Work from our laboratory has established that angiotensin II (Ang II) produces a greater enhancement of the nerve stimulation (NS)-induced release (overflow) of both norepinephrine (NE) and neuropeptide Y (NPY) and a greater increase in perfusion pressure of the mesenteric arterial bed obtained from the spontaneously hypertensive rat (SHR) compared to age-matched Wistar-Kyoto (WKY) or Sprague-Dawley rats. The enhancement of NS-induced NPY release was blocked by the AT1 receptor antagonist EMD 66684 and the AT2 receptor antagonist PD 123319. Both captopril and EMD 66684 decreased NPY and NE overflow from SHR mesenteric beds, suggesting an endogenous renin-angiotensin system (RAS) is active in the mesenteric artery. We also observed that the recently discovered new arm of the RAS, namely, angiotensin (1-7) (Ang-(1-7)), attenuated the NS-induced increase in NE and NPY release and the accompanied increased perfusion pressure. These inhibitory effects were greater in blood vessels obtained from SHR compared to WKY. We suggest that inhibition of sympathetic neurotransmission contributes to the mechanism(s) by which Ang-(1-7) acts to inhibit the vasoconstrictor effect of Ang II. Administration of the MAS receptor antagonist D-Ala(7)Ang-(1-7) attenuated the decrease in both NE and NPY release due to Ang-(1-7) administration. The AT2 receptor antagonist PD 123391 attenuated the effect of Ang-(1-7) on NE release without affecting the decrease in NPY release. We observed a shift in the balance between Ang II and Ang-(1-7) levels in the SHR with an increase in Ang II and a decrease in Ang-(1-7) in the blood and mesenteric artery. This appears to be due to an increase in angiotensin-converting enzyme (ACE) in the mesenteric artery of the SHR.

Renal denervation decreases arterial pressure (AP) in hypertensive rats and humans. This procedure destroys both afferent and efferent nerves. Several investigators have proposed that renal afferent nerves contribute to the elevated AP. We developed a procedure to selectively remove renal afferent nerves with capsaicin (1-100 mM) both topically on the nerve and in the renal pelvis. We examined the effects of renal deafferentation on the development of genetic and renal hypertension. We studied spontaneously hypertensive rats (SHR), and a model of renal hypertension, two kidney-one clip (2K1C) in Sprague-Dawley rats. SHR were treated at 3-4 weeks of age with capsaicin. Mean arterial pressure was recorded by tail cuff through 16 weeks of age. On week 17, rats were cannulated, allowed 3 days to recover then had their AP measured directly for 3 days (3 hrs/day). Rats with renal deafferentation (n=11) had lower arterial pressure weeks 9-16 (average reduction AP=10.1±1.4 mmHg, ANOVA, p=0.0049) compared to control (saline treated, n=6) although the final direct recording was not significantly different on week 17 (control AP=184.1±3.4 mmHg vs deafferented AP=173.9±4.3 mmHg, p=0.07). Substance P levels from the kidneys were reduced in deafferented rats compared to control (6.9±1.0 vs 17.3±5.2 pg/g protein, p=0.0009). In contrast, renal NE levels were not altered (307±19 vs 313±20 pg/g protein, p=0.428). In the second study, the left kidney in weanling Sprague-Dawley rats was exposed to capsaicin or saline. Rats were allowed to mature (>250 g BW) then subjected to left renal artery clipping (0.2mm) or sham clip. AP was recorded by tail cuff during development of 2K1C for 6 weeks before direct cannulation to record AP on week 7. Renal deafferentation prevented the development of hypertension in 13 rats compared to 9 saline treated rats (average reduction AP=16.9±2.7 mmHg, ANOVA, p=0.0031). Saline treated rats had a higher AP 7 weeks after clipping (147.1±10.2 vs 130.5±4.2 mmHg direct recording, p=0.02). The left kidney contained 48% SP compared to the right kidney (p=0.04). These data suggest that increased afferent renal nerve activity contributes to the elevation in AP in hypertension and contributes to essential hypertension in humans. Supported by USPHS DA017371.

