Thomas C. Westfall - US grants

There is now convincing evidence that in addition to muscarinic-cholinergic receptors there are also nicotinic-cholinergic receptors in the mammalian central nervous system. We and others have observed that activation of nicotinic-cholinergic receptors leads to a release of both dopamine and serotonin in the striatum and hypothalamus. The objective of the present proposal is to focus on three important aspects of the nicotine-induced activation of dopaminergic and serotonergic neurotransmission. Firstly, we will determine the mechanism(s) by which activation of nicotinic receptors produces a release of these neurotransmitters. The binding of radioactive ligands (3H-nicotine; 3H-acetyl-choline) to rat striatal membranes will be examined before and after lesioning of various neural pathways (dopamine, serotonin, glutamate) to help identify the site of nicotinic receptors. The release of endogenous dopamine and serotonin from slices and synaptosomes (by HPLC-EC analysis) in the absence or presence of various drugs, ions, or perturbations will provide information on various aspects of the release process (e.g. importance of propagated action potentials, exocytosis, intermediate neuromediators, desensitization, additivity with other mechanisms of inducing transmitter release, etc.). Secondly, the role of opioid drugs and peptides on the nicotine induced release of dopamine and serotonin from striatal slices will be rigorously analyzed in an attempt to determine if they exert a neuromodulatory role. Finally, the effect of chronic treatment of rats with nicotinic agonists and antagonists on norepinephrine, dopamine and serotonin turnover in vivo and release in vitro as well as on the number and density of nicotinic receptors in vitro will be examined. These studies will provide important information on the mechanism and modulation of the release of dopamine, serotonin and possibly glutamate in the striatum following activation of nicotinic receptors. In addition, these experiments should provide valuable information concerning the adaptation of neurotransmitter systems following continual exposure to nicotinic agonists and antagonists. These results should relate much better to the possible effects of tobacco and nicotine abuse in man.

The purpose of the present proposal is to generate useful new information on the role of adrenergic in the development and maintenance of hypertension in the Spontaneously Hypetensive Rat (SHR). The present application is a logical extension of ongoing studies and will focus on two important aspects of our previous observations. First, to investigate the functional significance of the alterations in the relesse of norepinephrine (NE) from blood vessels and central brain regions of the SHR and secondly, to further characterize, explore and define the mechanisms contributing to the alterations in the release of NE from blood vessels and brain regions of the SHR. The overall unifying hypothesis to be examined is, "There are defects at the level of noradrenergic nerve terminal in the periphery and the central nervous system resulting in alterations in transmitter release. Depending upon the neuroeffector junction in question, the increased release (blood vessels, posterior hypothalamus) and decreased release (anterior hypothalamus, A2 region of the NTS) of NE contributes to the development and/or maintenance of hypertension. Changes in the activity of prejunctional release modulatory receptors contribute to these alterations in transmitter release." The proposal has been divided into two parts. Part one deals with studies on peripheral noradrenergic transmission in the SHR and Part two deals with central noradrenergic transmission in the SHR. The Specific Aims are: 1) to investigate the functional significance of the enhanced release of NE from isolated blood vessels and the importance of an alteration in prejunctional receptor activity in contributing to the enhanced release; 2) to further characterize and define the mechanism(s) for the enhancement of NE release by angiotensin in blood vessels of SHR; 3) to further characterize and define the mechanism for the attenuation of the yohimbine induced enhancement of NE release in blood vessels of the SHR; 4) to investigate the functional significance of alterations in the evoked release of NE from the brain using push-pull perfusion; 5) to determine if there are alterations in the activity of prejunctional release modulatory receptors in the CNS of SHR; and 6) to examine the role of vasopressin as possible modulator of NE release from brain regions of normal and hyoertensive rats. The proposal should generate useful new information on several aspects of the role of noradrenergic neurons in the development and maintenance of hypertension in the SHR and other hypertensive models.

