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Biomedical subjects

R M Burch

Publications and source records attributed to R M Burch.

At least 91 records · Page 5Linked to original sources
PubMed1985

Solubilization of a thromboxane A2/prostaglandin H2 antagonist binding site from human platelets.

A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k-1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I]-PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation.

Blood Platelets↗
PubMed1985

Blockade of colchicine-induced inhibition of vasopressin-stimulated osmotic water flow: failure to influence microtubule formation.

Colchicine inhibits vasopressin-induced osmotic water flow across isolated toad urinary bladder. Concomitantly, colchicine has been shown to reduce the relative cytoplasmic volume fraction of microtubules in the apical granular cells of this epithelium that have been shown previously to mediate the hydroosmotic effect of vasopressin. Therefore, an intact cytoskeleton has been postulated to be a requirement for a full response to vasopressin. Since it has been demonstrated recently that cyclooxygenase inhibitors (meclofenamic acid) abrogate the inhibition by colchicine of vasopressin-stimulated water flow, we tested by stereological criteria the hypothesis that colchicine in the presence of meclofenamic acid does not prevent the polymerization of tubulin. Our results show that the relative cytoplasmic volume fraction of microtubules was reduced 75% by colchicine in the presence or absence of meclofenamic acid. An alternative explanation of the inhibitory action of colchicine is its ability in the toad urinary bladder to enhance the endogenous synthesis of and sensitivity to prostaglandin E, a potent negative modulator of vasopressin-stimulated water flow. An intact microtubular component of the cytoskeleton does not appear to be required for a maximal response to a physiological dose of vasopressin.

Animals↗
PubMed1985

Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to washed human platelets.

A binding site for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist [125I]9,11-dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 to washed human platelets was studied. 9,11-Dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 competitively antagonized aggregation of washed human platelets induced by the TXA2/PGH2 mimetic U46619. A Schild analysis of the pharmacologic study revealed a pA2 of 8.08 and a slope of -1.12. The pA2 value yielded a Kd of 8 nM. The association rate constant (k1) for [125I]PTA-OH was 6.6 X 10(6)M-1 min-1 and the dissociation rate constant (k-1) was 1.82 X 10(-1), yielding a kinetically determined Kd (k-1/k1) of 27 nM. Scatchard analysis of [125I]PTA-OH binding to washed human platelets revealed one class of binding sites with a Kd of 21 +/- 5 nM and maximum binding of 42 +/- 6.4 fmol/10(7) platelets (N = 5) (2530 +/- 380 binding sites/platelet). Several TXA2/PGH2 receptor agonists and antagonists competed with [125I]PTA-OH for binding. For the four antagonists used in this study, the rank order of potency for displacing the ligand from its binding site correlated (r = 0.93) with the rank order of potency for their ability to inhibit U46619-induced aggregation in human platelet-rich plasma. The antiaggregatory prostaglandins prostaglandin F2 alpha, prostaglandin D2, and Iloprost also displaced the ligand, but only at concentrations considerably higher than that required to produce their pharmacologic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1984

Alterations in extracellular calcium concentration differentially influence prostaglandin and thromboxane synthesis in epithelial cells from the toad urinary bladder.

The effects of alterations in extracellular calcium concentration on prostaglandin (PGE) and thromboxane (TXB2) syntheses were studied in isolated epithelial cells from the urinary bladder of the toad, Bufo marinus. In epithelial cells prepared using collagenase, basal iPGE synthesis was greater than iTXB2 synthesis. Increasing extracellular calcium from zero to 1 mM increased iPGE synthesis and decreased iTXB2 synthesis equivalently such that total conversion of endogenous arachidonate to these two metabolites was unaltered. Vasopressin stimulated iPGE and iTXB2 syntheses when the incubation buffer contained 1 mM calcium but had no effect in the presence of 0.4 microM calcium. In contrast, using an EDTA isolation method, basal iPGE and iTXB2 syntheses were equal in the presence of zero calcium. Increasing extracellular calcium concentration to 1 mM caused a greater enhancement in iTXB2 synthesis compared to iPGE. Increasing extracellular calcium to 2 mM was associated with a decline in iPGE and iTXB2 syntheses back to the levels observed with no calcium added to the medium. The effect of increasing the calcium concentration was greater in phosphate than in bicarbonate buffer. In a Tris buffer the effect of altered calcium was almost completely abrogated. These studies demonstrate that the choice of buffer and alterations in extracellular calcium concentration differentially alter basal arachidonic acid metabolism to prostaglandins and thromboxane in isolated toad urinary bladder cells. The results suggest that there may exist several endogenous pools of arachidonic acid which are differentially influenced by calcium. Furthermore, the pool sensitive to vasopressin has an absolute requirement for calcium.