Ang‐(1‐7) produces a significant decreasein the nerve stimulation (NS)‐induced release of the sympathetic nervous system cotransmitters NE and NPY, as well as perfusion pressure of the mesentericarterial bed of the rat. These effects are greater in mesenteric beds obtained from Spontaneously Hypertensive Rats (SHR) compared to its normotensive control, Wistar‐Kyoto rats (WKY). We examined the hypothesis that the Ang‐(1‐7) induced inhibition of sympathetic neurotransmission is mediated at least in part by prostacyclin (PGI2). Experiments were performed using mesenteric beds obtained from 10–12 week old SHR and WKY rats. The effect of Ang‐(1‐7) on the NS‐evokedrelease of NE and NPY was determined before and after the administration of the COX inhibitor Indomethacin (to prevent PGI2 synthesis) and the IP receptor antagonist CAY 10441. A reversal of the attenuation of NE and NPY release by Ang‐(1‐7) was observed in the presence of Indomethacin in both SHR and WKY. A trend was observed suggesting CAY10441 is partially reversing the attenuation of NE and NPY release from SHR mesenteric beds, but not WKY. The attenuation of perfusion pressure by Ang‐(1‐7) was reversed by both Indomethacin and CAY10441 in SHR, but not in WKY. These data suggest a role of PGI2 mediating vasodilation and the inhibition of sympathetic neurotransmission by Ang‐(1‐7). (Supported by HL60260 and NIGMS008306)

Noradrenaline, neuropeptide Y and adenosine triphosphate are co-stored in, and co-released from, sympathetic nerves. Each transmitter modulates its own release as well as the release of one another; thus, anything affecting the release of one of these transmitters has consequences for all. Neurotransmission at the sympathetic neurovascular junction is also modulated by non-sympathetic mediators such as angiotensin II, serotonin, histamine, endothelin and prostaglandins through the activation of specific prejunctional receptors. In addition, nitric oxide (NO) has been identified as a modulator of sympathetic neuronal activity, both as a physiological antagonist against the vasoconstrictor actions of the sympathetic neurotransmitters, and also by directly affecting transmitter release. Here, we review the modulation of sympathetic neurovascular transmission by neuronal and non-neuronal mediators with an emphasis on the actions of NO. The consequences for co-transmission are also discussed, particularly in light of hypertensive states where NO availability is diminished.

Ang‐(1–7) produces a significant decreasein the nerve stimulation (NS)‐induced release of both NE andNPY as well as perfusion pressure of the perfused mesentericarterial bed of the rat. This effect is greater in mesenteric beds obtained from Spontaneously Hypertensive Rats (SHR) compared to normotensive control. Two hypotheses were examined; 1. The Ang‐(1–7) induced inhibition of sympatheticneurotransmission is mediated at least in part by prostacyclin(PGI2) and 2. Ang‐(1–7) elicits greater effects in mesenteric beds of SHR because there is decreased Ang‐(1–7) present in the mesenteric artery and plasma of SHR. Experiments were carried out using mesenteric beds and plasmaobtained from 10–12 week old SHR and Wistar‐Kyoto (WKY) rats. The effect of Ang‐(1–7) on the NS‐evokedrelease of NPY was determined before and after the administrationof the COX inhibitor Indomethacin (to prevent PGI2 synthesis) and the PGI2 receptor blocker CAY 10441. A reversal in the attenuation of perfusion pressure by Ang‐(1–7) was observed in the presence of Indomethacin and CAY10441. The attenuation of NPY release by Ang‐(1‐7) also appears to be reversed by Indomethacin and CAY10441. Additionally, plasma and tissue concentration of Ang‐(1–7) were significantly lower in SHR, while those of Ang II were significantly higher. (Supported by HL60260 and NIGMS008306)

Thomas C. Westfall, PhD, is the William Beaumont Professor and Chair Department of Pharmacological and Physiological Science at the Saint Louis University School of Medicine. Contact: westfatc@slu.edu Introduction Over the years it has been customary at many medical schools in the United States and Canada to conduct live animal exercises or demonstrations (dogs, cats, pigs, etc.) to illustrate the physiological and pharmacological actions of autonomic and cardiovascular drugs. In fact several generations of medical students have learned practical aspects of autonomic and cardiovascular physiology and pharmacology from such traditional exercises. During these exercises, students would spend three to four hours injecting drugs, monitoring responses such as blood pressure, heart rate and respiration and discussing the various possible mechanisms of their observations. Although these exercises were thought to be very valuable and were of high educational impact numerous reasons have caused most medical schools, including Saint Louis University, to discontinue such live animal labs. These have included increasing costs of animals and equipment, shortage of qualified demonstrators and concerns about animal welfare issues. Over the last six years in the Principles of Pharmacology Course at Saint Louis University we have substituted such live animal demonstrations with the use of high fidelity human simulators in an operating room setting to illustrate, describe and discuss the physiological and pharmacological actions of cardiovascular and autonomic drugs. These exercises have been extremely popular with the students, carry a high educational impact and represent an exciting substitute to live animal exercises.

