Biphasic neurogenic vasodilatation in the bovine intraocular long posterior ciliary artery.
PhD thesis, University of Glasgow.
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1.Previous research in the bovine intraocular long posterior ciliary artery (LPCA) has suggested that its response to electrical field stimulation (EFS) is a uniphasic vasodilatation, mediated jointly by the neurotransmitters nitric oxide (NO) and calcitonin gene related-peptide (CGRP).
2.This study of the bovine intraocular LPCA, where a short period of EFS (10 s) was employed, demonstrated that the vasodilator response to EFS was in fact biphasic. The first and second components of the response peaked separately at 10 s and 50 s following the onset of stimulation.
3.Both components of vasodilatation to EFS were abolished by tetrodotoxin (TTX), confirming their neurogenic origin.
4.Inhibition of the first component of the vasodilatation to EFS by the nitric oxide synthase (NOS) inhibitor, L-NAME, and the inhibitor of soluble guanylate cyclase, ODQ, confirmed the involvement of NO from nitrergic nerves. Experiments involving treatment with capsaicin and the CGRP antagonist, CGRP8-37, failed to produce any evidence of involvement of CGRP in the second component of neurogenic vasodilatation.
5.A number of other potential neurotransmitter candidates including substance P, vasoactive intestinal polypeptide (VIP) and adenosine triphosphate (ATP) were investigated, but there was no convincing evidence to suggest that they play a role in mediating the second component of neurogenic vasodilatation.
6.As the transmitter responsible for mediating the second component of neurogenic vasodilatation could not be identified, the mechanism by which it operated was also investigated.
7.Although the first component of neurogenic vasodilatation was clearly mediated by NO, inhibitors of PDE5, PKG and the soluble guanylate cyclase/cGMP/NO pathway had no effect on the second component of vasodilatation.
8.Furthermore, various K+ channel blockers, or inhibitors of the PKA/cAMP pathway did not inhibit the second component of neurogenic vasodilatation. Therefore, neither the identity of the neurotransmitter responsible for the second component, nor its mechanism of action could be determined.
9.NG-substituted analogues of L-arginine are routinely used to inhibit the NOS family of enzymes. The first of these to be introduced, NG-methyl-L-arginine (L-NMMA), is generally reported to inhibit all three isoforms of NOS. Despite this, however, L-NMMA does not inhibit nitrergic nerve-mediated relaxation in other bovine tissues including the retractor penis muscle or penile artery.
10.In this study of the bovine intraocular LPCA, L-NMMA was found to inhibit the endothelium-dependent dilatation mediated by eNOS, but not the nitrergic vasodilatation by nNOS. Indeed, the ability of L-NMMA to protect nitrergic dilatation against blockade by L-NAME in a manner similar to L-arginine, suggests it might act as an alternative substrate for nNOS in this tissue.
11.Isoform-selective inhibitors of NOS are of great interest, both as investigational tools and potential therapeutic agents. Two nNOS-specific inhibitors have recently been identified: N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris (trifluoroacetate) salt (AAAN) and NG-propyl-L-arginine.
12.AAAN failed to affect vasodilatation induced either by nitrergic nerves or by bradykinin-induced, endothelium-derived NO in the bovine intraocular LPCA.
13.Although NG-propyl-L-arginine did inhibit nitrergic vasodilatation, it also blocked the bradykinin-induced, endothelium-dependent vasodilatation mediated by NO. It thus failed to exhibit the expected selectivity for nNOS over eNOS in the bovine intraocular LPCA.
14.These findings with AAAN and NG-propyl-L-arginine are a reminder that it is not always possible to extrapolate findings in biochemical assays to functional responses in intact tissues.
15.In conclusion, these findings show that neurogenic vasodilatation in the bovine LPCA involves two components: a fast, transient component mediated by NO from nitrergic nerves, and a second slower, more sustained component mediated by an as yet unidentified second neurotransmitter.
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