There is considerable evidence for the role of both the central nervous system and the sympathetic nervous system in the development and/or maintenance of hypertention in the SHR. The SHR is excessively responsive behaviorally and physiologically to a variety of stressful stimuli. Neuropeptide-Y(NPY) is a 36 amino acid peptide that has been shown to be co-localized with norepinephrine in peripheral noradrenergic neurons as well as with norepinephrine or epinephrine in central catecholamine neurons. The Working Hypothesis of this proposal is that, "NPY may be released together with norepinephrine upon nerve stimulation; that NPY may work together with norepinephrine in causing certain physiological responses such as contraction of vascular smooth muscle, presynaptic inhibition of norepinephrine release and centrally mediated decreases in blood pressure; and that alterations in the actions of NPY may contribute to the development and maintenance of hypertension." The purpose of the present investigation will be to carry out a systematic study of several aspects of NPY's actions at the vascular neuroeffector junction and central catecholamine neurons and to examine the interaction between NPY and catecholamine systems in normotensive and hypertensive animals in which stress is known to play an important role. Experiments will be carried out in Sprague-Dawley rats, Spontaneously Hypertensive rats and Wistar-Kyoto normotensive rats. Six Specific Aims will be addressed: 1) to determine and characterize the effects of NPY on the isolated caudal artery and perfused mesentery arterial bed in vitro and in situ; 2) to determine the effect of NPY on noradrenergic neurotransmission at the vascular neuroeffector junction. Similar preparations as in Aim One will be used; 3) to assess the release of NPY from noradrenergic neurons innervating blood vessels, release of NPY will be measured from caudal arteries in response to field stimulation; the perfused mesenteric bed to periarterial nerve stimulation and into the plasma in pithed rats in which the sympathetic outflow is stimulated at the level of the spinal cord; 4) to characterize the effect of NPY on blood pressure following microinjection into specific regions of the hypothalamus or brainstem; 5) to determine the effect of NPY in the evoked release of catecholamines from slices of hypothalamus or brainstem or following push-pull perfusion; and 6) to assess the release of NPY from these same brain regions following stimulation. It is thought that this investigation will better define and characterize the physiological and pathophysiological role of NPY in circulatory control mechanisms, stress and hypertension.

There is convincing evidence to suggest that dopamine autoreceptors are present on dopaminergic nerve terminals in the striatum which regulate the release and the synthesis of this neurotransmitter. The major objectives of the present proposal are first, to gain an understanding of pharmacological differences between autoreceptor and postsynaptic receptors; and second, to gain an understanding of the mechanism(s) by which activation of dopamine autoreceptors results in an inhibition of dopamine release and synthesis. One major strategy is to examine the relative potency of selected agonists and antagonists in activating or antagonizing autoreceptors or postsynaptic receptors. Dopamine autoreceptors will be studied by examining the effect of selective dopamine agonists and antagonists on the evoked release of endogenous dopamine (measured by high performance liquid chromatography coupled to electrochemical detection) or the synthesis of dopamine (measured by the incorporation of 3H-tyrosine into 3H-dopamine) in striatal slices or synaptosomes. Postsynaptic receptor function will be studied by examining the effect of the same drugs on the evoked release of 3H-acetylcholine, 3H-glutamate and endogenous glutamate. The second major strategy will be to examine the mechanism by which activation of dopamine autoreceptors cause a release of dopamine utilizing several probes and perturbations. These probes and perturbations will assess the role of calcium ions, Na+ K+ ATPase, cyclic nucleotides (cAMP, cGMP) and the recruitment of varicosities vs. electrosecretion coupling as possible mechanisms of the inhibition of dopamine release. These studies should provide a better understanding of dopamine autoreceptors. This is of obvious importance since the use of dopamine autoreceptor agonists may represent a novel approach to various diseases such as schizophrenia, Huntington's Disease and tardive dyskinesia where overactivity of central dopamine neurons may be etiological. Moreover, the dopamine blocking action of neuroleptic drugs may be achieved by various combinations of pre- and postsynaptic effects and the relative importance may vary from one class of neuroleptics to another. These studies will provide a better understanding of the physiology and pharmacology of central dopaminergic neurotransmission.

biomedical equipment purchase;

