Elsevier

Neuroscience

Volume 101, Issue 2, 7 November 2000, Pages 283-287
Neuroscience

Nitric oxide synthase inhibitors unmask acetylcholine-mediated constriction of cerebral vessels in the in vitro isolated guinea-pig brain

https://doi.org/10.1016/S0306-4522(00)00365-1Get rights and content

Abstract

The control of arterial vascular tone by acetylcholine contributes to the regulation of cerebral blood flow. We analysed the effects of intraluminal application of acetylcholine (1 μM) on the cerebral vascular tone by measuring changes in resistance to perfusion pressure in an isolated guinea-pig brain preparation maintained in vitro by arterial perfusion under constant flow. Acetylcholine induced a reproducible, fast-onset dilation that was prevented by the nitric oxide scavenger Methylene Blue (10 μM) and by the muscarinic receptor antagonist atropine (0.1 μM). Prolonged arterial perfusion with the nitric oxide synthase inhibitors N-nitro-l-arginine (1 mM) and N-nitro-l-arginine methyl ester (30–100 μM) induced a slowly developing increase of 25.9±13.44 mmHg in vascular tone and blocked the acetylcholine-induced vasodilation. In these experimental conditions, the dilation determined by the nitric oxide donor nitroprusside (0.1 μM) was unaffected. In five experiments, the blockade of dilation unmasked a slow acetylcholine-mediated vasoconstriction (14.40±3.85 mmHg) that was antagonized by atropine.

The results demonstrate that acetylcholine exerts two simultaneous and opposite effects on guinea-pig cerebral vessels, characterized by a slow direct constriction concealed in physiological conditions by a fast vasodilation mediated through the release of nitric oxide by endothelial cells. Acetylcholine-mediated increase in vascular tone may play a role in aggravating cerebral perfusion when endothelial cell damage occurs during brain ischemia.

Section snippets

Experimental procedures

The experiments were carried out on brains isolated from young adult guinea-pigs (150–200 g) maintained in vitro by arterial perfusion. The procedure for brain isolation has been described extensively elsewhere.14., 16., 31., 34. In brief, during anesthesia with pentobarbital (20 mg/kg, i.p.), the brain was extracted under hypothermic conditions and transferred to an incubation chamber, where a micro-cannula was inserted in the basilar artery to ensure arterial perfusion with a complex saline

Results

The experiments were performed on 21 isolated guinea-pig brains. The arterial basal resistance to perfusion pressure was 34.26±8.15 mmHg (n=7) and 33.36±10.80 mmHg (n=5) at 15 and 32°C, respectively. No changes in vascular resistance were observed in untreated brains up to 6–7 h (n=5). At the onset of the experiment, the functional viability of the vessels was tested by intraluminal perfusion with U46619 (30–100 nM), a potent vasoconstrictor that activates the thromboxane A2 receptors on cerebral

Discussion

The present study demonstrates that ACh influences cerebral vessel tone via two, possibly simultaneous, effects, both mediated by muscarinic receptors: a fast NO-dependent dilation and a slower vasoconstriction. The vasodilatory effect of ACh is dependent on the release of NO from the endothelium, as demonstrated by its abolition following arterial perfusion with NOS inhibitors and the NO scavenger Methylene Blue. Recently, Methylene Blue has been shown to possess additional pharmacological

Conclusions

The present study demonstrates, for the first time, a direct constrictory effect of physiological concentrations of ACh on undamaged cerebral vessels in which the NO-dependent dilation has been functionally impaired. The experiments confirm that the in vitro isolated guinea-pig brain is a suitable preparation to study the brain vascular system in a condition of preservation of the neuronal activity, vascular responsiveness and endothelial function. The combined evaluation of the interactions

Acknowledgements

The study was partially supported by Schering-Plough (Milan, Italy) and by the Italian Health Ministry. We would like to thank Dr Carmen Balsamo for the technical contribution in the early phase of the study.

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