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Biomedical subjects

H A Singer

Publications and source records attributed to H A Singer.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation.

Elevations in guanosine 3',5'-cyclic monophosphate concentration ([cGMP]) are proposed to induce arterial smooth muscle relaxation by either 1) decreasing myoplasmic [Ca2+] ([Ca2+]i), 2) decreasing the [Ca2+]i sensitivity of phosphorylation, or 3) uncoupling force from myosin phosphorylation. We evaluated the importance of each of these mechanisms by measuring changes in [cGMP], aequorin- and fura-2-estimated [Ca2+]i, myosin light chain phosphorylation, and stress in histamine-stimulated swine carotid arteries. In tissues submaximally stimulated with 3 microM histamine, nitroprusside (NP) induced a proportional decrease in myoplasmic [Ca2+] and myosin phosphorylation, suggesting that the relaxation was at least partially induced by decreases in [Ca2+]i without a change in the [Ca2+]i sensitivity of phosphorylation. In tissues maximally stimulated with 10 microM histamine, NP and nitroglycerin produced significant relaxations that were not associated with significant sustained reductions in [Ca2+]i or myosin phosphorylation. With both submaximal and maximal histamine stimulation, nitrovasodilators produced more substantial relaxation than that expected from the nitrovasodilator-induced reduction in myosin phosphorylation. These results suggest that nitrovasodilators relax histamine-stimulated swine arterial smooth muscle by at least two mechanisms: 1) reducing [Ca2+]i, an effect observed in submaximally stimulated tissues, and 2) uncoupling of stress from myosin phosphorylation.

Animals↗
PubMed1992

Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+.

Elevations in extracellular [Mg2+] ([Mg2+]o) relax vascular smooth muscle. We tested the hypothesis that elevated [Mg2+]o induces relaxation through reductions in myoplasmic [Ca2+] and myosin light chain phosphorylation without changing intracellular [Mg2+] ([Mg2+]i). Histamine stimulation of endothelium-free swine carotid medial tissues was associated with increases in both Fura 2- and aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and force. Elevated [Mg2+]o decreased myoplasmic [Ca2+] and force to near resting values. However, elevated [Mg2+]o only transiently decreased myosin phosphorylation values: sustained [Mg2+]o-induced decreases in myoplasmic [Ca2+] and force were associated with inappropriately high myosin phosphorylation values. The elevated myosin phosphorylation during [Mg2+]o-induced relaxation was entirely on serine 19, the Ca2+/calmodulin-dependent myosin light chain kinase substrate. Myoplasmic [Mg2+] (estimated with Mag-Fura 2) did not significantly increase with elevated [Mg2+]o. These results are consistent with the hypothesis that increased [Mg2+]o induces relaxation by decreasing myoplasmic [Ca2+] without changing [Mg2+]i. These data also demonstrate dissociation of myosin phosphorylation from myoplasmic [Ca2+] and force during Mg(2+)-induced relaxation. This finding suggests the presence of a phosphorylation-independent (yet potentially Ca(2+)-dependent) mechanism for regulation of force in vascular smooth muscle.

Aequorin↗
PubMed1992

Angiotensin-II-binding sites on hepatocyte nuclei.

