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Biomedical subjects

E E Reynolds

Publications and source records attributed to E E Reynolds.

36 records · Page 2Linked to original sources
PubMed1990

Role of thromboxane A2/prostaglandin H2 receptor in the vasoconstrictor response of rat aorta to endothelin.

Endothelin stimulates the release of arachidonic acid in vascular smooth muscle cells, raising the possibility that eicosanoid metabolites of arachidonic acid may be involved in mediating the contractile effects of endothelin. We have investigated the hypothesis that endothelin-1 stimulates formation of thromboxane A2 in rat aorta and that this thromboxane A2 mediates part of the contractile response to endothelin-1. Preincubation of rat aorta with 10 microM SQ 29,548, a "selective" antagonist of thromboxane A2/prostaglandin H2 (TP) receptors, caused a rightward shift in the dose-response curve and a reduction in the maximal response to endothelin-1. Preincubation with SQ 29,548 had no effect on the dose-response curves to either norepinephrine or KCl. In rings of rat aorta that had been precontracted with 1 nM endothelin-1, addition of either 10 microM SQ 29,548 or 10 microM indomethacin (cyclooxygenase inhibitor) caused a relaxation of 50-65% of developed tension. The inhibitory effect of SQ 29,548 on endothelin-induced contraction was greater when it was added after endothelin than when added before endothelin, suggesting that activation of the TP receptor may be more important in the maintenance of developed tension than in the initiation of contraction. Finally, 100 nM endothelin-1 stimulated the indomethacin-sensitive formation of thromboxane A2 in rings of rat aorta. These results suggest that a significant portion of the sustained contraction induced by endothelin in rat aorta requires the persistent activation of the TP receptor by a cyclooxygenase product, most likely thromboxane A2 or prostaglandin H2.

Animals↗
PubMed1989

Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells.

Endothelin-stimulated [3H]-inositol phosphate formation and [3H]-arachidonic acid release were measured in cultured vascular smooth muscle cells from rabbit renal artery. Both responses were partially inhibited by pretreatment with pertussis toxin, indicating the involvement of pertussis toxin-sensitive guanine nucleotide binding regulatory proteins in the coupling processes. Pretreatment with the phorbol ester PMA inhibited endothelin-stimulated [3H]-inositol phosphate formation, but potentiated endothelin-stimulated [3H]-arachidonic acid release, suggesting that these two coupling processes occur in a parallel and independent manner in vascular smooth muscle cells.

Animals↗
PubMed1989

Endothelin is a positive inotropic agent in human and rat heart in vitro.

We have investigated the response to endothelin of isolated atrial and ventricular trabeculae from failing human hearts obtained at transplant. Results indicate that endothelin exerts a significant positive inotropic effect on human atrial and ventricular tissue, with increases in developed tension of 74.6 +/- 14.1% (+/- SEM) and 9.9 +/- 4.0%, respectively. Further studies on rat cardiac muscle demonstrate that the greater inotropic effect on atrial than ventricular muscle is also exhibited by the rat heart in vitro, with 39.9 +/- 10.7% and 17.1 +/- 5.9% increases in developed tension for atria and papillary muscle, respectively. Studies in rat atria also provide no evidence for an effect of endothelin on the frequency of spontaneous contractions. These results suggest that the potential exists for regulation of cardiac function in humans and rats by endothelial-derived factors such as endothelin, possibly via augmentation of atrial systole.

Adolescent↗
PubMed1989

Long-term suppression of the cerebral spread of a memory: effects of idazoxan and clonidine.

Bitemporal injections of puromycin consistently induce amnesia of aversive maze-learning in mice when administered within 3 days of training. These bitemporal puromycin injections lose their amnestic effectiveness if the latency between training and injection is extended beyond 6 days. Consistent with other evidence, we believe that memory (in our task) "spreads" during the 6 days following training. Since previous experiments have indicated that the central noradrenergic system is involved in this process of "memory spread," we have examined the effect of stimulation or blockade of the alpha 2-receptor. To this end, we administered a single dose of the alpha 2-adrenoceptor antagonist, idazoxan, or the alpha 2-agonist, clonidine. Idazoxan (1 mg/kg, SC) had no effect on engram spread. Clonidine (25 micrograms-125 ng/kg, SC), by contrast, suppressed engram spread for at least 30 days after treatment. When mice were tested at 60 and 90 days after treatment, spontaneous recovery (i.e., engram spread) was evident in only about 50% of the clonidine treated mice. Coadministration of idazoxan with clonidine blocked the effects of clonidine on "memory spread."

Animals↗
PubMed1988

Novel kinetics of single cell Ca2+ transients in stimulated hepatocytes and A10 cells measured using fura-2 and fluorescent videomicroscopy.

