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