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Biomedical subjects

R A Murphy

Publications and source records attributed to R A Murphy.

At least 127 records · Page 7Linked to original sources
PubMed1986

Two different heavy chains are found in smooth muscle myosin.

Two putative myosin heavy chains designated SM1 and SM2 were detected on a 3.5% polyacrylamide-sodium dodecyl sulfate gel electrophoresis system loaded with homogenates of several mammalian smooth muscles. The two polypeptides were present in nearly equal amounts in all smooth muscle tissues tested and in myosin purified from swine carotid media and stomach. Both proteins were equally stained by smooth muscle-specific myosin antibodies. The smaller of the polypeptides had a mobility nearly identical to that of the single heavy chain observed in purified fast-twitch skeletal myosin. Electrophoresis of pyrophosphate extracts from swine carotid media, swine stomach, rabbit thoracic aorta, and guinea pig taenia coli on nondenaturing pyrophosphate gels revealed a single protein band. When subsequently electrophoresed on a sodium dodecyl sulfate gel, the native bands from swine tissue extracts revealed the two putative heavy chains in nearly equal amounts, as well as a large amount of a higher molecular weight peptide whose properties reflect those of filamen. Sodium dodecyl sulfate gel analysis of the myosin band from pyrophosphate gels of purified swine stomach myosin showed exclusively the two heavy chains in a nearly 1:1 ratio. Smooth muscle myosin migrates homogeneously on pyrophosphate gels, and the virtual equality of the two heavy chains may reflect the presence of large amounts of a myosin isoenzyme, which is a heavy-chain heterodimer.

Animals↗
PubMed1986

Estimates of activation in arterial smooth muscle.

We have previously described the onset of a "latch" state in the swine carotid media after K+ depolarization. This state was characterized by maintained stress after a decrease in shortening velocities and in the level of cross-bridge phosphorylation. The present experiments were designed to determine whether there were changes in other mechanical properties in swine carotid media associated with the onset of the latch state. Medial strips (less than 500 microM thick), incubated in physiological salt solution (PSS) at 37 degrees C at their optimal length (Lo), were subjected to ramp stretches (5.86 mm/s) of 5% Lo. The active stress (Sa) response to stretch was computed by subtraction of the passive element contribution (as determined from identical stretches after 30 min incubation in Ca2+-free PSS) from the total response in the activated muscle. Transitions in the total and active stress responses to stretch were observed in strips stimulated with 109 mM K+ for 1 min or longer and were interpreted as yielding of the contractile apparatus. Active dynamic stiffness (dS/dLo) calculated from the initial 1% Lo portion of the stretch response, correlated linearly with active stress over a wide range. Maximal stress and dynamic stiffness were reached by 1 min and were maintained for at least 30 min in K+-depolarized preparations. However, yield stress increased significantly between 1 and 10 min, and there was a large increase in the length at which yield was observed (1.09 +/- 0.06 to 1.86 +/- 0.10% Lo; n = 9). These increases were maintained between 10 and 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Determinants of Ca2+-dependent stress maintenance in skinned swine carotid media.

Ca2+-dependent stress maintenance without proportional myosin light chain (MLC) phosphorylation was demonstrated in the detergent-skinned swine carotid media when [Ca2+] was reduced from high to intermediate concentrations (Chatterjee, M., and R. A. Murphy. Science Wash. DC 221: 464-466, 1983). In this study of the same preparation, we examined the influence of the initial [Ca2+] and MLC phosphorylation levels on the formation of a stress maintaining state (the "latch" state). Fibers were contracted with 0.82, 1.3, 2.1, or 7.2 microM Ca2+ and then exposed to a lower [Ca2+] to determine the magnitude and Ca2+ sensitivity of stress maintenance. MLC phosphorylation levels were measured in all strips. The magnitude of the stress that rapidly developed was dependent on the initial activating [Ca2+]. The Ca2+ sensitivity of stress maintenance appeared to be independent of the initial levels of stress and MLC phosphorylation. However, the magnitude of the maintained stress was dependent on the initial levels of Ca2+. Only two values for half-maximal responses were evinced in all Ca2+-dependent stress curves: 1.4 X 10(-6) M Ca2+ for rapid stress development and 3.1 X 10(-7) M for stress maintenance. Cumulative [Ca2+]-response curves were shown to result in a decreased maximal stress response and an increase in the apparent Km compared with curves determined by responses to single [Ca2+] exposures. This was associated with a time- and stress-related deterioration of the preparation. The latter was not prevented by exogenous calmodulin or leupeptin (an effective inhibitor of an endogenous Ca2+-dependent protease shown to disrupt the cellular cytoskeleton).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Myoplasmic calcium, myosin phosphorylation, and regulation of the crossbridge cycle in swine arterial smooth muscle.