The kinetics of agonist-induced increases in cytosolic free Ca2+ have been measured in single A10 vascular smooth muscle cells and rat hepatocytes using fluorescent videomicroscopy with fura-2 as a Ca2+ indicator. At high agonist concentrations there was no difference in the kinetics of the Ca2+ transient measured in vasopressin-stimulated single A10 cells or in cell populations. However, stimulation of single A10 cells with concentrations of vasopressin below 0.5 nM produced characteristic Ca2+ transients composed of two distinct peaks. The two peaks appeared to represent a temporal separation between release of intracellular Ca2+ and influx of extracellular Ca2+. The double transient was not observed in single rat hepatocytes stimulated with low concentrations of vasopressin or phenylephrine. In both A10 cells and hepatocytes, the initial rate of increase in Ca2+ concentrations in response to submaximal agonist concentrations was faster in single cells than in cell populations. This difference was due to asynchrony of the cellular response, where there was a latent period of variable length before onset of a rapid increase in Ca2+ concentration. The duration of the latent period was dependent on the agonist concentration, higher concentrations of agonist giving a reduced latent period. The hormone-stimulated Ca2+ transient measured in single hepatocytes with fura-2 was different from the series of transient spikes as previously reported using aequorin as the Ca2+ indicator, suggesting that fura-2 and aequorin may report different aspects of the Ca2+ response in stimulated cells. Collectively, these results demonstrate that measurement of Ca2+ transients in single cells provide novel information concerning the nature of the Ca2+ transient that is not apparent from studies with cell populations.

Animals↗
PubMed1988

Neuropeptide Y receptor-effector coupling mechanisms in cultured vascular smooth muscle cells.

Primary cultures of rabbit pulmonary artery (RPA) vascular smooth muscle (VSM) were utilized to determine the coupling of neuropeptide Y (NPY) receptors to several effector systems in VSM. NPY inhibited forskolin-stimulated adenylate cyclase by 65%, with an EC50 of 0.3 nM. However, NPY did not stimulate phosphoinositide (PI) hydrolysis or the elevation of cytosolic calcium, (Ca+2)i, in cultured RPA-VSM cells, nor did it potentiate norepinephrine-induced PI hydrolysis or elevation of (Ca+2)i. These results suggest that NPY-induced vasocontraction is not mediated by PI hydrolysis or the modulation of (Ca+2)i.

Animals↗
PubMed1988

Effect of Na+/H+ exchange inhibitors on agonist-induced contraction of rat aorta.

A number of vasoconstrictor agonists activate the Na+/H+ antiport system in vascular smooth muscle, leading to alkalinization of the cytosol and influx of Na+. It is believed that agonist-induced Na+/H+ exchange may play an important role in contraction. We have evaluated this hypothesis by determining the effect of inhibition of Na+/H+ exchange on phenylephrine (PE)-induced contraction of rat aorta. Preincubation of rat aorta with the Na+/H+ exchange inhibitor amiloride (0.5 mM) inhibited subsequent PE-induced contraction. However, the more potent and specific Na+/H+ exchange inhibitor hexamethylene amiloride (10 microM) did not inhibit PE-induced contraction. Inhibition of Na+/H+ exchange by removal of extracellular Na+ and substitution with N-methyl-D-glucamine only moderately reduced PE-induced contraction. Hexamethylene amiloride (10 microM) and methyl-isobutyl amiloride (30 microM), another potent and specific inhibitor of Na+/H+ exchange, caused a slow, sustained and reversible contraction of rat aorta which was 114.7 and 86.6%, respectively, of maximal PE-induced contraction. Hexamethylene amiloride-induced contraction was dose-dependent, dependent on extracellular calcium and inhibited by nifedipine. We conclude that the vasorelaxant effects of amiloride are unrelated to inhibition of Na+/H+ exchange, and may be mediated by inhibition of kinases involved in contraction. These results demonstrate that inhibition of Na+/H+ exchange has no major effect on agonist-induced contraction of rat aorta, and suggest that vasoconstrictor activation of Na+/H+ exchange does not play a major role in agonist-induced contraction of rat aorta.

Amiloride↗
PubMed1987

Histamine-induced calcium release and phorbol antagonism in cultured airway smooth muscle cells.

Primary cultures of airway smooth muscle cells were exposed to histamine, and intracellular free calcium transients were measured by the calcium-sensitive dye fura-2. Stimulation with 100 microM histamine resulted in a rise in intracellular calcium from an unstimulated level of 178 +/- 25 to 497 +/- 154 nM Ca2+ (SE; n = 14) and a return to base-line free calcium concentration within 1 min of stimulation. Pretreatment of cells with the H1 receptor blocker pyrilamine (2.5 microM) abolished the response; however, the calcium transient was not altered by pretreatment with the H2 blocker cimetidine (50 microM), by chelation of external calcium, or by pretreatment with 2 mM Co2+ or 5 microM nifedipine. Activation of protein kinase c by 200 nM phorbol 12-myristate 13-acetate (PMA) resulted in no detectable rise in cytosolic calcium but completely blocked the release of internal calcium by histamine. We conclude that 1) histamine causes a transient rise of cytosolic calcium in airway smooth muscle, 2) the rise in cytosolic calcium is mediated by H1 receptor coupling that triggers release of internal calcium stores, and 3) activation of protein kinase c blocks the histamine-induced release of intracellular calcium.

