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Biomedical subjects

R R Gorman

Publications and source records attributed to R R Gorman.

At least 55 records · Page 3Linked to original sources
PubMed1984

12S,20-dihydroxyicosatetraenoic acid: a new icosanoid synthesized by neutrophils from 12S-hydroxyicosatetraenoic acid produced by thrombin- or collagen-stimulated platelets.

A new metabolite of arachidonic acid, formed during interaction between thrombin- or collagen-stimulated platelets and unstimulated neutrophils, has been demonstrated by both thin-layer radiochromatography and high-performance liquid chromatography. Production of the 3H-labeled metabolite in combined suspensions containing [3H]arachidonate-labeled platelets and unlabeled neutrophils from aspirin-treated donors suggested that platelet 3H-labeled 12S-hydroxy-5,8-cis,10-trans,14-cis-icosatetraenoic acid (12-HETE) was the precursor. This was confirmed by identification of the same product when purified 12-[3H]HETE was added directly to unstimulated neutrophils. Hydrogenation and oxidation of the isolated product, followed by gas chromatography-mass spectrometry showed the structure to be 12S,20-dihydroxyicosatetraenoic acid. These experiments further show that platelet stimuli known to occur in vivo may initiate metabolic interactions between different cell types via the arachidonic acid pathway.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1984

Acetylglycerylether phosphorylcholine-(AGEPC) and leukotriene B4-stimulated cyclic AMP levels in human polymorphonuclear leukocytes.

It seems paradoxical that AGEPC induces a transient rise in cyclic AMP, yet the preincubation of neutrophils with agents that elevate cyclic AMP actually inhibits AGEPC-induced aggregation. However, similar transient elevations in cyclic AMP are observed using other stimulators of PMN function such as fmet-leu-phe, C5a (22), immune complexes (24), and phagocytosable particles (11). Elevations in cyclic AMP by PGE1, PGI2, dibutyryl cyclic AMP, and phosphodiesterase inhibitors, before the addition of an agonist, also blocked subsequent neutrophil activation in the above studies. Thus, these observations are not unique to AGEPC. However, the finding that the cyclooxygenase inhibitor indomethacin enhanced AGEPC-stimulated cyclic AMP accumulation is a novel observation and suggests that some oxygenated derivative of the 5-lipoxygenase pathway is responsible for the increase in cyclic AMP. The evidence for the association of the spike in cyclic AMP and the 5-lipoxygenase is strengthened by the observation that the 5-lipoxygenase inhibitor U-60257 attenuates the AGEPC-induced spike in cyclic AMP. It should be noted that U-60257 does not antagonize LTB4-stimulated cyclic AMP accumulation and has no direct influence on the neutrophil adenylate cyclase. The final correlation of the spike in cyclic AMP and the 5-lipoxygenase is made by the fact that LTB4 itself stimulates cyclic AMP levels in intact neutrophils as well as the adenylate cyclase in cell homogenates. As is the case with AGEPC, the transient spike in cyclic AMP induced by LTB4 is coincident with the onset of neutrophil aggregation. However, it is clear that the spike in neutrophil cyclic AMP induced by AGEPC can be dissociated from neutrophil aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1983

Agonist specific desensitization of leukotriene C4-stimulated PGI2 biosynthesis in human endothelial cells.

Leukotriene C4 (LTC4) and, to a lesser extent, leukotriene D4 (LTD4) concentration dependently stimulate prostacyclin (PGI2) biosynthesis in cultured human umbilical vein endothelial cells. PGI2 biosynthesis was quantitated by radioimmunoassay and its structure confirmed by gas chromatography/mass spectrometry. Preincubation of endothelial cells with LTC4 resulted in desensitization to subsequent LTC4 stimulation. However, PGI2 biosynthesis in response to thrombin, PGH2 and arachidonic acid was not inhibited by preincubation with LTC4. The C-6-sulfidopeptide leukotriene receptor level antagonist FPL-55712 attenuates LTC4, but not thrombin-stimulated PGI2 biosynthesis. These data suggest that human umbilical vein endothelial cells have a C-6-sulfidopeptide leukotriene receptor, and that stimulation of this receptor results in PGI2 biosynthesis.

Blood Vessels↗
PubMed1983

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3',5'-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4.

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I2, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B4 synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B4 itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B4 is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B4 synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B4 can limit their own activity in neutrophils.