DESCRIPTION (provided by applicant): This application is for a renewal of the Training Program in the Pharmacological Sciences to support seven outstanding predoctoral students who will be working for a Ph.D. degree in Pharmacological and Physiological Science or the program of their mentor with research emphasis on cellular communication and control exerted through the endocrine, cardiovascular and nervous system. This is a broadly based, multidisciplinary effort which involves 28 faculty from six departments in the School of Medicine at Saint Louis University. These include: Pharmacological and Physiological Science;Biochemistry and Molecular Biology;Chemistry;Internal Medicine;and Ophthalmology. Students will be selected from among candidates entering the Pharmacological and Physiological Science or the program of their mentor after successfully completing the one year Core Program in Basic Biomedical Science, M.D./Ph.D. students who have completed the basic science curriculum and now wish to obtain the Ph.D. degree in Pharmacological and Physiological Science or the program of their mentor and finally students who enter the Pharmacological and Physiological Science Program with an advanced standing. During the first year of study, all traditional Ph.D. students will participate in the interdisciplinary Core Graduate Program in Biomedical Sciences. This program has been designed to provide students with a strong foundation in all aspects of basic biomedical science and the freedom to explore diverse research opportunities. The curriculum combines lectures, small group discussion and seminars. Students completing the Core Program, M.D./Ph.D. students or students with M.S. degrees will enter the program in Pharmacological and Physiological Science or the program of their mentor and take advanced courses, journal clubs and seminars. Subsequent training for all Ph.D. candidates will concentrate on the development of research and teaching competence in a specific area of inquiry under the mentorship of one or more members of the Pharmacological Sciences Training Faculty. The mentors and laboratories participating in this program are well equipped to provide state-of-the-art research training. In addition, core and shared facilities for advanced technologies are available for enhancement of the research training of the participating candidates. The overall objectives of this training program are to provide individuals with the opportunity to achieve a high degree of competence in the area of pharmacological sciences thus preparing them for teaching and research careers.

biomedical equipment purchase;

biomedical equipment purchase;

DESCRIPTION: (Adapted from the 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.

DESCRIPTION (provided by applicant): Neuropeptide Y (NPY) is known to be co-localized, with norepinephrine (NE) and adenosine triphosphate (ATP) in vascular sympathetic neurons where it is thought to play a role as a co-transmitter/co-modulator. Recent evidence has established that NPYplays a physiological role in sympathetically mediated vasoconstriction by acting on postjunctional Y1 receptors. NPY is also known to exert prejunctional effects leading to inhibition of NE and most likely ATP release and an increase in NPY-ir release via Y2 receptors as well as inhibition of NE synthesis via Y3 and Y5 receptors. These results suggest that the process of NE synthesis and release of sympathetic co-transmitters may be differentially modulated by NPY suggesting an additional level of control in the prejunctional regulation of sympathetic neurotransmission. However the physiological role of this action has not been completely established. We have obtained evidence that an important mechanism for the prejunctional effects of NPY is through inhibition of Ca 2+channels. There is also evidence for a role of SNARE proteins in the preferential release and modulation of transmitter releaseby auto and heteroreceptors. The purpose of the present proposal is to further investigate the physiological role (Aims 1 and 2) on the NPY induced modulation of sympathetic co-transmitter release and NE release and the mechanisms involved (Aim 3) in the prejunctional modulation by NPY and other mediators. The rationale for studies proposed in Aim 1 is as follows: If there is endogenous modulation of transmitter release by NPY, then agonists and antagonists specific for the prejunctional NPY autoreceptor should alter evoked transmitter release in a manner consistent with the receptors being activated by endogenous agonist (e.g. released NPY). In other words, the effect of exogenously administered agonists and antagonists should vary with the biophase concentration of endogenous NPY. The response of the effector cell should be consistent with inhibition or stimulation ofNPY release. It would also seem necessary to demonstrate functional receptors in vivo. A similar rationale exists for Aim 2. In Aim 1 we examine the effect of a series of selective Y1 and Y2 agonists and antagonists on the nerve stimulation evoked release of NE, NPY-ir and ATP as well as 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 3) after the NPY concentration has been reduced by depletion of tissue levels acutely or chronically. We will also examine the in vivo effect of agonists and antagonists in the pithed rat preparation. In Aim 2 similar experiments will evaluate the effect of NPY drugs on the nerve stimulation evoked increase in NE synthesis as measured by DOPA accumulations after decarboxylase inhibition. In Aim 3 we will determine if interruption of SNARE proteins by Botulinum neurotoxins (BoNTs) inhibit the evoked release of NE, NPY-ir and ATP as a mechanism for preferential release or differentiated modulation. We will also examine if signaling through SNARE proteins contributes to the prejunctional modulation by NPY. These studies will provide useful new information on the functional role and mechanism of action of NPY in the prejunctional regulation of sympathetic neurotransmission.