We have observed that Ang‐(1‐7) produces a significant decrease in the nerve stimulation (NS)‐induced release of both NE and NPY as well as perfusion pressure of the perfused mesenteric arterial bed of the rat. In the present study we examined the hypothesis that the Ang‐(1‐7) induced inhibition of sympathetic neurotransmission is mediated at least in part by prostacyclin (PGI 2 ), which is well known to inhibit sympathetic neurotransmission and to mediate part of the vasodilator action of Ang‐(1‐7). Experiments were carried out using the mesenteric arterial bed obtained from 10–12 week old Sprague Dawley and Spontaneously Hypertensive Rats (SHR). The effect of Ang‐(1‐7) on the NS‐evoked release of NE and NPY was determined before and after the administration of indomethacin to block PGI 2 synthesis and before and after the administration of the PGI 2 receptor blocker CAY 10441. Results obtained to date support our hypothesis that PGI 2 mediates the actions of Ang‐(1‐7). (Supported by HL60260 and NIGMS008306)

Our lab has observed that dopaminochrome (DAC), an oxidized form of dopamine, is cytotoxic to the mesencephalic cell line MN9D and induces an inflammatory response in the microglial cell line BV2. Dopamine is highly susceptible to oxidation and may form DAC when exposed to an oxidizing environment, leading to polymerization of DAC into neuromelanin, of which DAC is a major component. We believe the formation of DAC and its subsequent aggregation to neuromelanin may play a role in the progression of Parkinson's disease due to its proinflammatory and cytotoxic properties. In this study, we examined the effect of DAC in vivo. DAC (10 nmoles) was stereotaxically injected into the substantia nigra pars compacta (SNpc) of Sprague Dawley rats. Five, ten, and twenty days post‐surgery, the brains were harvested and assessed for inflammation and neurodegeneration. Results to date suggest that acute administration of 10 nmoles DAC is sufficient to induce activation of SNpc resident microglia without any apparent neurodegeneration. The profile of the resulting inflammation and the effect of microglial inhibition prior to DAC injection will be further assessed. (Supported in part by NIGMS 008306)

Neuropeptide Y (NPY) is a cotransmitter with norepinephrine (NE) and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin-angiotensin system (RAS), as well as a role for NPY in the development of hypertension in experimental animal models and in humans. Angiotensin II (ANG II) is known to facilitate sympathetic neurotransmission, an effect greater in spontaneously hypertensive rats (SHR) than normotensive Wistar-Kyoto (WKY) rats. A newly discovered product of the RAS is angiotensin-(1-7) [ANG-(1-7)]. There is evidence suggesting that ANG-(1-7) opposes the actions of ANG II, resulting in hypotensive effects. The objective of this study was to investigate the role of ANG-(1-7) on the nerve-stimulated overflow of NE and NPY from the mesenteric arterial bed of SHR and the mechanisms involved in mediating any effects produced. ANG-(1-7) (0.001, 0.01, 0.1 microM) decreased nerve-stimulated NE and NPY overflow, as well as perfusion pressure in preparations obtained from SHR. This effect was greater in preparations of SHR than WKY controls. In addition, ANG-(1-7) decreased NE overflow to a greater extent than NPY overflow. Administration of the Mas receptor antagonist, D-Ala(7) ANG-(1-7), attenuated the decrease in both NE and NPY overflow due to ANG-(1-7) administration. However, the angiotensin type 2 receptor antagonist, PD-123391, attenuated the effect of ANG-(1-7) on NE overflow without affecting the decrease in NPY overflow. Moreover, in the presence of N(G)-nitro-L-arginine methyl ester, ANG-(1-7) decreased NPY overflow, but not NE overflow. ANG-(1-7) decreases the nerve-stimulated overflow of NE and NPY in preparations of SHR, whereas ANG II enhances it. Therefore, ANG-(1-7) may counteract the effects of ANG II by acting on ANG type 2 and Mas receptors.