Angiotensin-II (Ang II) stimulates gene expression and cell growth in several cell types. Studies that have shown localization of Ang II to nuclei of myocytes and hepatic nuclear Ang II binding suggest that these actions may be mediated by nuclear receptors. We characterized Ang II binding to rat liver nuclei, which were free of plasma membrane based on enzyme analysis and electron microscopy. At 18 C, specific binding of 0.1-0.3 nM [125I]Ang II to nuclei and nuclear envelopes reached equilibrium by 2 h. Unlabeled Ang II inhibited [125I]Ang II binding to nuclei with an IC50 of 1.4 +/- 0.2 nM (+/- SE; n = 6). In half of the nuclear preparations, a lower affinity site (IC50, 50.4 +/- 23.6 nM), which accounted for 7-32% of specific Ang II binding, was detected by Scatchard analysis. Results similar to these were obtained with nuclear envelopes. Other Ang peptides competed for binding in the rank order: Ang III (IC50, 2.1 nM) greater than Ang I (IC50, 33) greater than [Des-Phe8]Ang II (IC50, 362) greater than [Des-Asp1-Des-Arg2]Ang II (IC50, 736). Losartan (DuP 753), an AT1 receptor antagonist, inhibited binding (IC50, 10.9 +/- 0.9 nM), whereas the AT2 receptor antagonist PD123177 did not. The pH optimum for binding to nuclear envelopes was 7, with binding more sensitive to low (5 and 6) than high (8 and 9) pH. Nonhydrolyzable GTP analogs accelerated displacement of bound [125I]Ang II by 10(-5) M Ang II. Differences were noted in pH sensitivity, time course, binding affinity for Ang I, II, and III, and rate of dissociation between nuclei or nuclear envelopes and plasma membrane Ang II binding. These results suggest that nuclear envelopes have a G-protein-coupled Ang II-binding site, which belongs to the AT1 class of Ang II receptors, with properties different from the plasma membrane receptor.

Angiotensin II↗
PubMed1990

Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle.

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.

Animals↗
PubMed1990

Phorbol ester-induced stress and myosin light chain phosphorylation in swine carotid medial smooth muscle.

The mechanisms by which activators of protein kinase C (PKC) stimulate contractile responses in arterial smooth muscle is not known. In this study, we assessed the relative contribution of CA(++)-dependent and independent pathways in mediating phorbol ester-induced 20 kdalton myosin light chain (MLC)-phosphorylation and force in medial smooth muscle strips from swine carotid artery. Phorbol 12,13-dibutyrate (PDB; 10(-7)M)-stimulated stress development was associated with a significant increase in the fraction of phosphorylated MLC, from 0.08 +/- 0.02 to 0.24 +/- 0.02 after 30 min of stimulation. Under conditions of Ca++ depletion, which normally do not support Ca++/calmodulin-dependent activation of myosin light chain kinase (MLCK) by physiological stimuli, PDB-induced contractile responses were reduced significantly. However, after Ca2++ depletion, PDB (10(-6) M; 30 min) still caused an increase in MLC-phosphorylation from 0.10 +/- 0.02 at rest to 0.19 +/- 0.03. Preincubation with nifedipine (10(-7) M) had no significant effect on contractile responses to PDB, indicating that Ca++ influx through nifedipine-sensitive voltage channels did not contribute significantly to the observed Ca++ dependency of the PDB responses. Staurosporine (0.1-0.3 microM), a putative PKC inhibitor, significantly inhibited PDB-induced contractile and MLC phosphorylation responses. Tonic histamine (3 microM)- and KCl-induced contractile and MLC-phosphorylation responses were inhibited by the same concentrations of staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1989

Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine.

The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

Amino Acids↗
PubMed1989

Angiotensin II receptor-mediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocytes.

We have described previously positive inotropy and increased levels of inositol-l-phosphate as in vitro responses to angiotensin II in cardiac tissue. In this study, changes in cardiac myocyte-free cytosolic calcium stimulated by angiotensin II were monitored with the fluorescent calcium indicator dye Fura-2. There was an initial peak transient increase followed by a sustained increase in cytosolic-free calcium in response to angiotensin II (10(-9)-10(-6) M). The peak transient response in cytosolic-free calcium after addition of angiotensin II (10(-7) M) occurred at 23 +/- 4 sec and was stimulated 2.16-fold (332 +/- 56 nM) above basal levels (154 +/- 14.7 nM). The calcium response was blocked or reversed by addition of verapamil (10(-8) M), lanthanum (0.2 mM) and zero calcium buffer. Angiotensin II receptor-mediated stimulation of inositol phosphates was quantified after separation by high-performance liquid chromatography in cultured chick heart cells prelabeled with L-myo-[1,2-3H(N)]inositol. A time course indicated that the peak response of the angiotensin II (10(-8) M)-stimulated increase in inositol-1,4,5-trisphosphate was at 30 sec. Angiotensin II (10(-8) M) significantly stimulated inositol-1,4-diphosphate (45%) and inositol-1,4,5-trisphosphate (78%) above basal levels. Bordetella pertussis toxin treatment of myocyte cultures in doses (500 ng/ml, 24 hr) shown to fully ADP-ribosylate a toxin-sensitive 41 KD alpha-subunit, blocked completely the angiotensin II-stimulated increases in inositol 1,4-diphosphate, inositol-1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. The rise in cytosolic-free calcium in response to angiotensin II was not blocked or inhibited by toxin pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1988