Our objective was to test the hypothesis that changes in crossbridge phosphorylation in the swine carotid media are due to changes in the myoplasmic calcium concentration. The photoprotein aequorin was loaded intracellularly by incubation in a series of calcium-free solutions. This loading procedure did not affect subsequent stress development, myosin light chain phosphorylation, or ultrastructure. The time course of light production, myosin light chain phosphorylation, shortening velocity at zero load, and active stress were measured in three stimulus protocols: depolarization with 109 mM potassium chloride at 22 degrees C, 37 degrees C, and 37 degrees C, followed by a reduction in potassium chloride to 20 mM to induce stress maintenance with basal phosphorylation (latch). Light-predicted intracellular calcium concentration was found to correlate with myosin phosphorylation and unloaded shortening velocity. The calcium concentration required for half-maximal myosin phosphorylation was approximately twice that for stress maintenance. These estimates depend on many assumptions, but they compared favorably with the half-maximal myosin phosphorylation values obtained for the calcium-dependence of stress maintenance and phosphorylation in Triton X-100 skinned carotid media preparations. This supports the hypothesis that myoplasmic calcium is the determinant of myosin phosphorylation and mean crossbridge cycling rates in intact smooth muscle depolarized by potassium chloride.

Aequorin↗
PubMed1986

Steroids and triiodothyronine reduce nerve growth factor concentrations in medium conditioned by L-929 fibroblasts.

Thyroid hormone, testosterone, and glucocorticoids stimulate the production of nerve growth factor (NGF) in mouse submandibular glands, but little is known about the influences of these hormones on other sources of NGF. In this study we tested the effects of thyroid hormone and steroids on NGF levels in medium conditioned by L-929 fibroblasts (L-cells), a mouse cell line that secretes NGF in culture. NGF concentrations in conditioned medium were measured by RIA and were normalized for cell number. In the absence of hormones, NGF levels in medium averaged 5.57 ng/ml. T3, cortisone, and aldosterone decreased NGF levels below the sensitivity of the RIA (less than 0.37 ng/ml), with respective ID50 values of 1 X 10(-5), 1 X 10(-8), and 5 X 10(-8) M. Testosterone decreased NGF levels by 66%, with an ID50 of 9 X 10(-9) M, and 17 beta-estradiol decreased it by up to 50%, although the hormone's effects had not plateaued at 10(-6) M. NGF concentrations were unaffected by progesterone at doses as high as 10(-6) M. Cell viability was unaffected by hormone treatment, and the total amount of secreted protein in medium decreased only in cultures treated with high doses of cortisone and aldosterone. Qualitatively similar results were obtained by bioassay; steroids markedly reduced, but did not eliminate, the nerve growth-promoting effects of L-cells on embryonic chick dorsal root ganglia in cocultures. These data indicate that hormones that stimulate NGF production in salivary glands inhibit NGF production by L-cells in culture.

Aldosterone↗
PubMed1985

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion.

Transients in myoplasmic [Ca2+] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K+ -stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca2+ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an improved estimate of activation rates.

Animals↗
PubMed1985

Cellular and subcellular colocalization of nerve growth factor and epidermal growth factor in mouse submandibular glands.

