Neuroreceptor mediated vascular control mechanisms in the rat

The nervous control of blood flow is regulated by specific receptors located in the vascular smooth muscle and endothelial cells. The variability demonstrated by different vascular beds in response to nerve stimulation, both during development and in the adult, may be related to the heterogeneous expression of specific neuroreceptor subtypes. The primary aim of this thesis therefore, was to determine if such differences could be attributed to differences in receptor gene expression. Reverse transcription polymerase chain reaction (RT-PCR) was used to screen receptor mRNA expression in the basilar, mesenteric, pulmonary, tail and hepatic arterial beds of the rat. Receptor subtypes studied included the a r and a 2-adrenergic, neurokinin and muscarinic receptors. Results demonstrated that the pattem of gene expression was variable, with no two arteries having the same receptor profile. Overall, some receptors showed a consistently strong pattem of expression, while others exhibited more variable profiles across the different arteries. Additional findings from this study included the previously unreported detection of mRNA for the M5 muscarinic and NK3 neurokinin receptors in all arteries studied. The RT-PCR results examining receptor expression were consistent with the functional data of other investigators, however in order to directly correlate mRNA expression and functional protein expression in the same tissue, the hepatic arterial bed was used to further characterise neuroreceptor mediated vascular responses. A dense sympathetic innervation was demonstrated in this vascular bed, using catecholamine histochemistry and antibodies directed against the synaptic vesicle protein synaptophysin. Physiological experiments revealed that the principle receptors involved in the contractile response to nerve stimulation were the a 1A-adrenergic, P2X(1)-purinergic and neuropeptide Y Yj receptors, all of which showed strong mRNA expression as detected using RT-PCR. Additionally, neurotransmitter release was shown to be modulated by presynaptic a 2-adrenergic and possibly P2X-purinergic receptors. Sympathetic nerves were also shown to activate a dilatory response via postsynaptic ßadrenergic receptors. A dilatory response, mediated by calcitonin gene related peptide (CGRP) was also demonstrated, correlating with the detection of perivascular nerves immunoreactive for CGRP and the strong mRNA expression of the CGRP; receptor subtype. No role could be found for nitric oxide (NO) in nerve-mediated vasodilatory responses, however experiments did reveal an important regulatory role for NO released from the endothelium in response to vasoconstriction and increased vascular tone. Overall, the studies in the rat hepatic arterial bed demonstrated that complex interactions occur between vasoconstrictor and vasodilatory pathways and that these responses are further modulated by presynaptic mechanisms. The major postsynaptic neuroreceptor subtypes involved were well predicted by mRNA expression levels as determined using RT-PCR, confirming that this technique is a useful adjunct in studies aimed at identifying functional vascular neuroreceptor mediated responses. Experiments were then performed to determine if alterations in receptor mRNA expression could account for the physiological changes described in the rat mesenteric artery during development. Using RT-PCR as a screening tool, no changes were detected that could account for the maturation of the adult response, nor was there any link between the appearance of the nerves and the initial expression of mRNA for the receptors under study. This lack of positive correlation between the presence of nerves and receptor gene expression was confirmed with denervation studies, where the ingrowth of sympathetic and sensory nerve fibres was prevented over the critical first two postnatal weeks. The effectiveness of sympathectomy treatments was assessed using catecholamine histochemistry and it was interesting to note that nerve growth factor sensitive sympathetic neurones innervating the mesenteric vascular bed were resistant to permanent destruction by the compound 6-hydroxydopamine, in contrast to sympathetic neurones innervating rat iris arterioles. In summary, this thesis reviews neuroreceptor mediated vascular control mechanisms and examines in detail the expression of neuroreceptor genes in arteries of the rat, both in the adult and during development. The contribution of the different receptor subtypes to nerve-mediated vascular responses are characterised specifically in the hepatic arterial bed. It is concluded that individual arteries possess both common and unique neural control mechanisms, which may ultimately be utilised in the development of systemic and vascular bed specific therapeutic agents. PUBLICATIONS ARISING FROM DATA PRESENTED IN THESIS The following papers resulting from work presented in this thesis have been published or accepted for publication: Phillips, J.K. , Vidovic, M. & Hill, C.E. (1996). a-adrenergic, neurokinin and muscarinic receptors in rat mesenteric artery; An mRNA study during post-natal development. Mech. Ageing Dev. , 92, 235-246. Phillips, J.K. , Vidovic, M. & Hill, C.E. (1997). Variation in mRNA expression of alphaadrenergic, neurokinin and muscarinic receptors amongst four different arteries of the rat. J. Auton. Nerv. Syst. , 62, 85-93. Phillips, J.K. , McLean, A.J. & Hill, C.E. (1998). Receptors involved in nerve-mediated vasoconstriction in small arteries of the rat hepatic mesentery. Br. J. Pharmacol. , In