cyclic-gmp and 4-bromophenacyl-bromide

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate. 2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC50 being 13.7 microM and 15.8 microM, respectively. 3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC50=32.7 microM, NaNP and 25.0 microM, SNAP), while acetylsalicylic acid (up to 1000 microM) and diclofenac (up to 100 microM) were ineffective. 4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production. 5. Indomethacin (10 microM), indoprofen (30 microM), diclofenac (100 microM) and acetylsalicylic acid (1000 microM) showed a comparable efficacy in inhibiting platelet thromboxane B2 (TXB2) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase. 6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP. 7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.

Topics: Acetophenones; Aspirin; Blood Platelets; Cyclic GMP; Cyclooxygenase Inhibitors; Diclofenac; Humans; Indomethacin; Indoprofen; Nitric Oxide; Nitroprusside; Penicillamine; Phospholipases A; Thromboxane B2

We have used mixed- and co-cultures of endothelial and vascular smooth muscle cells to investigate the role of phospholipase activation and arachidonic acid metabolites in the production of endothelium-derived relaxing factor (EDRF). Inhibition of phospholipase A2 with para-bromophenacyl bromide, dexamethasone or quinacrine, alone or in combination, blocked arachidonate release by 50%-60% but had no effect on EDRF production as assessed by cyclic GMP accumulation in mixed- or co-cultures of endothelial and vascular smooth muscle cells. Inhibition of the phospholipase C-diacylglycerol (DAG) lipase pathway of arachidonate release by the DAG lipase inhibitor RHC-80267 also caused partial inhibition of arachidonate release and had no effect on EDRF. When both phospholipase A2 and phospholipase C pathways for arachidonate mobilization were inhibited (dexamethasone + RHC 80267), arachidonate release was totally inhibited while EDRF release remained intact. We conclude that neither phospholipase activation nor arachidonate mobilization is required for EDRF release from cultured bovine endothelial cells.

Topics: Acetophenones; Animals; Arachidonic Acids; Biological Products; Bradykinin; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Melitten; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Phospholipases; Vasodilator Agents

The alkylating agent para-bromophenacyl bromide (PBPB) has been reported to block endothelium-dependent relaxation of arteries, presumably by inhibiting phospholipase in the endothelium. We have investigated the site of action of PBPB-induced inhibition of melittin (MEL)- and bradykinin (BK)-stimulated endothelium-dependent relaxation and vascular smooth muscle (VSM) cyclic GMP (cGMP) accumulation. Studies were performed using short-term co-cultures of bovine pulmonary artery endothelial cells (EC) grown on microcarrier beads and rat aortic VSM. The culture system allowed separate pretreatment of ECs and VSM cells with PBPB (3 X 10(-5) M) before placement of the two cell types in co-culture. Because PBPB is an alkylating agent, it was felt that any blockade would endure after washout. Intracellular (VSM) concentrations of cGMP rose in response to MEL or BK stimulation only when EC were present, and were not decreased when only the EC had been pretreated with PBPB. cGMP accumulation induced by MEL or BK was significantly (P less than .01) inhibited in co-cultures where either the VSM alone or VSM and EC combined were pretreated with PBPB. The PBPB pretreatment adequately blocked EC phospholipase activity as measured by attenuation of the release of [3H]arachidonate from prelabeled EC by MEL. In parallel studies, when endothelium-derived relaxing factor was transferred by superfusion of EC to denuded rabbit aortic rings contracted with phenylephrine and pretreated with PBPB, the relaxation response induced by MEL or BK was inhibited. Similarly, when endothelium-derived relaxing factor was transferred to wells of cultured VSM pretreated with PBPB, cGMP accumulation was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Biological Factors; Bradykinin; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Melitten; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside

The mechanisms by which adenosine triphosphate, thrombin, and trypsin cause relaxation of vascular smooth muscle were investigated. Relaxation of the rat thoracic aorta with adenosine triphosphate, thrombin, and/or trypsin was associated with increased levels of cyclic guanosine monophosphate in both time- and concentration-dependent manners. Thrombin and trypsin did not alter cyclic adenosine monophosphate levels, whereas adenosine triphosphate increased cyclic adenosine monophosphate levels after significant relaxation occurred. Removal of the endothelium abolished adenosine triphosphate-, thrombin-, and trypsin-induced relaxation and the associated increased levels of cyclic nucleotides. Relaxation due to these agents was also inhibited by exposure to nordihydroguaiaretic acid, a lipoxygenase inhibitor, and eicosatetraynoic acid, a lipoxygenase and cyclooxygenase inhibitor. Indomethacin, a cyclooxygenase inhibitor, potentiated relaxation to these agents, whereas the increased levels of cyclic nucleotides due to adenosine triphosphate were unaltered. Bromophenacyl bromide, a phospholipase A2 inhibitor, decreased relaxation due to adenosine triphosphate, thrombin, and trypsin and the associated increased levels of cyclic nucleotides. Removal of extracellular calcium, which also presumably inhibits phospholipase A2, prevented the elevated levels of cyclic nucleotides and the inhibitory effects of adenosine triphosphate and trypsin on contraction. In contrast, sodium nitroprusside-induced relaxation and/or increased levels of cyclic guanosine monophosphate were unaltered by nordihydroguaiaretic acid, eicosatetraynoic acid, bromophenacyl bromide, and removal of extracellular calcium. After incubation of intact tissue with 32P-orthophosphate, the patterns of protein phosphorylation caused by adenosine triphosphate, thrombin, and trypsin were indistinguishable from those of acetylcholine, sodium nitroprusside and 8-bromo cyclic guanosine monophosphate. All these agents dephosphorylated myosin light chain. Thus, the present study supports the hypothesis that relaxation induced by adenosine triphosphate, thrombin, and trypsin is mediated through the formation of an endothelial factor which elevates cyclic guanosine monophosphate levels and causes cyclic guanosine monophosphate-dependent protein phosphorylation and dephosphorylation of myosin light chain.

Topics: Acetophenones; Adenosine Triphosphate; Animals; Aorta, Thoracic; Calcium; Cyclic AMP; Cyclic GMP; Endothelium; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroprusside; Norepinephrine; Phosphoproteins; Rats; Rats, Inbred Strains; Thrombin; Trypsin

The effects of prostaglandin (PG) F-2 alpha and E-2, cyclic GMP, testosterone and 2,4'-dibromoacetophenone (DBA) on rat seminiferous tubules in vitro were ascertained by measuring contractile frequency, size of contractions and tone (tubular diameter). PGF-2 alpha (10(-9)--10(-5) M) increased frequency and tone but not magnitude of contraction. Contraction frequency and tone were decreased by PGE-2 (10(-9)--10(-5) M) but the size of contractions was again unaffected. Cyclic GMP (10(-10)--10(-6) M) increased the frequency of contractions and tone and decreased size of contractions. Testosterone decreased the frequency and size of contractions and increased tone at concentrations of 10(-9) to 10(-7) but higher concentrations (10(-6) M) reversed these changes. DBA (10(-7)--10(-3) M) greatly decreased the frequency and size of contractions and tone and spontaneous contractions were abolished at 10(-3) M. Our data strongly suggest that PGs, cyclic nucleotides and testosterone are all important in vivo in modulating contractility of seminiferous tubules and as such would participate in sperm transport from the testes.

Topics: Acetophenones; Animals; Cyclic GMP; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Prostaglandins; Rats; Seminiferous Tubules; Testis; Testosterone