Immunocytochemical methods have been used to compare the cellular and subcellular distribution of nerve growth factor (NGF) and epidermal growth factor (EGF) in mouse submandibular glands. Rabbit antisera raised against purified proteins were characterized by immunoblot methods and were used to stain sections of salivary glands embedded in plastic. For light microscopy, antibodies were visualized by indirect immunofluorescence. For electron microscopy, thin sections were treated simultaneously with IgG against NGF and EGF coupled to colloidal gold particles of different size. Data indicate that NGF and EGF are present in all granular convoluted tubule cells and in no other cell type within the salivary gland. Ultrastructural analyses indicate that NGF and EGF are evenly distributed together within mature secretory granules, although a population of small granules was identified that is not immunoreactive for either protein. Taken together, the data suggest that granular convoluted tubule cells are homogeneous in the production and storage of NGF and EGF.

Animals↗
PubMed1985

Effects of epidermal growth factor on acid secretion from guinea pig gastric mucosa: in vitro analysis.

Epidermal growth factor (EGF) has been tested on guinea pig gastric mucosa mounted in Ussing chambers to investigate the suitability of using in vitro methods for examining EGF's effects on acid secretion. Epidermal growth factor reduced the rate of histamine-induced acid secretion to near basal levels when applied to the serosal gastric surface at nanomolar concentrations. Inhibitory effects were evident 10-15 min after EGF treatment and were maximal by 40 min. Cyclic adenosine monophosphate-induced secretion was also reduced by EGF, although the effect occurred more slowly than in histamine-treated tissues. Epidermal growth factor increased transmucosal resistance in histamine-treated, but not cyclic adenosine monophosphate-treated mucosa; potential difference was unaffected. Nerve growth factor had no effect when tested in the in vitro system. The EGF binding protein was found to enhance slightly the inhibitory activity of EGF on acid secretion. When applied to the luminal (mucosal) gastric surface, EGF inhibited secretion marginally but only at micromolar concentrations. These results indicate that EGF acts directly upon cells within the gastric mucosa, and is most effective when applied to the serosal gastric surface. They further suggest that in vitro preparations of intact gastric mucosa can be used for analyzing the inhibitory effects of EGF on gastric acid secretion.

Animals↗
PubMed1985

Alteration of amino acid metabolism in epileptogenic mice by elevation of brain pyridoxal phosphate.

A single intraperitoneal injection of pyridoxal-5'-phosphate (PLP) in a species of mouse, DBA/2J, that is normally susceptible to sound-induced convulsion exacerbated its epileptic condition. The effect of injection was most pronounced about 30 min after the administration and subsided gradually within the following 4 h. Correlated with this increased seizure susceptibility were enhanced levels of synaptosomal aspartate and glutamate, and a diminished gamma-aminobutyric acid (GABA) level. The concentrations of nonneuroactive amino acids remained unchanged. When stimulated with veratrine, synaptosomes prepared from PLP-injected mice showed an increased release of aspartate and glutamate and a decreased release of GABA compared to those prepared from control mice. The activity of glutamate decarboxylase in the brains of PLP-treated mice was lowered, whereas the activity of GABA-transaminase was enhanced. Finally, the epileptic condition of DBA mice could be ameliorated by maintenance on a diet composed of vitamin B6-deficient feed and cellulose.

Acoustic Stimulation↗
PubMed1985

Myosin phosphorylation and contraction of feline esophageal smooth muscle.

We tested the hypothesis that phosphorylation of the 20,000-Da light chain of myosin (LC 20) is related to mechanical activation of esophageal smooth muscle. Circular muscle layer strips of cat esophagus were taken from the lower esophageal sphincter (LES) and the distal esophageal body (EB). The LES strips developed tone spontaneously, and the EB strips were tonically contracted with carbachol. Both tissues relaxed in response to electrical-field stimulation. Phosphorylation of the LC 20 was determined in tissues quick-frozen during relaxation and during stress redevelopment after cessation of field stimulation. Stress and phosphorylation levels were low after 30 s of field stimulation, and a rapid contraction followed field stimulation. Phosphorylation in the LES increased from 0.043 +/- 0.029 to 0.328 +/- 0.043 mol Pi/mol LC 20 within 10 s after stimulation of the inhibitory nerves was terminated, while stress was still rising rapidly. Phosphorylation in the LES then declined to a steady-state value of 0.162 +/- 0.034 mol Pi/mol LC 20 after 10 min. Isotonic shortening velocities at a constant afterload following a quick release showed changes with time that were proportional to the level of phosphorylation. This was also true for values of maximal shortening velocity estimated for zero external load and for the rate of stress redevelopment after a step shortening. Comparable measurements were made in the carbachol-contracted EB. These results indicate that visceral smooth muscles, which normally function tonically (LES) or phasically (EB), exhibit an initial rapid mechanical activation associated with myosin phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Alterations in the pituitary-thyroid axis and 5'-deiodinase activity in mice with muscular dystrophy.