Animals↗
PubMed1986

Agonist-induced calcium transients in cultured smooth muscle cells: measurements with fura-2 loaded monolayers.

Elevation of cytosolic Ca2+ in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDT1, A10, rabbit aorta) loaded with the either quin-2 or fura-2, and assayed either in suspension or in monolayer cultures attached to plastic cover slips. Agonists (norepinephrine, vasopressin) induced both the release of intracellular Ca2+ and the influx of extracellular Ca2+. Agonist-induced Ca2+ influx was not blocked by dihydropyridines, and depolarization did not induce Ca2+ influx. However, in fura-2 loaded monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive Ca2+ influx. Thus cultured smooth muscle cells appear to express receptor-operated Ca2+ channels, but not functional voltage-operated Ca2+ channels.

Aminoquinolines↗
PubMed1986

Down regulation of beta adrenergic receptors in S49 lymphoma cells induced by atypical agonists.

The ability of the atypical agonists celiprolol and pindolol to induce sequestration and down regulation of beta adrenergic receptors was investigated in S49 lymphoma cells. Sequestration was measured as the loss of binding sites for [3H]CGP-12177, a hydrophilic radioligand that binds only to surface beta adrenergic receptors at 6 degrees C. Down regulation was measured as the loss of binding sites for [125I]iodopindolol, a lipophilic radioligand which at 37 degrees C binds to both surface and sequestered receptors. Pindolol and celiprolol do not stimulate adenylate cyclase in membranes from wild-type (WT) S49 cells or do they induce the sequestration of beta adrenergic receptors on intact cells. Incubation of WT S49 lymphoma cells with isoproterenol for 24 hr resulted in the loss of 75% of total cellular beta adrenergic receptors (down regulation). Exposure of WT S49 cells to pindolol or celiprolol for 24 hr resulted in the loss of approximately half of the total cellular beta adrenergic receptors. In mutant S49 cells [cyc- (variant of S49 lymphoma cells which lacks Ns activity) and UNC (variant of S49 lymphoma cells in which Ns is present but cannot interact with beta adrenergic receptors)] in which interaction of beta adrenergic receptors with the stimulatory guanine nucleotide binding regulatory protein (Ns) does not occur, a 24 hr incubation with isoproterenol caused the loss of approximately half of the beta adrenergic receptors, whereas pindolol and celiprolol caused no change in the number of receptors. These results suggest that there are two mechanisms of down regulation of beta adrenergic receptors in S49 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1985

Activation of calcium mobilization and calcium influx by alpha 1-adrenergic receptors in a smooth muscle cell line.

Alpha 1-adrenergic receptor (alpha 1R) mediated increases in the cytosolic levels of free Ca+2 and the inositol phosphates were measured in a smooth muscle cell line, DDT1. Norepinephrine (NE) stimulated a rapid increase in cytosolic Ca+2 by two distinct components: 1) release of Ca+2 from intracellular sites (mobilization), and 2) influx of extracellular Ca+2. The mobilization component was not affected by removal of extracellular Ca+2 or addition of La+3 or Co+2 to the buffer. The influx component was abolished by EGTA, La+3, or Co+2, but was not affected by the voltage-operated Ca+2 channel blockers diltiazem or nifedipine. Depolarization of DDT1 cells with 100 mM KCl or with gramicidin did not induce Ca+2 influx. NE also increased inositol trisphosphate to 78% over basal levels within 1 minute. These results suggest that alpha 1R on DDT1 cells are coupled to both the mobilization of intracellular Ca+2 and to receptor-operated Ca+2 channels in the plasma membrane, and that polyphosphoinositide hydrolysis may play a role in these phenomena.

Aminoquinolines↗
PubMed1984

Agonist-induced changes in the properties of beta-adrenergic receptors on intact S49 lymphoma cells. Time-dependent changes in the affinity of the receptor for agonists.

