Cannabidiol (CBD) has gained a lot of interest in recent years for its purported medicinal properties. CBD has been investigated for the treatment of anxiety, depression, epilepsy, neuroinflammation, and pain. Recently there has been an interest in CBD as a possible treatment for age-related disorders such as Alzheimer's disease and related disorders (ADRD). Here we tested the hypothesis that chronic CBD administration would improve learning and memory in the SAMP8 mouse model of Alzheimer's disease. SAMP8 mice aged 11 months (at the start of the study) were administered vehicle or CBD (3 or 30 mg/Kg) daily via oral gavage for 2 months. Vehicle-treated young SAMP8 mice (age 3 months at the start of the study) served as unimpaired controls. After 30 days of treatment (4 and 12 months of age), learning and memory, activity, anxiety, strength and dexterity were assessed. High dose CBD treatment significantly improved learning and memory of the 12-month-old mice in the T maze. Novel object recognition memory was also improved by CBD in aged CBD treated mice. Aged CBD treated mice also displayed less anxiety in the elevated plus maze test compared to controls. However, activity and strength levels were similar between groups. Biochemical analysis revealed decreased markers of oxidative stress, providing a possible mechanism by which CBD treatment impacts learning, memory, and anxiety. These results highlight the potential use of CBD as a therapeutic for age related cognitive impairment and dementia.

Abstract Background: TNBC patients with residual disease after neoadjuvant chemotherapy (NAC) have high recurrence rates. Targetable mechanisms likely responsible for NAC resistance must therefore be identified to identify new therapeutic options. Alterations in the PI3K/mTOR pathway as well as expression of the immune checkpoint PD-L1 have emerged as potential targets, with significant frequency of alteration in TNBC. Importantly, the AKT inhibitor ipatasertib (ipat) and the anti-PD-L1 antibody atezolizumab (atezo) have demonstrated activity against TNBC. Recent data suggests that the presence of circulating tumor cell-free DNA (cfDNA) following NAC correlates with residual disease and a higher recurrence risk. We have hypothesized that combination therapy with ipat and atezo will target micrometastatic disease, as determined by the presence of cfDNA after NAC, in TNBC patients. Trial design:Open label single-arm phase II study to evaluate combination therapy with ipat and atezo, in TNBC patients with detectable cfDNA after completion of NAC, definitive surgery, and adjuvant radiation and/or chemotherapy. Eligible patients will receive: atezo [840mg IV days 1 and 15 and ipat [400 mg orally daily on days 1-21, followed by one week off] in a 28-day cycle for 6 cycles; cfDNA will be evaluated after 3 and 6 cycles. Biomarkers including PD-L1 expression on tumor cells or infiltrating immune cells in the primary tumor or PD-L1 expression on circulating tumor cells will be assessed. Eligibility criteria:Patients ≥ 18 yrs of age with pathologically confirmed residual invasive TNBC (ER and PR negative defined as <10% of cells expressing ER/PR by local assessment; HER2 negative according to ASCO/CAP guidelines) following NAC with evidence of cfDNA after completion of all local and systemic neoadjuvant and adjuvant therapy. Patients must enroll within 12 months of last therapy (definitive breast surgery, radiation and/or all intended adjuvant therapy). Prior treatment with immunotherapeutic agents is allowed. Specific aims:The primary objective is to evaluate the efficacy of 6 cycles of ipat + atezo in reducing micrometastatic disease (detectable cfDNA) in patients with residual breast and/or axillary disease after NAC and completion of all locoregional and/or systemic adjuvant therapy. Secondary objectives include: evaluating efficacy of ipat + atezo in reducing micrometastatic disease after 3 cycles; determining the recurrence risk after treatment with ipat + atezo; and determining the safety and tolerability of the combination. Correlative objectives include determining whether: 1) pretreatment circulating markers (mutations or copy number changes in PTEN/PI3K/AKT) are associated with response; 2) PD-L1 expression on tumor cells or infiltrating tumor cells in the primary tumor is associated with response; 3) PD-L1 expression on circulating tumor cells has utility as a pharmacodynamic biomarker; and 4) stool microbiome profiles are associated with response and/or survival. As an exploratory objective, patient attitudes and experience surrounding testing for tumor cfDNA and, for those testing positive, participation in a trial targeting cfDNA, will be assessed. Statistical methods:The primary objective is to determine the response rate defined as the proportion of patients with detectable cfDNA who become undetectable. We anticipate that 30% of patients screened will be tumor cfDNA-positive, thus anticipate screening ~ 120 patients to enroll 40. With 40 patients enrolled (assuming a one-sided alpha of 0.05), we will have 80% power to detect a 19.0% (81% positive versus 93% positive) clearance rate using a one-sample binomial exact test. Target Accrual:40 patients Contact:A. DeMichele (angela.demichele@pennmedicine.upenn,edu) Clinicaltrials.gov #: NCT04434040 Citation Format: Elizabeth Mittendorf, Sara Tolaney, Paul Wileyto, Michelle DeMeo, Hope Rugo, Rita Nanda, Ingrid Mayer, Ben Park, Heather MacArthur, Angela DeMichelle. Combination ipatasertib and atezolizumab to prevent recurrence in triple negative breast cancer(TNBC): A phase II single arm trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-33-01.