Mice with hereditary muscular dystrophy have reduced levels of serum T3. To determine possible causes of T3 deficits, we evaluated pituitary thyrotroph ultrastructure by electron microscopy, thyroid gland morphology by light microscopy, and T4 to T3 conversion by measuring iodothyronine 5'-deiodinase activity. Differences were not evident between dystrophic and normal littermates in either the structure of pituitary thyrotrophs or thyroid tissues. Dystrophic mice, however, had only 50% the normal hepatic 5'-deiodinase activity. Cerebral 5'-deiodinase, which does not appear to contribute significantly to serum T3, was similar in normal and dystrophic mice. Submandibular gland concentrations of nerve growth factor and epidermal growth factor are reduced in dystrophic mice but can be increased by T4 treatment. To distinguish whether growth factor deficits are due to reductions in serum T3 or to deficiencies in T4 5'-deiodinase activity and subsequent T3 utilization within the salivary gland, we measured submandibular deiodinase activity. Gland homogenates were active in the deiodinase assay, but no differences were detected between normal and dystrophic mice. In order to evaluate tissue responses to reductions in circulating T4, we treated mice with methimazole. Structural analyses revealed that thyrotrophs in dystrophic mice were less stimulated than thyrotrophs in similarly treated normal littermates. Likewise, thyroid follicular cells appeared less active, and thyroid weights increased only 40-50% as much as in normals. Liver 5'-deiodinase activity decreased in both normal and dystrophic mice. Cerebral 5'-deiodinase activity increased more than 4-fold in normal females but only 2-fold in dystrophic females; 2- to 3-fold increases occurred in both normal and dystrophic males. In summary, the structure of pituitary and thyroid glands in dystrophic mice is similar to that of tissues from normal littermates, but hepatic conversion of T4 to T3 is reduced. When challenged by methimazole-induced reductions in serum T4, pituitary and cerebral tissues in dystrophic mice respond abnormally.

Animals↗
PubMed1984

Actin and tropomyosin variants in smooth muscles. Dependence on tissue type.

Actin was found to be the major source of myofibrillar protein heterogeneity in smooth muscles. Three isoelectric variants, alpha-smooth muscle (alpha-SM), beta-non-muscle (beta-NM), and gamma-actins (gamma-SM and gamma-NM) were measured in 15 different smooth muscles, alpha-SM and gamma-actin contents displayed an inverse relationship in a given smooth muscle, some of which contained primarily alpha-SM actin while gamma-actins dominated in others. alpha-SM actin and gamma-actin distributions were tissue-specific, independent of species. A greater proportion of alpha-SM actin appears to be associated with tissues having a high degree of tonic activity. beta-Nonmuscle actin was a significant, and relatively constant, component of all smooth muscle tissues. The high NM-actin content of these tissues may reflect the importance of proliferative, synthetic, or secretory activities in smooth muscle, because the alpha-SM actin disappeared in tissue culture with a time course paralleling the modulation of phenotype from a contractile to a proliferative cell. Two tropomyosin subunits were present in approximately equal amounts in all smooth muscle tissues studied. One tropomyosin subunit exhibited identical mobility on two-dimensional gel electrophoresis, while the other was characterized by some species-specific variation which was unrelated to actin variant distribution. No variants of the 20,000-dalton regulatory light chain of myosin were observed. These results suggest that SM-specific actin variants are associated with functional diversity among smooth muscles.

Actins↗
PubMed1984

Assessment of vascular smooth-muscle mechanisms using isolated segments of the vessel wall.