The binding of the antagonist [125I]iodopindolol to beta-adrenergic receptors on intact wild-type S49 mouse lymphoma cells and mutants that have impaired abilities to generate cyclic AMP in response to catecholamines was studied. The binding of [125I]iodopindolol is of high affinity (KD = 35 pM), rapid, stable over 90 min, and rapidly reversible (t1/2 = 8 to 11 min). Nonspecific binding was very low (less than 5% of total binding at the KD). Kinetic and competition experiments performed under steady-state and non-equilibrium conditions revealed that the binding characteristics for agonists were very different in intact cells and in membranes. The interactions of antagonists, on the other hand, appeared to be identical in studies carried out with intact cells and membranes. In intact cells, the affinity of the receptor for agonists was observed to decrease rapidly within the first 5 min of exposure of the cells to an agonist. Competition experiments revealed that at least 80% of the receptor-agonist complex was in a high-affinity state when studies were carried out using short incubation times (0.5-1 min). Under equilibrium conditions, about 80% of the complex in wild-type, uncoupled, and kinase-deficient cells was of a low affinity. At equilibrium, only low-affinity binding was seen with coupling protein-deficient cells. This rapid, time-dependent decrease in the affinity of receptors for agonists was seen with most agonists although not with zinterol. The phenomenon was not due to differences in the kinetics of the interactions of agonists and [125I]iodopindolol with the receptor, and it is likely that the receptor undergoes a conformational change upon exposure to agonists. This effect was not observed in membranes and was not related to the presence of a functional guanine nucleotide-binding protein or to the production of cyclic AMP. Furthermore, hydrophilic agonists and antagonists, under short-term incubation conditions, did not fully compete for the binding sites labeled with the lipophilic radioligand [125I]iodopindolol, although this binding was fully and stereospecifically competed for by lipophilic antagonists. This suggests that in untreated cells a small but significant fraction of the receptors is sequestered in an environment not accessible to hydrophilic ligands.

Adenylyl Cyclases↗
PubMed1984

Relationship between intrinsic activities of agonists in normal and desensitized tissue and agonist-induced loss of beta adrenergic receptors.

The intrinsic activities of 14 agonists at beta adrenergic receptors were determined in membranes prepared from control L6 muscle cells or from cells preincubated in the presence of isoproterenol for 20 min. Several drugs that were full agonists when studied with membranes from naive cells were partial agonists in membranes prepared from desensitized cells. Prolonged incubation of cells with agonists resulted in a 70 to 80% decrease in the density of beta adrenergic receptors. This effect was observed after incubation of cells with either full or partial agonists, suggesting that the extent of receptor loss is independent of the intrinsic activity of an agonist. The rate of receptor loss was related, however, to the intrinsic activity of an agonist. The rate of receptor loss correlated with the intrinsic activities of agonists in desensitized (r = 0.94) and control (r = 0.87) membranes.

Adenylyl Cyclases↗
PubMed1982

Adenosine 5'-triphosphate independent secretion from PC12 pheochromocytoma cells.

PC12 is a clonal line of rat pheochromocytoma. The cells secrete dopamine and acetylcholine by a Ca2+-dependent process. The requirement for adenosine 5'-triphosphate (ATP) in secretion from PC12 was investigated. Cellular levels of ATP were decreased by brief treatments with inhibitors of glycolysis or oxidative phosphorylation. In order to deplete ATP stores below 40% of control values during a 5-min treatment with these compounds, it was necessary to inhibit both glycolysis and oxidative phosphorylation. Depletion of ATP pools to as little as 10% of control values did not result in any inhibition of dopamine or acetylcholine release evoked by 55 mM K+ or by carbachol. Rather, in many cases spontaneous and evoked releases were significantly increased under these conditions. The release observed in ATP-depleted cells was Ca2+ dependent. The data indicate that, unlike the situation with most other secretory cells, secretion from PC12 is independent of ATP. In PC12 some proteins involved in the secretion process may be altered so as to make the process independent of ATP; this may be due to the fact that PC12 is a tumor cell line. Phosphotyrosine-containing proteins, which are present in some other tumor cells, were not found in PC12.

Acetylcholine↗
PubMedDate not supplied

Truncated analogues of endothelin and sarafotoxin are selective for the ETB receptor subtype.

The sarafotoxins (SRTXs) are a family of 21-amino acid bicyclic peptides that are structurally similar to the potent vasoconstrictors, the endothelins (ETs). SRTX-6c exhibits high selectivity for the ETB receptor subtype over the ETA subtype. This selectivity may be a result of a modification at positions 9 or 13, which are Glu9 and Asn13 in SRTX-6c and Lys9 and Tyr13 in ET-1. The truncated linear endothelin [Ala11,15]ET-18-21 was also on ETB receptor-selective ligand with 6-nM binding affinity. Therefore, a series of truncated endothelin/sarafotoxin (8-21) hybrid analogues were prepared, varying the amino acid at residues 9 and/or 13. These peptides were assayed for binding in tissues selectively expressing either the ETA or the ETB receptor subtypes. All linear analogues showed poor ETA binding, while ETB affinity was sequence dependent. Monocyclic 11-15 disulfide-containing analogues failed to bind to either ETA or ETB receptors at concentrations of up to 10 microM.

Amino Acid Sequence↗