While vascular tone is affected by multiple mediators released from various component tissues, it has been a long‐held belief that the mechanical act of vascular smooth muscle contraction induces ATP release regardless of the stimulus that initiated vasoconstriction. Contrary to this theory, here we present evidence that the release of ATP by vascular smooth muscle is both agonist‐ and sex‐dependent. By utilizing a near‐real time luminometric technique developed by our lab, we found that while methoxamine, 5‐hydroxytryptamine, and angiotensin II all induced vasoconstriction in the rat mesenteric arterial bed, only angiotensin II caused ATP release. Furthermore, this response was only seen in preparations from male rats, whereas those from female rats showed no ATP release in response to any of the three vasoactive agents tested, indicating a sex difference in this process.

The control of vascular caliber relies on mediators released from multiple components of the vascular neuroeffector junction. These mediators include ATP, which is released from the sympathetic nerves from vesicles that also contain norepinephrine, as well as from the vascular smooth muscle (VSM) via pannexin-1 (panx-1) channels. We hypothesize that VSM-derived ATP plays a critical role in balancing the vasoactive effects of mediators at the vascular neuroeffector junction. Panx-1 channel opening on VSM has been reported to be stimulated by caspase cleavage, cytoplasmic Ca 2+ , and extracellular K + , but the effects of VSM GPCR activation are not as well characterized. Here we utilize a near-real time luminometric technique to quantify the release of ATP from the rat mesenteric arterial bed in response to vasoconstrictor agonists. We observed disparate effects of three physiologically relevant vasoactive agents, and these do not correlate with the relative changes in perfusion pressure. Whereas angiotensin II caused a robust release of ATP (228.6 ± 31.94 nM), no significant release was observed with either methoxamine (an α 1 agonist; 44.41 ± 31.94 nM) or 5-HT (0 ± 0 nM) . These results indicate that GPCR-mediated smooth muscle contraction is not sufficient to induce ATP release in this vascular bed. This is an important first step toward understanding the role of ATP in the vasculature.

Central neuropathic pain (CNP) a significant problem for many people, is not well-understood and difficult to manage. Dysfunction of the central noradrenergic system originating in the locus coeruleus (LC) may be a causative factor in the development of CNP. The LC is the major noradrenergic nucleus of the brain and plays a significant role in central modulation of nociceptive neurotransmission. Here, we examined CNS pathophysiological changes induced by intraperitoneal administration of the neurotoxin DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bro mobenzylamine hydrochloride). Administration of DSP-4 decreased levels of norepinephrine in spinal tissue and cerebrospinal fluid (CSF) and led to the development of thermal and mechanical hyperalgesia over 21 days, that was reversible with morphine. Hyperalgesia was accompanied by significant increases in noradrenochrome (oxidized norepinephrine) and expression of 4-hydroxynonenal in CSF and spinal cord tissue respectively at day 21, indicative of oxidative stress. In addition, spinal levels of pro-inflammatory cytokines (interleukins 6 and 17A, tumor necrosis factor-alpha), as well as the anti-inflammatory cytokine interleukin10 were also significantly elevated at day 21, indicating that an inflammatory response occurred. The inflammatory effect of DSP-4 presented in this study that includes oxidative stress may be particularly useful in elucidating mechanisms of CNP in inflammatory disease states. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Melanocortin neurons conserve body mass in hyper-or hypo-caloric conditions by conveying signals from nutrient sensors into areas of the brain governing appetite and metabolism. In mice, melanocortin-3 receptor (MC3R) deletion alters nutrient partitioning independently of hyperphagia, promoting accumulation of fat over muscle mass. Enhanced rhythms in insulin and insulin-responsive metabolic genes during hypocaloric feeding suggest partial insulin resistance and enhanced lipogenesis. However, exactly where and how MC3Rs affect metabolic control to alter nutrient partitioning is not known. The behavioral phenotypes exhibited by MC3R-deficient mice suggest a contextual role in appetite control. The impact of MC3R-deficiency on feeding behavior when food is freely available is minor. However, homeostatic responses to hypocaloric conditioning involving increased expression of appetite-stimulating (orexigenic) neuropeptides, binge-feeding, food anticipatory activity (FAA), entrainment to nutrient availability and enhanced feeding-related motivational responses are compromised with MC3R-deficiency. Rescuing Mc3r transcription in hypothalamic and limbic neurons improves appetitive responses during hypocaloric conditioning while having minor effects on nutrient partitioning, suggesting orexigenic functions. Rescuing hypothalamic MC3Rs also restores responses of fasting-responsive hypothalamic orexigenic neurons in hypocaloric conditions, suggesting actions that sensitize fasting-responsive neurons to signals from nutrient sensors. MC3R signaling in ventromedial hypothalamic SF1(+ve) neurons improvesmetabolic control, but does not restore appetitive responses or nutrient partitioning. In summary, desensitization of fasting-responsive orexigenic neurons may underlie attenuated appetitive responses of MC3R-deficient mice in hypocaloric situations. Further studies are needed to identify the specific location(s) of MC3Rs controlling appetitive responses and partitioning of nutrients between fat and lean tissues.