Identification and characterization of guinea pig angiotensin II ventricular and atrial receptors: coupling to inositol phosphate production.

Angiotensin II elicits a receptor-mediated positive inotropic response in cardiac tissue from most mammalian species by activating voltage-sensitive slow Ca2+ channels. In the guinea pig, we confirm there is no in vitro contractile force development in bioassay systems using isolated hearts or left atrial tissue in response to angiotensin peptides. However, 125I-angiotensin II binding sites that have the characteristics of a membrane receptor were identified in ventricular (myocardial) and atrial membrane preparations from guinea pigs. In ventricles, saturation-binding data yielded an optimal fit to a two-site model with a high-affinity site Kd1 = 3.6 +/- 0.7 nM and a low-affinity site Kd2 = 433 +/- 126 nM and binding capacities of 66 +/- 10 and 821 +/- 49 fmol/mg protein, respectively. In atria, saturation binding data yielded an optimal fit to a two-site model with a high-affinity site Kd1 = 1.6 nM and a low-affinity site Kd2 = 300 nM and capacities of 145 and 752 fmol/mg protein, respectively. The ventricular binding of 125I-angiotensin II was stimulated approximately twofold in the presence of the divalent cations calcium and magnesium (10 mM). Nonhydrolyzable analogues of guanosine triphosphate increased the dissociation rate of the bound 125I-angiotensin II and decreased hormone binding to the receptor at equilibrium. Competition for 125I-angiotensin II binding by an agonist-antagonist analogue series correlated with previous studies obtained in the rabbit, a mammal in which inotropic responses to angiotensin II were demonstrated. The data indicate the presence of angiotensin II myocardial and atrial receptors and a G-type coupling protein in guinea pig. Although this species lacks an inotropic response to angiotensin peptides, there is a dose-dependent increase in inositol-1-phosphate production in response to angiotensin II, and this response is blocked by the selective angiotensin II antagonist [Sar1,Ile8]angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Role of calcium in endothelium-dependent relaxation of arterial smooth muscle.

Endothelium-dependent relaxation was studied in rings of rabbit thoracic aorta. Relaxation responses were induced with methacholine, the calcium ionophore A23187 and maitotoxin before and after removal of Ca++ from the external medium; in the presence of calcium-channel entry blockers (verapamil and nifedipine); or with trifluoperazine. Deletion of Ca++ greatly impaired responses to all 3 agonists while trifluoperazine only blocked cholinergic-induced relaxation. The calcium-channel blockers had effects that were concentration- and time-dependent, but their action included blockade of A23187. Cytosolic-free Ca++ concentrations were measured in cultured endothelial cells after incubation of the cells with 10 microM Fura-2/AM or 50 microM Quin 2/AM. Bradykinin (1 X 10(-10) to 1 X 10(-7) M) and melittin (0.5 to 5 micrograms/ml) caused dose-dependent increases in intracellular Ca++ with maximal responses at 3 X 10(-8) M and 3 micrograms/ml, respectively. Both agents were able to induce an increase in cytosolic-free Ca++ in the presence of EGTA (1.5 X 10(-3) M) or verapamil (1 X 10(-5) M). The plateau phase of the Ca++ transient appeared to be modified slightly by verapamil, while the peak responses and plateau were attenuated by '0' Ca++/EGTA. To assess a function of the endothelium, production of endothelium-derived relaxing factor (EDRF) was studied in cells grown on microcarrier beads superfused in a column, and the column effluent was bioassayed on aortic rings.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Apparent dissociation between myosin light chain phosphorylation and maximal velocity of shortening in KCl depolarized swine carotid artery: effect of temperature and KCl concentration.