Adenylyl Cyclases↗
PubMed1983

The effects of continuous infusions of prostacyclin-Na (epoprostenol-sodium) on platelet counts, ADP-induced aggregation, and cyclic AMP levels in normal volunteers.

Six male volunteers received either 0 (buffer), 2.5 or 5.0 ng/kg/min PGI2 X Na for 72 hrs. Various platelet parameters were monitored for an additional 72 hrs. Each morning, for seven consecutive days, and +1 and +6 hrs after the termination of the infusion, blood was drawn and platelet rich plasma (PRP) was prepared. The PRP was immediately exposed to 100 ng/ml PGI2 X Na, and the subsequent increase in platelet cyclic AMP was measured by radioimmunoassay. Aggregation in response to 2 or 4 microM ADP was measured simultaneously. Three volunteers returned for a second 72 hr infusion of 5.0 ng/kg/min PGI2 X Na. After 72 hrs, the infusion rate was gradually "tapered off" over a 12 hr period at which time the infusion was terminated. The sensitivity of the PRP to ADP-induced aggregation was recorded before, during, and after the "tapering off" regimen. Platelet counts were not altered by any of the infusions. The responsiveness of the platelet adenylate cyclase to exogenous PGI2 X Na was inversely related to the concentration of PGI2 X Na infused. Desensitization occurred and was more severe after 72 hrs of infusion than after either 24 or 48 hrs. For example, after 72 hrs at 5.0 ng/kg, platelets lost approximately 50% of their responsiveness to PGI2. ADP-induced aggregation was not significantly inhibited ex vivo by the infusion of 2.5 ng/kg/min PGI2. During the infusion of 5.0 ng/kg/min PGI2, ADP-induced aggregation was inhibited at 24 and 48 hrs, but by 72 hrs, the platelets began to respond to ADP more like control cells even though the PGI2 X Na infusion was continuing. When the infusion was abruptly terminated a hyperaggregable response (rebound) to exogenous ADP was observed. In subjects where the 5.0 ng/kg/min infusion was gradually "tapered off" over a 12 hr period, there was no evidence of platelet hyperaggregability at the time points studied.

Adenosine Diphosphate↗
PubMed1983

Inhibition of platelet thromboxane A2 synthase activity by sodium 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate.

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinylmethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro, as well as in rhesus monkey platelets ex vivo. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 to 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation. Similar effects were obtained by oral administration of 1-5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxygenase inhibitors and equal to PGI2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cyclooxygenase inhibitors suggesting that its efficacy depended in part on endogenous PGI2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of pathophysiological states.

Animals↗
PubMed1983

Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis and cyclic AMP accumulation in human polymorphonuclear leukocytes.

Both LTB4 and AGEPC stimulate PMN aggregation in a concentration-dependent manner. The lipoxygenase inhibitors ETYA and NDGA block AGEPC-induced but not LTB4-induced PMN aggregation. Agents that elevate PMN cyclic AMP levels block both LTB4-and AGEPC-induced aggregation. Paradoxically, shortly after aggregation is initiated by either AGEPC or LTB4, a transient spike in cyclic AMP levels is observed. Subsequent experiments show that LTB4 can directly elevate cyclic AMP in PMNs. Reverse-phase high-pressure liquid chromatography, coupled with selective ion gas chromatography/mass spectrometry, shows that AGEPC stimulates neutrophils to synthesize LTB4. These data suggest that AGEPC-induced neutrophil aggregation is mediated by LTB4.

Cell Aggregation↗
PubMed1982

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets. Evidence for thromboxane A2 dependence.

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.

Blood Platelets↗
PubMed1982

Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis in human polymorphonuclear leukocytes.

Acetyl glyceryl ether phosphorylcholine (AGEPC) and leukotriene B4 (LTB4) induce concentration-dependent neutrophil aggregation. On a molar basis, LTB4 is approximately 10 to 100 times more potent than AGEPC. AGEPC-induced aggregation is attenuated by two inhibitors of arachidonate lipoxygenation, eicosatetraynoic acid and nordihydroguaiaretic acid, and to a lesser extent by the cyclooxygenase inhibitor, indomethacin. LTB4-induced aggregation is not readily reduced by the above inhibitors of arachidonic acid metabolism. Reverse phase high performance liquid chromatography, coupled with selective ion gas chromatography/mass spectrometry, shows that AGEPC stimulates neutrophils to synthesize sufficient LTB4 to account for the AGEPC response. In addition, the rate of LTB4 biosynthesis in response to AGEPC correlates well with the rate of AGEPC- and/or LTB4-induced neutrophils aggregation, and desensitization experiments indicate that AGEPC and LTB4 cross-desensitize. These data suggest that AGEPC-induced neutrophil aggregation may be mediated by LTB4.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1981

Analysis of the biological activity of azoprostanoids in human platelets.

