Inhibition of endothelium-dependent relaxations by phorbol myristate acetate in canine coronary arteries: Role of a pertussis toxin-sensitive G-protein

Activation of protein kinase C by phorbol esters inhibits the endothelium-dependent relaxations evoked by certain stimuli. The release of endothelium-derived relaxing factor can be evoked by a number of distinct subcellular processes, including activation of a pertussis toxin-sensitive G-protein. The aim of the present study was to determine whether or not the inhibitory effect of phorbol esters on endothelial function was associated with inhibition of the pertussis toxin-sensitive pathway. Rings of canine coronary artery were suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution, gassed with 95% O2-5% CO2 (37°C). Treatment of arterial rings with pertussis toxin (100 ng/ml) or with phorbol myristate acetate (PMA, 10-8 M) inhibited the endothelium-dependent relaxations produced by UK 14,304, an alpha-2 adrenergic agonist, leukotriene C4 or by NaF, a direct activator of G proteins, but did not affect the endothelium-dependent relaxations produced by bradykinin or by A23187. If the arterial rings were first treated with pertussis toxin, PMA (10-8 M) no longer inhibited the endothelium-dependent relaxations to NaF. Increasing the concentration of PMA (to 3 x 10-8 and 10-7 M) caused inhibition of responses to bradykinin. At higher concentrations, PMA (3 x 10-7 and 10-6) also inhibited the relaxations evoked by A23187. The endothelium-independent relaxations evoked by nitroglycerin were not affected by PMA (10-8 to 10-6) These results suggest that in canine coronary arteries, endothelial leukotriene and alpha-2 adrenergic receptors are linked to a pertussis toxin-sensitive G-protein that stimulates the release of endothelium-derived relaxing factor(s). At low concentrations, PMA inhibits selectively this pertussis toxin-sensitive pathway, possibly by inhibiting the function of the pertussis toxin-sensitive G-protein in the endothelial cells.link_to_subscribed_fulltex

Loss of endothelial pertussis toxin-sensitive G protein function in atherosclerotic porcine coronary arteries

Pertussis toxin, an irreversible inhibitor of some G proteins, inhibits endothelium-dependent relaxations to certain agonists in porcine coronary arteries. In the present study, the effects of the toxin were examined on endothelium-dependent and -independent relaxations of hypercholesterolemic and atherosclerotic porcine coronary arteries to assess the functional state of the endothelial pertussis toxin-sensitive G protein. Male Yorkshire pigs were maintained on either a regular diet (control group, n = 7) or a 2% high-cholesterol diet (cholesterol-fed group, n = 7) for 10 weeks. After the initial 2 weeks of maintenance, animals in both groups underwent balloon catheter removal of the endothelium of the left anterior descending or left circumflex coronary arteries. Endothelium-dependent responses were examined in vitro after 10 weeks of maintenance; at this time, a full lining of endothelial cells in both left coronary arteries was confirmed histologically. In arteries with endothelium of the control group (normal responses), pertussis toxin significantly inhibited the endothelium-dependent relaxations to serotonin, UK14304 (a selective α2-adrenergic receptor agonist), and thrombin but not those to ABP, bradykinin, or the calcium ionophore A23187. In previously denuded arteries of the control group (effects of endothelial regeneration alone) or intact arteries of the cholesterol-fed group (effects of hypercholesterolemia alone), the relaxations to serotonin, UK14304, and thrombin were impaired significantly; those relaxations were impaired further in previously denuded arteries of the cholesterol-fed group (effects of atherosclerosis). The inhibitory effects of pertussis toxin were significantly reduced after endothelial regeneration and in hypercholesterolemia and were almost absent in atherosclerosis. Direct relaxations of coronary vascular smooth muscle evoked by nitric oxide or sodium nitroprusside were not significantly affected by either hypercholesterolemia or atherosclerosis. These results indicate that the function of endothelial pertussis toxin-sensitive G protein is impaired in regenerated endothelial cells or hypercholesterolemia and is almost absent in atherosclerosis, accounting in part for the endothelial dysfunction under those pathological conditions.link_to_subscribed_fulltex

Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries: Possible dysfunction of a pertussis toxin-sensitive G protein