Stimulation of swine carotid artery medial fibers with 110 mM KCl at 37 degrees C results in increases in myosin light chain (MLC) phosphorylation levels and maximal shortening velocity (Vo) during the period of stress development. During the period of stress maintenance, MLC phosphorylation levels and Vo are not maintained, but fall to suprabasal levels, resulting in a correlation between MLC phosphorylation and Vo and suggesting that MLC phosphorylation regulates Vo. This study identifies other conditions of KCl depolarization of swine carotid medial fibers in which this relationship between MLC phosphorylation and Vo is altered. A decrease in temperature from 37 degrees to 23 degrees C results in a similar magnitude of stress maintenance in response to 110 mM KCl and similar levels of MLC phosphorylation, but a reduction in Vo by approximately 50%. This differential effect of temperature on Vo and MLC phosphorylation results in a downward shift in the slope of the regression line describing the relationship between these two parameters. Decreasing [KCl] to 40 mM in the stimulating solution results again in similar magnitudes of maintained stress. MLC phosphorylation levels are not transient, but maintained at a constant value and Vo is transient at levels approximately 50% of those at 110 mM KCl at 37 degrees C. This results in a complete lack of correlation between MLC phosphorylation and Vo. Thus, stress can be developed to equal magnitudes under differing activation conditions with dissimilar patterns of MLC phosphorylation and Vo. Therefore, there is not a strict relationship between MLC phosphorylation and Vo in all cases.

Animals↗
PubMed1987

Maximal rates of activation in electrically stimulated swine carotid media.

The kinetics of crossbridge phosphorylation and cellular activation rates in arterial smooth muscle are unknown, and the response rates are usually limited by agonist diffusion rates. In this study, electrical field stimulation (10-20 V AC, 60 Hz) was used to activate strips of swine carotid artery smooth muscle. Pretreatment with 0.5 microM phenoxybenzamine (PBZ) for 30 minutes significantly inhibited field stimulated stress due to norepinephrine released from adrenergic nerves. Addition of 1-10 mM tetraethylammonium ion (TEA) to PBZ-pretreated tissues allowed direct stimulation of muscle cells and significantly potentiated the response to field stimulation. Activation in field stimulated tissues pretreated with PBZ and TEA was strongly inhibited by the Ca2+-channel antagonist verapamil (0.5 microM for 15 minutes). Activation was rapid with myosin light chain (MLC)-phosphorylation values rising to greater than 50% of total MLC, with a half-time of approximately 1 second. Stress and stiffness development was biphasic and lagged with an estimated half-time for the first phase of 3 seconds. Stress and stiffness continued to slowly rise after 5 seconds of stimulation, while MLC phosphorylation declined significantly between 5 and 10 seconds to a nearly constant value of 35-40% for up to 60 seconds. Unloaded shortening velocity was maximal (0.067 Lo/sec) at the earliest time point examined (0.5 second), prior to significant stress development and at submaximal phosphorylation. Significant decreases in unloaded shortening velocity were observed after 10 seconds. These results show that the kinetics of phosphorylation and dephosphorylation and the rates of activation are very rapid in this arterial smooth muscle.

Animals↗
PubMed1987

Calcium dependence of phorbol 12,13-dibutyrate-induced force and myosin light chain phosphorylation in arterial smooth muscle.

