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Effects of endothelial removal and hypoxia on responses to adenosine, 5'-(N-ethylcarboxamido)-adenosine (NECA), 2-chloroadenosine, N6-cyclohexyladenosine (CHA), sodium nitroprusside, and acetylcholine were examined in guinea pig aortic rings. Rings contracted with 2 microM prostaglandin F2 alpha (PGF2 alpha) relaxed in a dose-dependent manner in response to all drugs. The order of potency of adenosine compounds was NECA greater than 2-chloroadenosine greater than adenosine greater than CHA. Endothelial rubbing potentiated the PGF2 alpha response by 11 +/- 3%, eliminated the acetylcholine (ACh) response, but had no effect on nitroprusside and CHA responses. Responses to adenosine, NECA, and 2-chloroadenosine were significantly depressed by rubbing (P less than 0.05). Oxyhemoglobin (5 microM) and metyrapone (0.1 mM) reduced ACh responses in intact rings but had no effect on the adenosine and nitroprusside responses. Indomethacin treatment (10 microM) did not alter ACh, nitroprusside, or adenosine responses in intact rings. Hypoxia (10% O2) potentiated maximal responses to adenosine (+26 +/- 3%) and nitroprusside (+28 +/- 4%) in intact and rubbed rings and reduced the maximal response to ACh in intact rings (-28 +/- 3%). It is concluded that 1) adenosine mediates relaxation in guinea pig aorta by endothelial-dependent and -independent mechanisms, 2) receptors involved in both endothelial-dependent and -independent relaxations are characteristic of the A2 adenosine subtype, 3) the endothelial response appears unrelated to EDRF or prostanoid release, and 4) the adenosine response is significantly potentiated by hypoxia.

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.

The present study investigated the mechanism by which thromboxane A2/prostaglandin H2 (TXA2/PGH2) stimulates platelet activation. Previous studies in isolated platelet vesicles have suggested that TXA2/PGH2 functions to release calcium from intraplatelet stores. On this basis, we investigated whether TXA2/PGH2 causes mobilization of calcium in intact platelets. Calcium redistribution was measured using the fluorescent probe, chlortetracycline (CTC), and a photon-counting microspectrofluorometer. Human platelet-rich plasma was incubated with CTC (50 microM) for 40 min at 25 degrees C. Shape change was induced with arachidonic acid (AA, 100 microM) or ADP (0.75-1.0 microM). It was found that AA addition resulted in a significant release of intraplatelet calcium. This release was blocked by inhibition of the cyclooxygenase with indomethacin (20 microM) or the specific TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA, 100 microM). On the other hand, neither indomethacin nor 13-APA had any effect on calcium release stimulated by ADP. However, prostacyclin (13 nM) inhibited both AA- and ADP-induced calcium release. These findings provide evidence that cyclooxygenase products of AA, i.e., TXA2 and/or PGH2 directly caused the mobilization of intraplatelet calcium. Furthermore, this calcium mobilization appears to be mediated through a specific TXA2/PGH2 receptor interaction.

We previously demonstrated that thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2), ADP, and A23187 cause calcium mobilization in intact human platelets. Other studies have also shown that platelet shape change and aggregation induced by a variety of platelet agonists can be reversed by specific antagonists. In the present study, we used the fluorescent calcium probe chlortetracycline to evaluate whether the reversal of platelet activation involves a resequestration of intraplatelet calcium. It was found that the TXA2/PGH2 receptor antagonist 13-azaprostanoic acid (13-APA) reversed calcium mobilization and shape change induced by AA but not that induced by ADP. A similar specificity of action was observed using the specific ADP receptor antagonist, ATP, in that ATP only reversed ADP-induced calcium release and shape change. In contrast, prostacyclin reversed both AA and ADP-induced calcium redistribution and shape change. In the latter experiments, a net calcium sequestration was actually observed on prostacyclin addition. These findings indicate that the resequestration of released calcium leads to platelet deactivation. Furthermore, there appear to be at least two mechanisms by which a reduction in cytosolic calcium can be produced: specific interruption of the agonist-receptor interaction, for example, 13-APA antagonism of TXA2/PGH2; and stimulation of platelet adenosine 3',5'-cyclic monophosphate production by prostacyclin and consequent calcium sequestration.