Animals↗
PubMed1984

ADH or theophylline-induced changes in intracellular free and membrane-bound calcium.

Ca2+ is thought to play a role in the enhancement of water permeability of toad urinary bladder epithelial cells by antidiuretic hormone (ADH) or theophylline. This study examined the effects of ADH and theophylline on intracellular free Ca2+ ([Ca2+]i) and total cellular exchangeable Ca2+ in isolated toad bladder epithelial cells. ADH or theophylline enhanced water permeability maximally by 15-25 min after a 4-min lag. 45Ca2+ efflux, a probe for total cellular exchangeable (plasma membrane plus intracellular) Ca2+, was enhanced by ADH within 2 min and returned to control by 8 min. Chlortetracycline fluorescence, a probe for intracellular Ca2+ only, was not affected, suggesting that ADH released only plasma membrane-bound Ca2+. Theophylline enhanced 45Ca2+ efflux and decreased chlortetracycline fluorescence, suggesting release of Ca2+ from intracellular sources. Both agents decreased [Ca2+]i as assessed by quin-2 fluorescence with a time course similar to the enhancement in water permeability. The results suggest that the changes in membrane-bound Ca2+ and [Ca2+]i induced by ADH and theophylline may play a role in the enhanced permeability to water in response to these agents.

Aminoquinolines↗
PubMed1983

Vasopressin stimulates thromboxane synthesis in isolated hamster hepatocytes: relation to hepatocyte calcium content.

This study was designed to determine if vasopressin stimulates the formation of thromboxane B2 in freshly isolated hamster hepatocytes and if so, whether this thromboxane synthesis plays a role in the vasopressin-induced efflux of 45Ca++ from these cells. Thromboxane synthesis was found to be linear for 15 minutes, to be stimulated by arachidonic acid, and to be inhibited by indomethacin and by 7-(1-imidazolyl) heptanoic acid (7IHA), a selective inhibitor of thromboxane synthetase. Vasopressin stimulation of thromboxane synthesis was dose-related. Pretreatment with 7IHA and meclofenamate significantly increased basal 45Ca++ uptake by the cells. Neither 7IHA nor meclofenamate inhibited the ability of vasopressin to reduce the 45Ca++ content of the isolated hepatocytes. These results indicate that vasopressin stimulates thromboxane synthesis in hepatocytes but suggest that thromboxane does not play a role in the vasopressin-induced reduction in 45Ca++ content in this cell type.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

45Ca fluxes in isolated toad bladder epithelial cells: effects of agents which alter water or sodium transport.

Vasopressin enhances osmotic water flow and sodium transport across the toad urinary bladder by mechanisms involving cyclic AMP and calcium. It is believed that changes in intracellular calcium concentration or in its binding to membranes may in part mediate the effects of vasopressin. In addition, several agents which alter the response of the toad bladder to vasopressin may also act by altering cellular calcium metabolism. The effects of vasopressin and several agents which modify its effects in the toad bladder were studied on 45Ca fluxes in isolated epithelial cells from the toad bladder. Compartmental analysis of 45Ca exchange revealed three components. Vasopressin reduced the amount of calcium in the most rapidly exchanging pool from 1.67 +/- 0.20 to 0.86 +/- 0.12 nmol/mg of protein (P less than .025) and the most slowly exchanging pool from 2.72 +/- 0.26 to 1.90 +/- 0.34 nmol/mg of protein (P less than .001), while not affecting the intermediate pool. Theophylline, which mimics the natriferic and hydroosmotic effects of vasopressin, also mimicked the effects on 45Ca exchange by vasopressin. Exogenous cyclic AMP and the prostaglandin endoperoxide analog U46619, which mimic the hydroosmotic effect of vasopressin, also reduced the amount of calcium in the most slowly exchanging pool. Prostaglandin E1, which inhibits the hydroosmotic effect of vasopressin at the concentrations used increased the size of the most slowly exchanging pool. These studies suggest that prostaglandins and other agents which alter the effect of vasopressin in the isolated toad bladder may elicit their effects in part by influencing the calcium concentration at some critical site.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1983

Prostaglandin-independent inhibition of calcium transport by nonsteroidal anti-inflammatory drugs: differential effects of carboxylic acids and piroxicam.