Phorbol dibutyrate (PDB) is an activator of protein kinase C and has been observed to cause a slow developing contraction in vascular smooth muscle. The mechanism of phorbol ester-induced contraction is unknown. We studied the Ca++-dependence of, and the degree of myosin light chain phosphorylation (MLC-P), during PDB-induced contractions in rabbit aortic rings. PDB elicited concentration-dependent contractions (3 X 10(-8) to 10(-6) M) in rabbit aortic rings incubated in normal (1.6 mM Ca++) physiologic salt solution (PSS). Addition of the Ca++-channel blocker nifedipine (0.1 microM) to PSS or removal or Ca++ from PSS significantly reduced the contractile responses to PDB. Depletion of Ca++ by repeated washes in O Ca++-PSS containing 10(-3) M ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid reduced, but did not eliminate, the responses to PDB. In PSS, PDB significantly increased the fraction of phosphorylated MLC/total MLC to 0.33 from a resting value of 0.20. Ca++ depletion reduced the resting fraction (MLC-P/MLC) to 0.14. PDB-stimulated contractions in Ca++-depleted tissues occurred in the absence of significant increases in MLC-P. Sodium nitroprusside partially relaxed PDB-induced contractions by approximately 50% whether elicited in the presence of 1.6 mM Ca++ or after Ca++ depletion. In both cases relaxation occurred in the absence of statistically significant decreases in MLC phosphorylation. Ca++-dependent MLC phosphorylation may account for a component of the PDB contractile response in rabbit aorta. Studies in the absence of Ca++ suggest that PDB may activate contraction without concomitant MLC-P.

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↗
PubMed1984

Effects of cytochrome P-450 inhibitors on endothelium-dependent relaxation in rabbit aorta.

Endothelium-dependent relaxation in rabbit aorta was inhibited by exposure to two cytochrome P-450 inhibitors, metyrapone and SKF-525A. Aortic rings were contracted to a stable plateau by addition of phenylephrine (10(-7) M). Relaxation was elicited by the cumulative addition of methacholine (3 X 10(-8) - 3 X 10(-6) M) or A23187 (10(-8) - 10(-6) M). Exposure to metyrapone (500 microM) or SKF-525A (10 micrograms/ml) was found to inhibit relaxation in response to concentrations of methacholine exceeding 10(-7) M. Maximal relaxation was inhibited 73% by metyrapone. Relaxation stimulated by concentrations of A23187 exceeding 10(-7) M was also found to be inhibited by both metyrapone and SKF-525A exposure. Maximum A23187-induced relaxation (55% of the phenylephrine contractile response) was inhibited 40% by metyrapone and 55% by SKF-525A. Arachidonic acid (10-100 microM) also elicited endothelium-dependent relaxation in rings pretreated with indomethacin (10 micrograms/ml) and contracted with phenylephrine. This relaxation response was abolished by exposure to metyrapone or SKF-525A. These results suggest that cytochrome P-450 may be involved in endothelium-dependent relaxation responses, perhaps by metabolizing arachidonic acid to active products.

Animals↗
PubMed1983

Endothelium-dependent relaxation of rabbit aorta. I. Relaxation stimulated by arachidonic acid.

Vascular responses to the addition of arachidonic acid (AA) were characterized in rings of rabbit thoracic aorta. In preparations which were precontracted to a stable plateau by 10(-7) M phenylephrine, AA (10-100 microM) elicited transient relaxation if the endothelium was intact. The same concentrations of AA only contracted tissues in which the endothelium was purposely disrupted. Indomethacin pretreatment (14 microM, 30 min) potentiated the relaxation response in intact rings. Under these conditions, relaxation in response to 100 microM AA was 32(+/- 3)% of the tension stimulated by 10(-7) M phenylephrine. Preincubation with 5,8,11,14-eicosatetraynoic acid (50 microM) or nordihydroguaiaretic acid (25 microM) completely prevented AA-induced relaxation. Contractile responses to AA were examined in rings lacking pre-existing active tension. AA (3-100 microM) elicited rapidly developing and somewhat transient contractions in rings with endothelium. Slow developing contractions which were significantly smaller in magnitude were observed in rings lacking endothelium. Indomethacin or 5,8,11,14-eicosatetraynoic acid pretreatment inhibited AA-stimulated contractions both in intact and de-endothelialized tissues. Nordihydroguaiaretic acid pretreatment had no effect on contractile responses in rings lacking endothelium and slightly potentiated responses in intact rings. We conclude that endothelium-dependent relaxation of rabbit aorta by AA is mediated by a noncyclooxygenase metabolite(s). Contractile responses are mediated by cyclooxygenase metabolites, which in intact rings are derived largely from the vascular endothelium.

5,8,11,14-Eicosatetraynoic Acid↗