A structure-activity analysis of seven different azoprostanoids in human platelets and rat aortas has shown that discrete changes in the prostanoid skeleton induce marked changes in biological activity. The 15S-hydroxyl group is important for agonist activity in the platelet (pro-aggregatory) and in the aorta (vasoconstriction). Removal of the 15S-hydroxyl results in a compound that inhibits the thromboxane synthetase and platelet aggregation, and is a much weaker constrictor of the aorta. The 13,14 double bond is more important for agonist activity on the aorta than the 5,6 double bond of the prostanoid skeleton. Completely saturated molecules that are 15-deoxy still retain considerable activity on the aorta, but are two orders of magnitude less active as inhibitors of the thromboxane synthetase and aggregation than the azoprostanoid with both 5,6 and 13,14 double bonds. Compounds that exhibit no agonist activity in platelets still retain considerable agonist activity on the aorta, suggesting that the thromboxane A2 receptor in platelets may be different from the thromboxane A2 receptor in blood vessels.

Blood Platelets↗
PubMed1981

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2).

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.

Animals↗
PubMed1981

Intravenous infusion of prostacyclin sodium in man: clinical effects and influence on platelet adenosine diphosphate sensitivity and adenosine 3':5'-cyclic monophosphate levels.

Clinical tolerance, inhibition of platelet aggregation and intracellular platelet adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were evaluated in normal volunteers given i.v. infusions of prostacyclin sodium at rates up to 15 ng/kg/min. Short-term infusions (30 and 60 minutes) were tolerated at rates up to 10.0 ng/kg/min; higher rates produced headaches, anxiety, nausea and vomiting. Six-hour and 24-hour infusions were tolerated at rates up to only 4.0 ng/kg/min. Twenty-four hour infusions at 4 ng/kg/min produced a consistent 4-7 microM shift to the right in the platelet ADP dose-response curve; this platelet inhibitory activity did not diminish during the infusion. Prostacyclin sodium infusion elevated intracellular cyclic AMP levels, the increases corresponding to the onset of measurable inhibition of ADP-induced aggregation, although the magnitude of the increase did not necessarily reflect the degree of inhibition. Increased template bleeding times were seen with a greater than 10-microM shift in the ADP dose-response curve. We conclude that although prostacyclin sodium has a narrow safety margin, the drug does produce platelet inhibition at infusion rates generally tolerated by healthy volunteers.

Adenosine Diphosphate↗
PubMed1981

Regulation of endothelial cell cyclic nucleotide metabolism by prostacyclin.

An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Agonist-specific desensitization of PGI2-stimulated cyclic AMP accumulation by PGE1 in human foreskin fibroblasts.

Agonist-specific desensitization of prostaglandin I2-stimulated (PGI2)1 adenosine 3':5'-monophosphate (cyclic AMP) accumulation can be demonstrated in intact human foreskin fibroblasts (HFF) following a single exposure to PGE1 or a stable PGI2 analog (nitrilo-PGI2). A single PGI2-stimulation of HFF cells does not result in desensitization. Continuous re-addition of PGI2 over a 4 hr period does induce desensitization to subsequent PGI2-stimulation. HFF cells that are desensitized to PGI2 are also desensitized to PGE1 or nitrilo-PGI2 stimulation indicating that these agonists share a common adenylate cyclase complex. Desensitization to PGI2 can be measured after a 60 min, but not after a 30 min, exposure to PGE1 or nitrilo-PGI2. Once HFF cells are desensitized, a 12-24 hr period is required for the recovery of PGI2 sensitivity. The adenylate cyclase in membranes prepared from intact cells that were preincubated with PGE1 is also desensitized to subsequent PGI2-stimulation. Preincubation of cells with PGI2 does not induce desensitization of PGI2-stimulated adenylate cyclase. These data suggest that HFF cells must be constantly exposed to a biologically active prostaglandin for desensitization to occur. The intrinsic chemical lability of PGI2 may be a biochemical protection mechanism against desensitization in cells that normally respond to PGI2.

Adenylyl Cyclases↗