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Flow modulates endothelial function in the rabbit carotid artery

Lasse E. Rasmussen


Shear stress is believed to be the most important stimulating mechanism for production of endothelial NO; however the impact of increases in flow and shear stress on the basal and stimulated release of the endothelial autacoids in large arteries is uncertain. Thus experiments were designed to investigate the influence of shear stress and pressure on endothelial function in the rabbit carotid artery. Increases and decreases in isometric force were compared in static rings and perfused (5 or 50 ml/min) segments of the same arteries, in the presence and absence of endothelium. The α1-adrenoceptor agonist phenylephrine and the muscarinic agonist acetylcholine were applied as vasoconstrictor and vasodilator stimuli, respectively. Experiments were designed to measure how immediate onset and how sustained flow influenced the level of contraction of rabbit carotid artery with regards to both the basal and stimulated release of endothelial autacoids.

Immediate onset of flow: The changes in tension of acute onset of flow occurred in three phases; an immediate short lasting relaxation (within the first minute), which presumably was mediated by a rapid cooling as flow was initiated; a second phase contraction (next 10 minutes), which was endothelium-dependent and inhibited by L-NAME plus indomethacin; a tertiary phase of flow-mediated relaxation, which was endothelium independent, and not observed if the vasoconstrictor was present in both the perfusate and superfusate, an if the temperature was controlled. Only minor flow-mediated effects were observed when the perfusate temperature was controlled and the vasoconstrictor was added to both the luminal and abluminal side of the artery, but if acetylcholine was added as well, onset of flow mediated a contraction. Start of bubbling the salt-solution with CO2 in air during constant flow in segments contracted with phenylephrine and relaxed with acetylcholin, evoked a contraction similar to that observed by onset of flow. It is concluded that the changes in tension evoked by the onset of flow can be the result of changes in
temperature and changes in the cellular micro-environment with regards of vasoconstrictor bioavailability, and content of O2 and CO2.

Sustained flow: Continuous flow (5 and 50 ml/min) reduced the cGMP content and shifted the concentration-response curve to phenylephrine to the left compared to non-perfused static rings. Removal of the endothelium abolished the differences in cGMP content and the sensitivity to phenylephrine between static rings and perfused segments. No difference in sensitivity to phenylephrine was observed in tissues treated with N-ω-nitro-L-Arginine Methyl ester (L-NAME). Acetylcholine-evoked relaxations were increased in perfused segments. L-NAME nearly abolished the acetylcholine-evoked relaxation in static rings, whereas about half of the relaxation remained in segments exposed to flow. This remnant relaxation was blocked by inhibition of endothelial small and intermediate conductance calcium activated potassium channels (SKCa and IKCa) by apamin plus 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34). Release of EDHF was evident only if the NO-synthase was inhibited by L-NAME, since Apamin plus TRAM-34 in the presence of indomethacin was unable to influence the acetylcholine-evoked relaxation. These experiments demonstrate that continuous flow increases the constriction evoked by α1-adrenergic activation in the rabbit carotid artery through a reduced influence of basally released endothelial NO, and further that luminal flow unmasks an ability of the endothelium to release a non-NO, non-cyclooxygenase vasodilator, presumably endothelium-derived hyperpolarising factor (EDHF).