Nonsteroidal antiinflammatory drugs (NSAID) alter calcium fluxes in several tissues. The present study was designed to assess simultaneously the effects of NSAID on ATP-dependent 45Ca accumulation into rat and hamster liver mitochondria, microsomes and plasma membrane vesicles and prostaglandin (PG) and thromboxane synthesis in these organelles. The carboxylic acid NSAID, indomethacin, meclofenamate and naproxen inhibited 45Ca accumulation by mitochondria and microsomes while plasma membrane 45Ca transport was not inhibited. Similarly, none of the NSAID inhibited 45Ca transport by human erythrocyte ghosts. Addition of exogenous PGE2, PGH2 or a thromboxane mimetic [U46619 [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid] ) did not reverse the inhibition by NSAID. ID50 values for inhibition of 45Ca uptake into mitochondria and microsomes were identical, 25 to 30 microM for meclofenamate and 80 to 90 microM for indomethacin. The ID50 values for inhibition of PGE synthesis were less than 1 microM for meclofenamate and 4 to 5 microM for indomethacin. Dose-response relationships for inhibition of 45Ca uptake were identical with controls in mitochondria isolated from normal or essential fatty acid-deficient Long-Evans rats even though PGE synthesis in essential fatty acid-deficient was only 20% of that in normal rats. In contrast to the other NSAID tested, piroxicam, a member of a new class of NSAID, the oxicams, did not affect mitochondrial or microsomal 45Ca transport at concentrations up to 300 microM, while the ID50 for inhibition of PGE synthesis was 0.35 microM. The mechanism for inhibition of 45C transport by NSAID is not known but does not appear to be related to inhibition of fatty acid cyclooxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Verapamil inhibition of vasopressin-stimulated water flow: possible role of intracellular calcium.

The effects of the organic calcium antagonists verapamil and methoxyverapamil (D-600) on vasopressin-stimulated water flow in response to an osmotic gradient were investigated. When toad bladders were exposed to either verapamil or D-600 for 60 min, vasopressin-stimulated water flow was inhibited. In the presence of verapamil (100 microM), water flow decreased from 38 +/- 3 to 26 +/- 3 mg/min/hemibladder (P less than .05, n = 5) and in the presence of D-600 (100 microM) from 48 +/- 3 to 32 +/- 4 mg/min/hemibladder (P less than .02, n = 5). When preincubated with hemibladders for 90 min they also inhibited basal 45Ca uptake into isolated toad bladder epithelial cells (from 1.58 +/- 0.20 to 1.02 +/- 0.14 nmol/mg of protein per 5 min, P less than .05, n = 6). Addition of verapamil to the bathing media during recovery from a vasopressin stimulus to block reuptake of calcium into the epithelial cells inhibited water flow in response to a subsequent incubation with vasopressin to a greater extent than the inhibition of water flow when verapamil was present only before exposure to vasopressin. Inhibition was also greater in the presence of lower extracellular calcium concentrations. The results are consistent with the notion that the calcium antagonists inhibit vasopressin-stimulated water flow by virtue of depleting a critical pool of intracellular calcium rather than preventing the simultaneous entry of calcium from extracellular sites during the exposure to vasopressin.

Animals↗
PubMed1982

Vasopressin stimulates prostaglandin and thromboxane synthesis in toad bladder epithelial cells.

The effects of arginine vasopressin (AVP) and its nonpressor analogue desamino-8-arginine vasopressin (dDAVP) on immunoreactive prostaglandin E and thromboxane B2 synthesis from endogenous arachidonic acid by epithelial cells isolated from toad urinary bladders were investigated. In epithelial cell suspensions prepared using a collagenase treatment, AVP (5 mU/ml) stimulated prostaglandin E synthesis from 0.27 +/- 0.05 to 0.53 +/- 0.09 pmol . min-1 . mg protein-1 (P less than 0.01, n = 6) and stimulated thromboxane A2 synthesis, as assessed by measurement of its stable metabolite thromboxane B2, from 0.032 +/- 0.004 to 0.054 +/- 0.009 pmol . min-1 . mg protein-1 (P less than 0.02, n = 6). dDAVP (130 nM) also stimulated immunoreactive prostaglandin E and thromboxane B2 synthesis (P less than 0.05, n = 6). In cells prepared by EDTA treatment or scraping, aVP did not alter prostaglandin or thromboxane synthesis. This study demonstrates that AVP and dDAVP stimulate both prostaglandin and thromboxane synthesis in epithelial cells from the toad urinary bladder and suggests that the antidiuretic activity of these peptides is associated with this effect. The results are consistent with previous observations that the synthesis of prostaglandin E and thromboxane by epithelial cells results in a negative and positive modulation, respectively, of the action of AVP on water transport.

Animals↗
PubMed1980

Vasopressin-stimulated water flow is decreased by thromboxane synthesis inhibition or antagonism.

