was nondialyzable and did not contain IgG or human chorionic gonadotropin.

Cells, Cultured↗
PubMed1982

Tonic force maintenance with reduced shortening velocity in arterial smooth muscle.

The isotonic shortening velocity of swine carotid media tissues contracting in response to high K+, histamine, norepinephrine, or AC electrical stimulation rapidly increased to a maximum value and then declined to a steady-state level while force was still increasing or steady. The maximum shortening velocity calculated for no external load on the tissue (Vo) also decreased during the course of contractions when active stress remained constant. The fall in velocity with time was not Ca2+ dependent, because reductions in the [Ca2+] in high K+ solutions that significantly reduced the maximum stress (Fo) had no effect on Vo in the steady state. On washout of high K+ solutions, the ability of the tissue to shorten on isotonic quick release fell rapidly to low levels before isometric stress exhibited significant declines. The data indicate that cross-bridge cycling rates are modulated in this tissue. We suggest that this reflects the formation of attached, noncycling cross bridges (termed latch bridges), which constitute an internal load on the contractile system during tonic contractions.

Animals↗
PubMed1982

Role of Ca2+ and myosin light chain phosphorylation in regulation of smooth muscle.

The time course of phosphorylation of the 20,000-dalton myosin light chain (LC 20) was determined during contraction and relaxation in K+- and histamine-stimulated medial strips of swine carotid arteries. Resting LC 20 phosphorylation levels of 0.15 mol P/mol LC 20 rapidly increased to peak values of 0.6-0.7 mol P/mol LC 20 after stimulation and then declined significantly, although stress continued to rise to a stable steady-state maximum. LC 20 dephosphorylation after agonist washout preceded the decline in isometric stress. Over the entire contraction-relaxation cycle, phosphorylation was correlated with shortening velocity and not with developed stress. The maximum shortening velocity with no external load (Vo) was directly proportional to LC 20 phosphorylation (r = 0.986). The data indicate that LC 20 phosphorylation is necessary for cross-bridge cycling leading to shortening or stress development but that stress can be maintained by additional mechanisms. We suggest that dephosphorylation of an attached cross bridge in the presence of Ca2+ arrests the cycle, forming an attached, noncycling cross bridge.

Animals↗
PubMed1982

High force development and crossbridge attachment in smooth muscle from swine carotid arteries.

In experiments designed to achieve maximal activation, the active force/cell cross-sectional area in tissues prepared from the swine carotid media was 6.7 +/- 0.3 (sd) X 10(5) N/m5. This value exceeds that reported for other vertebrate muscle cells and is striking because of the low smooth muscle myosin content. The hypothesis that high force generation may, in part, reflect an increase in the crossbridge duty cycle, i.e., the fraction of the cycle during which force is generated, was tested by determining the rate of force redevelopment after a step shortening and the ration of the load-bearing capacity of the contractile system to the developed stress during the course of isometric contractions. Maximal crossbridge cycling rates estimated by the rate of force redevelopment occurred 30 seconds after the onset of a high K+-induced contraction, and decreased thereafter, although the load-bearing capacity or maximum active stress was maintained. These results from isometric experiments support the hypothesis and provide further evidence that attached, non-cycling crossbridges contribute to force maintenance in tonically contracting arterial smooth muscle.

Animals↗
PubMed1981

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.

Actins↗
PubMed1981

Submandibular glands in mice with muscular dystrophy: studies with nerve growth factor.

Experiments have been carried out to examine the submandibular glands in mice with hereditary muscular dystrophy. Radioimmunoassay data confirm biological studies which show that submandibular glands in mice with muscular dystrophy contain less nerve growth factor (NGF) than glands of normal animals. Male dystrophics have half as much submandibular NGF as unafflicted mice, while females have only 10% of control levels. Gel filtration and electrophoretic studies detect no differences in the molecular properties of NGF in gland extracts from normal and dystrophic mice. Furthermore, NGF from both sources show equal activity in the sensory ganglion bioassay. Together, these results suggest that NGF deficits in submandibular glands of dystrophic mice are not due to measurement artifacts arising from alterations in the structure of the molecule. Morphological studies have uncovered a cytological basis for chemical deficits within submandibular glands of dystrophic mice. Stereological analysis of light and electron microscopic sections revealed that growth factor containing granular tubule cells (GTC) take up a smaller portion of the total gland volume, are smaller in size, and contain fewer secretory granules than comparable cells in glands from controls. Furthermore, the ultrastructure of GTC in dystrophic animals suggests that the cells are less active in producing secretory protein than GTC in glands from normal animals. These results are consistent with the idea that growth factor deficits arise from cellular abnormalities in the granular tubule segment of the gland.

Animals↗
PubMed1981

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.

Actomyosin↗
PubMed1980

Tight binding of arterial myosin to skeletal F-actin.

A study of the K+-activated myosin ATPase activity, which was measured at high ionic strength in the absence of divalent cations, permitted estimates of the actin-myosin interaction under conditions where (i) myosin-myosin interactions were prevented, (ii) the actin-myosin interaction could be studied in the presence of ATP, and (iii) variation in myosin light chain phosphorylation did not alter smooth muscle myosin ATPase activity. A comparison of myosins isolated from swine carotid arteries and mixed (leg and back) rabbit skeletal muscle was conducted in the presence and absence of rabbit skeletal actin. It was found that (i) arterial myosin, like skeletal myosin, exhibited hyperbolic kinetics for ATP hydrolysis, (ii) specific ATPase activities were significantly higher for skeletal myosin, (iii) saturating concentrations of actin appear to totally inhibit the arterial myosin ATPase activity, but only partially inhibit skeletal myosin activity, (iv) the free actin concentration required for half-maximal inhibition was significantly lower for the arterial myosin ATPase activity than for the skeletal myosin activity; (v) unlike skeletal actomyosin, arterial actomyosin exhibits tight binding characteristics in the presence of ATP, (vi) the binding stoichiometry for arterial myosin to skeletal F-actin was 2 mol of actin monomer/mol of myosin. These observations reveal differences in the interaction of arterial and skeletal myosin with actin, and may in part, explain the high force-generating characteristics of arterial smooth muscle.

Actins↗
PubMed1980

Partitioning of staphylococcal delta haemolysin.

The behaviour of the biologically active components present in crude delta haemolysin was followed during various fractionation procedures utilised in the purification of delta haemolysin. Most chromatographic techniques yielded multiple peaks of delta haemolysin. None of the procedures completely separated delta haemolysin from the other components of crude culture filtrates. Efforts to purify delta haemolysin should be renewed.

Carboxymethylcellulose Sodium↗