Isolated segments of the blood vessel wall in the form of strips or rings offer advantages for the assessment of vascular smooth-muscle contractile function and its contribution to the properties of blood vessels. Several criteria must be met to obtain these advantages. These requirements are outlined in the context of an analytical framework based on the sliding filament/crossbridge model. The applicability of this model to smooth muscle is discussed, and the concept of a "functional unit" in the vascular wall is introduced. A functional unit is defined as all the smooth-muscle cells that are mechanically linked in series and bear equal stress. Several factors contribute to heterogeneity among functional units. The constraints imposed on the estimation of the static and dynamic mechanical properties of the smooth muscle are discussed. Various experimental strategies to minimize the effects of inhomogeneities in functional units can enhance the value of mechanical measurements.

Animals↗
PubMed1984

Myosin phosphorylation and cyclic adenosine 3',5'-monophosphate in relaxation of arterial smooth muscle by vasodilators.

Recent evidence indicates that contraction of vascular smooth muscle may be regulated by two calcium-dependent mechanisms: activation of myosin kinase, and calcium binding to a second, unknown regulatory site. This hypothesis implies that vasodilators could modify vascular tone by several mechanisms, including inactivation of myosin kinase. Since relaxation of the carotid artery following agonist removal may occur when myosin phosphorylation is at resting levels, we could determine whether dephosphorylation of myosin is necessarily involved in the molecular mechanisms mediating relaxation in response to vasodilators. The relaxant effects of adenosine, 3-isobutyl-1-methylxanthine, forskolin, sodium nitroprusside, and 8-bromo-cGMP were tested under conditions where myosin phosphorylation was at basal levels (0.08 +/- 0.02 mol Pi/mol light chain). All of these agents increased the rate of relaxation in nonsteady state experiments where relaxation was induced by stimulus washout. Steady state dose-response curves were obtained for forskolin and 8-bromo-cGMP in the presence of basal myosin phosphorylation. Forskolin caused a dose-dependent increase in cAMP levels at a rate consistent with a cause and effect relationship between relaxation and total tissue cAMP content. Both drugs relaxed the muscles, with no detectable change in myosin phosphorylation. Therefore, dephosphorylation of myosin is not a necessary event in the molecular mechanism of several vasodilators, including some which presumably act via cyclic nucleotides.

Animals↗
PubMed1984

Contamination of dental unit cooling water with oral microorganisms and its prevention.

Each time the handpiece turbine is stopped while the bur is still in the patient's mouth, almost 1 ml of microbe-laden oral fluids may be aspirated into the average dental unit water line by the retraction valve present in the dental unit. This fluid may contain an average in excess of 54,000 microorganisms per milliliter, including both facultative and obligate anaerobic bacteria of medium to high virulence. Other infectious agents including hepatitis, herpes, influenza, and other upper respiratory viruses may also be present. This germ-laden water may then be sprayed into the mouth of the next patient, possibly initiating an oral or upper respiratory tract infection. Sterilizing the handpiece between appointments, although of great significance in the prevention of disease transmission, will not totally eliminate the problem discussed here as almost 95% of the oral fluid aspirated into the dental unit went past the handpiece and into the handpiece cooling water line. Complete elimination of the retraction valve in the unit is the most effective means of solving this problem, but, with present dental unit designs, this may result in water dripping onto the patient. A viable alternative is the installation of a check valve (anti-retraction valve) downstream from the retraction valve. Installation of a check valve resulted in an almost 4,000-fold decrease in this contamination. The attachment of a check valve will have no effect on normal less virulent bacteria already present in tap water. These bacteria colonize the water lines of the dental unit and are then continually shed into the water in numbers often as high as 1,000,000 CFU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1983

Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle.

Stress development depended on calcium-stimulated myosin phosphorylation in an arterial smooth muscle preparation in which the concentration of calcium was controlled. However, developed stress was maintained at a concentration of calcium that did not support phosphorylation. These results, in conjunction with other evidence, suggest that the interaction of two regulatory mechanisms with different calcium sensitivities regulate both stress and the rate and energetics of contraction.

Animals↗