Parkinson disease is a specific form of neurodegeneration characterized by a loss of nigra-striatal dopaminergic neurons in the midbrain of humans. The disease is also characterized by an increase in oxidative stress and a loss of glutathione in the midbrain region. A potential link between all these factors is the oxidation of dopamine to dopaminochrome (DAC). Using the murine mesencephalic cell line MN9D, we have shown that DAC [50-250 microM] leads to cell death in a concentration-dependent manner, whereas oxidized l-dopa, dopachrome [50-250 microM], is only toxic at the highest concentration used. Furthermore, chronic exposure of MN9D cells to low concentrations of DAC [50-100 microM] is cytotoxic between 48 and 96 h. DAC also increases superoxide production within MN9D cells as indicated by dihydroethidium fluorescence, that can be prevented by co-administration with the antioxidant, N-acetylcysteine [5 mM]. Moreover, the cytotoxicity induced by DAC can also be prevented by administration of N-acetylcysteine [1-5mM]. Finally, depletion of reduced glutathione in MN9D cells by buthionine sulfoximine [50-100 microM] administration significantly enhances the cytotoxic effect of low concentrations of DAC [50-100 microM] and DAC [175 microM] itself reduces the proportion of oxidized glutathione in total glutathione within 30 min of administration in MN9D cells. Overall, we have shown that DAC causes MN9D cell death in an oxidatively dependent manner that appears closely linked with a rapid loss of reduced glutathione. These findings have implications for understanding the pathogenesis of neurodegenerative pathways in Parkinson disease.

NPY is a cotransmitter with NE and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin‐angiotensin system (RAS) as well as a role for NPY in the development of hypertension in experimental animal models and in humans. Angiotensin II (Ang II) is known to facilitate sympathetic neurotransmission; an effect greater in Spontaneously Hypertensive Rats (SHR) than normotensive Wistar‐Kyoto (WKY) rats. A newly discovered product of the RAS is the peptide Angiotensin 1–7 (Ang 1–7). There is evidence suggesting that Ang 1–7 opposes the actions of Ang II resulting in hypotensive effects. The objective of this study was to investigate the role of Ang 1–7 on the nerve stimulated overflow of NE and NPY from the mesenteric arterial bed of SHR and the receptors mediating any effects produced.Ang 1–7 (0.001, 0.01, 0.1μM) decreased nerve stimulated NE and NPY overflow, as well as perfusion pressure in preparations obtained from SHR. This effect was greater in preparations of SHR than WKY controls. Administration of the Mas Receptor Antagonist (MRA), D‐Ala Ang 1–7, and the Angiotensin Type 2 Receptor Antagonist (AT2A), PD12339 attenuated this effect.Ang 1–7 decreases the nerve stimulated overflow of NE and NPY in preparations of SHR, while Ang II enhances it. Therefore, Ang 1–7 may counteract the effects of Ang II by acting on AT2 and Mas Receptors. (Sponsored by NIH HL60260 and AHA)

Current evidence suggests that hyperactivity of the sympathetic nervous system and endothelial dysfunction are important factors in the development and maintenance of hypertension. Under normal conditions the endothelial mediator nitric oxide ( NO) negatively modulates the activity of the norepinephrine portion of sympathetic neurotransmission, thereby placing a "brake" on the vasoconstrictor ability of this transmitter. This property of NO is diminished in the isolated, perfused mesenteric arterial bed taken from the spontaneously hypertensive rat (SHR), resulting in greater nerve-stimulated norepinephrine and lower neuropeptide Y (NPY) overflow from this mesenteric preparation compared with that of the normotensive Wistar-Kyoto rat (WKY). We hypothesized that increased oxidative stress in the SHR contributes to the dysfunction in the NO modulation of sympathetic neurotransmission. Here we demonstrate that the antioxidant N-acetylcysteine reduced nerve-stimulated norepinephrine and increased NPY overflow in the mesenteric arterial bed taken from the SHR. Furthermore, this property of N-acetylcysteine was prevented by inhibiting nitric oxide synthase with N-omega-nitro-L-arginine methyl ester, demonstrating that the effect of N-acetylcysteine was due to the preservation of NO from oxidation. Despite a reduction in norepinephrine overflow, the nerve-stimulated perfusion pressure response in the SHR mesenteric bed was not altered by the inclusion of N-acetylcysteine. Studies including the Y-1 antagonist BIBO 3304 with N-acetylcysteine demonstrated that this preservation of the perfusion pressure response was due to elevated NPY overflow. These results demonstrate that the reduction in the bioavailability of NO as a result of elevated oxidative stress contributes to the increase in norepinephrine overflow from the SHR mesenteric sympathetic neuroeffector junction.

