application) Neuropeptide Y (NPY) is known to be co-localized with norepinephrine (NE) and adenosine triphosphate (ATP) in vascular sympathetic neurons where it may play a role as a co-transmitter/co-modulator. Recent evidence has established that NPY plays a physiological role in sympathetic mediated vasoconstriction by acting on postjunctional Y1 receptors. NPY is also known to exert prejunctional effects leading to inhibition of NE, NPY and ATP release via Y2 receptors and inhibition of catecholamine (CA) synthesis via Y3 receptors. These results suggest that the process of CA synthesis and release may be differentially modulated by NPY suggesting an additional level of control in the prejunctional regulation of sympathetic neurotransmission; however, the physiological role of these actions has yet to be established. Our investigations into the mechanisms of these actions suggest that NPY receptor activation inhibits voltage-gated Ca2+ channels although direct evidence has not yet been obtained. The purpose of the present proposal is to investigate the physiological role (Aim 1 and 2) and the mechanism(s) of action (Aim 3) of the NPY-induced inhibition of CA synthesis and release. The rationale for studies proposed in Aim 1 is as follows: If NPY normally exerts an inhibitory autoregulation on transmitter release then an antagonist for the receptor in question should interrupt the feedback circuit and increase transmitter release. The prejunctional effects of both exogenously administered agonists and antagonists should vary with the biophase concentration of endogenous NPY. Furthermore, the response of the effector cell should be consistent with inhibition or stimulation of NPY release. Finally, it would seem necessary to demonstrate functional receptors in vivo as well as in vitro. A similar rationale exists for Aim 2.
In Aim 1, the effect of a series of selective Y1 and Y2 agonists and antagonists will be examined on the release of NE, NPYir and sometimes ATP evoked by nerve stimulation, as well as measurements of perfusion pressure in the mesenteric arterial bed. This will be done before and after the endogenous NPY concentration is elevated by: 1) increasing the frequency of nerve stimulation or 2) decreasing the perfusion rate or after the NPY concentration has been reduced by depletion of tissue levels. The in vivo effect of agonists and antagonists will also be examined in the pithed rat preparation.
In Aim 2, similar experiments will evaluate the effect of NPY analogs on the nerve stimulation evoked increase in NE synthesis as measured by DOPA accumulation after decarboxylase inhibition.
In Aim 3, voltage-clamp recordings will be accomplished in NGF-differentiated PC12 cells to directly determine if activation of Y2 and Y3 receptors can decrease Ca2+ current. Whether this is mediated by inhibition of Ca2+ influx through N-type and L-type voltage activated Ca2+ channels will be assessed, as will whether inhibition of L-type Ca2+ channels also involves the action of PKC. These studies are designed to provide useful new information on the functional role and mechanism of action of NPY in the prejunctional regulation of sympathetic neurotransmission.
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