The purposes of the present study were to examine the natural course of the impairment of endothelium-dependent relaxations during a regeneration and tissue repair process after balloon endothelium removal and to elucidate the cellular mechanism(s) underlying it. Twenty-three male Yorkshire pigs underwent balloon endothelium removal along the proximal portion of either the left anterior descending or circumflex coronary artery and were then maintained on a regular chow for 4, 8, 16, or 24 weeks. Endothelium-dependent responses were examined in vitro in rings taken from the control and previously denuded arteries studied in parallel. Morphometric analysis revealed that intimal thickening developed only at the previously denuded area. In the previously denuded arteries with regenerated endothelium, the endothelium-dependent relaxations to UK 14304 (a selective α2-adrenergic agonist), serotonin, and aggregating platelets were impaired 4 weeks after endothelium removal and remained so throughout the study. The endothelium-dependent relaxations to thrombin and adenosine diphosphate became depressed 8 weeks after endothelium removal and those to bradykinin became depressed 16 weeks after endothelium removal, while those to the calcium ionophore A23187 were maintained throughout the study. Endothelium-dependent relaxations to all vasoactive agents were unaltered in the control arteries. In the control arteries, pertussis toxin, an inhibitor of certain G proteins, markedly inhibited the endothelium-dependent relaxations to UK 14304 and serotonin and partially inhibited those to thrombin and aggregating platelets. The responses inhibited by the toxin in control arteries were significantly reduced in the previously denuded arteries with regenerated endothelium. The inhibitory effect of pertussis toxin was markedly reduced in those arteries with regenerated endothelium. In quiescent rings, the presence of normal endothelium inhibited the contractions caused by serotonin and aggregating platelets; this endothelium-dependent depression was markedly impaired in the previously denuded arteries throughout the study. Direct relaxation of the coronary smooth muscle to nitric oxide or sodium nitroprusside or direct contraction to KCl or serotonin were comparable between the control and previously denuded arteries. These experiments indicate that endothelium-dependent relaxations progressively worsen after regeneration of the endothelium and that the dysfunction of a pertussis toxin-sensitive G protein partly account for the endothelial dysfunction in the chronic regenerated state.link_to_subscribed_fulltex

Endothelium-dependent inhibition of ergonovine-induced contraction is impaired in porcine coronary arteries with regenerated endothelium

The inhibitory effects of the endothelium against ergonovine-induced contraction were examined in isolated porcine coronary arteries under normal conditions and after endothelial regeneration. Endothelium-dependent responses were examined in vitro in normal Yorkshire pigs (n = 16) and in pigs that had undergone balloon endothelium removal of the left anterior descending coronary artery (LAD) 4 weeks before the study (n = 10). The presence of a complete endothelial lining was confirmed histologically. In rings from normal arteries contracted with prostaglandin F(2α) in the presence of indomethacin and ketanserin (a 5-HT2-serotonergic blocker), ergonovine caused endothelium-dependent relaxations. They were attenuated by rauwolscine (an α2-adrenergic blocker), inhibited by methiothepin (a combined 5-HT1- and 5-HT2-serotonergic blocker) or by pertussis toxin (an inhibitor of several G proteins) and abolished by oxyhemoglobin (a selective inactivator of endothelium-derived relaxing factor). In quiescent rings from normal arteries, ergonovine caused contractions that were inhibited by the presence of the endothelium; this endothelium-dependent inhibition was ablished by oxyhemoglobin. The direct contractions were not affected by prazosin (an α1-adrenergic blocker), rauwolscine, 6-hydroxydopamine (an agent causing chemical sympathectomy), or diphenhydramine (an H1-histaminergic blocker) but were inhibited by ketanserin. In rings with regenerated endothelium contracted with prostaglandin F(2α), the endothelium-dependent relaxations to ergonovine were reduced significantly and were not inhibited by pertussis toxin. In quiescent rings with regenerated endothelium, the endothelium-dependent inhibition of ergonovine-induced contraction was less. Oxyhemoglobin caused endothelium-dependent contractions in quiescent rings (an indirect index of basally released endothelium-derived relaxing factor) that were reduced significantly in quiescent rings with regenerated endothelium. These results indicate that 1) the endothelium exerts its inhibitory action against ergonovine-induced contractioins by the release of endothelium-derived relaxing factor under basal conditions and upon stimulation by ergonovine, 2) endothelium-dependent relaxations to ergonovine are mediated mainly by 5-HT1-serotonergic receptors, whereas the direct contractions are mediated by 5-HT2-serotonergic receptors with little contribution of α-adrenoceptors, 3) the inhibitory role of the endothelium is impaired significantly in the regenerated state because of the reduced ability to release the relaxing factor, and 4) endothelial pertussis toxin-sensitive G protein may be involved in the synthesis of the relaxing factor upon stimulation by ergonovine, and dysfunction of the G protein may account partly for the dysfunction of regenerated endothelium.link_to_subscribed_fulltex