NPY is a cotransmitter with NE and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin‐angiotensin system (RAS) as well as a role for NPY in the development and maintenance of hypertension in experimental animal models and in humans. Angiotensin II (Ang II) is known to facilitate sympathetic neurotransmission, and evoke the release of NE. This effect of Ang II is enhanced in blood vessels obtained from Spontaneously Hypertensive Rats (SHR). The objective of this study was to investigate the role of Ang II on the basal and nerve stimulated overflow of NPY from the perfused mesenteric arterial bed of SHR as hypertension develops.We observed that Ang II (0.01; 0.1 μM) facilitates basal and nerve stimulated overflow of NPY from the mesenteric arterial bed; an effect that is greater in preparations obtained from 4–6, 10–12 and 18–20 week old SHR than age matched normotensive Wistar‐Kyoto (WKY) or Sprague Dawley (SD) rats. Preparations obtained from prehypertensive (4–6 week old) SHR appear to behave similarly to those of 10–12 week old SHR with respect to Ang II induced changes in nerve stimulated NPY overflow. This facilitatory effect of Ang II appears to be mediated by the AT1 receptor. In addition, Captopril administration resulted in decreased NE overflow from SHR preparations, suggesting that the local RAS is active in this model. (Sponsored by NIH HL60260 and AHA)

The sympathetic nervous system and renin-angiotensin system are both thought to contribute to the development and maintenance of hypertension in experimental models such as the spontaneously hypertensive rat (SHR). We demonstrated that periarterial nerve stimulation (NS) increased the perfusion pressure (PP) and neuropeptide Y (NPY) overflow from perfused mesenteric arterial beds of SHRs at 4-6, 10-12, and 18-20 wk of age, which correspond to prehypertensive, developing hypertensive, and maintained hypertensive stages, respectively, in the SHR. NS also increased PP and NPY overflow from mesenteric beds of Wistar-Kyoto (WKY) normotensive rats. NS-induced increases in PP and NPY were greater in vessels obtained from SHRs of all three ages compared with WKY rats. ANG II produced a greater increase in PP in preparations taken from SHRs than WKY rats. ANG II also resulted in a greater increase in basal NPY overflow from 10- to 12-wk-old and 18- to 20-wk-old SHRs than age-matched WKY rats. ANG II enhanced the NS-induced overflow of NPY from SHR preparations more than WKY controls at all ages studied. The enhancement of NS-induced NPY overflow by ANG II was blocked by the AT1 receptor antagonist EMD-66684 and the angiotensin type 2 receptor antagonist PD-123319. In contrast, ANG II greatly enhanced norepinephrine overflow in the presence of PD-123319. Both captopril and EMD-66684 decreased neurotransmitter overflow from SHR mesenteric beds; therefore, we conclude that an endogenous renin-angiotensin system is active in this preparation. It is concluded that the ANG II-induced enhancement of sympathetic nerve stimulation may contribute to the development and maintenance of hypertension in the SHR.

In rat pheochromocytoma (PC12) cells the dopamine D(2) receptor agonists apomorphine (APO) and n-propylnorapomorphine (NPA) produced a concentration dependent inhibition of K(+)-evoked neuropeptide Y release (NPY-ir). The effect of APO was blocked by the dopamine D(2)-receptor antagonist, eticlopride, but not the D(1)/D(3) or the D(4)/D(2) antagonists, SCH23390 or clozapine, respectively. The D(1)/D(5) receptor agonist, SKF38393 or the D(3) agonists PD128907 and 7-OH DPAT had no effect. Selective N and L-type voltage gated Ca(2+) channel blockers, omega-conotoxin GVIa (Ctx-GVIa) and nifedipine, respectively, produced a concentration dependent inhibition of NPY-ir release but were not additive with APO. The Ca(2+)/calmodulin-dependent protein kinase (CaM kinase) II inhibitor KN-62 produced a concentration-dependent inhibition of NPY-ir release but the combination of KN-62 and APO produced no further inhibition. PMA-mediated protein kinase C stimulation significantly increased both basal and K(+)-evoked release of NPY-ir, and in the presence of PMA APO had no inhibitory effect. The PKC antagonist, chelerythrine, inhibited K(+)-evoked NPY-ir release but was not additive with APO. Neither forskolin-mediated adenylate cyclase activation and the active cAMP analog Sp-cAMPS, nor the adenylate cyclase inhibitor SQ 22536, and the competitive inhibitor of cAMP-dependent protein kinases Rp-cAMPS, had any significant effect on K(+)-evoked NPY-ir release. This suggests the inhibitory effect of APO on K(+)-evoked release of NPY-ir from PC12 cells is most likely mediated through activation of dopamine D(2) receptors leading to direct inhibition of N and L-type voltage gated Ca(2+) channels, or indirect inhibition of PKC, both of which would reduce [Ca(2+)](i) and inactivate CaM kinase.