The time course of vasopressin stimulation of water flow and immunoreactive thromboxane B2 (iTXB2) and prostaglandin E (iPGE) biosynthesis was studied in the isolated toad urinary bladder. Vasopressin (25 mU/ml) significantly stimulated iTXB2 synthesis within 8 min, synthesis reaching a maximum rate by 17 min. iPGE synthesis was significantly stimulated within 8 min, remaining unchanged for 24 min. Maximum vasopressin-stimulated water flow was reached between 16 and 24 min. 7-(1-Imidazolyl)-heptanoic acid (7IHA), a thromboxane synthetase inhibitor, inhibited both vasopressin-stimulated water flow and iTXB2 synthesis in a dose-dependent fashion, but did not affect iPGE synthesis. Vasopressin-stimulated water flow and iTXB2 synthesis were significantly correlated (r = 0.75, n = 24, P less than 0.001). 13-Azaprostanoic acid (13APA), a thromboxane antagonist, inhibited vasopressin-stimulated water flow in a dose-dependent fashion. Inhibition of arachidonic acid metabolism abolished the effects of 7IHA and 13APA on vasopressin-stimulated water flow. 7IHA and 13APA had no effect on cAMP-stimulated water flow. These results confirm that vasopressin stimulates TXA2 and PGE synthesis and support the hypothesis that TXA2 is a positive modulator of vasopressin-stimulated water flow in the toad urinary bladder.

Animals↗
PubMed1980

Thromboxane and stable prostaglandin endoperoxide analogs stimulate water permeability in the toad urinary bladder.

The effects of thromboxane B(2) and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9alpha - 11alpha - (epoxymethano)prosta - 5Z,13E - dienoic acid (U44069) and (15Z)-hydroxy -11alpha,9alpha-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A(2) synthesis, thromboxane B(2) stimulated water flow in a dose-dependent manner. U44069 (1 muM) stimulated water flow from 3.6+/-0.8 to 12.4+/-1.2 mg/min per 10 cm(2) hemibladder surface area, while U46619 (1 muM) stimulated water flow from 2.8+/-1.0 to 21.8+/-2.0 mg/min per 10 cm(2). The prostaglandin endoperoxide/thromboxane A(2) antagonist trans- 13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B(2)- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 muM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P < 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A(2)-like compounds stimulate water flow in the toad urinary bladder.

Animals↗
PubMedDate not supplied

Bradykinin receptor antagonists.

Bradykinin and its active metabolites are produced at the sites of their actions by kallikreins. They potently elicit a variety of biological effects: hypotension, bronchoconstriction, gut and uterine contraction, epithelial secretion in airway, gut, and exocrine glands, vascular permeability, pain, connective tissue proliferation, and eicosanoid formation. These effects are mediated by at least two broad classes of receptors. The most common is the B2 subtype. The Stewart and Vavrek peptides characterized by a DPhe7 substitution have provided powerful tools for study of bradykinin's actions by competitively and specifically blocking bradykinin B2 receptors. The significance of kinins in certain human diseases is being explored using these new tools and potential therapeutic agents. At present, human clinical trials are underway to test the usefulness of bradykinin receptor antagonists in the symptoms of the common cold and in the pain associated with severe burns. Trials for use in asthma will be initiated in 1990.

Animals↗
PubMedDate not supplied

N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-L-leucine, NPC 15669, prevents neutrophil adherence to endothelium and inhibits CD11b/CD18 upregulation.

NPC 15669, a member of a new class of antiinflammatory agents termed leumedins, blocks inflammation in several animal models, including contact dermatitis and Arthus reaction, by inhibiting recruitment of neutrophils and lymphocytes into inflammatory lesions. These compounds do not block lipid metabolic enzymes, nor do they antagonize receptors for various chemoattractants, including LTB4, PAF, C5a, and fMLP. This report demonstrates that in vitro, pretreatment of stimulated neutrophils with NPC 15669 results in the dose-dependent inhibition of adherence to cultured human umbilical vein endothelial cells or to the protein substrate keyhole limpet hemocyanin. Adherence of HL-60 cells (a promyelocytic line) is unaffected when stimulated endothelial cells are pretreated with NPC 15669. Flow cytometric analysis of adhesion molecules expressed on neutrophils revealed that pretreatment of neutrophils with NPC 15669 prior to activation inhibits the increase in expression of the CD11b and CD18 adhesion molecule subunits. We conclude that (1) NPC 15669 acts on neutrophils to block activation-induced adherence, and (2) NPC 15669 inhibits the upregulation of the CD11b/CD18 (Mac-1) adhesion receptor.

Adult↗