Background: Sympathetic nerves are known to release three neurotransmitters: norepinephrine, ATP, and neuropeptide Y that play a role in controlling vascular tone. This paper focuses on the co-release of norepinephrine and ATP from the mesenteric arterial sympathetic nerves of the rat.New method: In this paper, a quantification technique is described that allows simultaneous detection of norepinephrine and ATP in a near-real-time fashion from the isolated perfused mesenteric arterial bed of the rat. Simultaneous detection is enabled with 3-D printing technology, which is shown to help integrate the perfusate with different detection methods (norepinephrine by microchip-based amperometery and ATP by on-line chemiluminescence).Results: Stimulated levels relative to basal levels of norepinephrine and ATP were found to be 363 nM and 125 nM, respectively (n = 6). The limit of detection for norepinephrine is 80 nM using microchip-based amperometric detection. The LOD for on-line ATP detection using chemiluminescence is 35 nM.Comparison with existing method: In previous studies, the co-transmitters have been separated and detected with HPLC techniques. With HPLC, the samples from biological preparations have to be derivatized for ATP detection and require collection time before analysis. Thus real-time measurements are not made and the delay in analysis by HPLC can cause degradation.Conclusions: In conclusion, the method described in the paper can be used to successfully detect norepinephrine and ATP simultaneously and in a near-real-time fashion. (C) 2016 Elsevier B.V. All rights reserved.

Objective: Appetitive responses to weight loss are mediated by a nutrient-sensing neural network comprised of melanocortin neurons. The role of neural melanocortin-3 receptors (MC3R) in mediating these responses is enigmatic. Mc3r knockout mice exhibit a paradoxical phenotype of obesity and reduced feeding-related behaviors in situations of nutrient scarcity. Here we examined whether MC3Rs expressed in mesolimbic neurons regulate feeding-related motivational responses.Methods: Interactions between Mc3r genotype, cognitive function and energy balance on food self-administration were assessed using operant conditioning with fixed-and progressive ratio (FR1/PR1) settings. Inhibition of Mc3r transcription by a loxP-flanked transcriptional blocker (TB) in C57BL/6JN mice (Mc3rTB/TB) was reversed in mesolimbic neurons using DAT-Cre (DAT-MC3R).Results: Caloric restriction (CR) caused 10-15% weight loss and increased motivation to acquire food rewards during training sessions. c-Fos-expression in the nucleus accumbens was increased 1 h following food presentation. While exhibiting weight loss, total food self-administration, enhanced motivation to self-administer food rewards in training sessions held during CR and c-Fos-activation in the nucleus accumbens following re-feeding were all markedly attenuated in Mc3rTB/TB mice. In contrast, cognitive abilities were normal in Mc3rTB/TB mice. Total food self-administration during FR1 sessions was not rescued in DAT-MC3R mice, however enhanced motivational responses to self-administer food rewards in PR1 conditions were restored. The nutrient-partitioning phenotype observed with Mc3r-deficiency was not rescued in DAT-MC3R mice.Conclusions: Mesolimbic MC3Rs mediate enhanced motivational responses during CR. However, they are insufficient to restore normal caloric loading when food is presented during CR and do not affect metabolic conditions altering nutrient partitioning. (C) 2016 The Author(s). Published by Elsevier GmbH.

Parkinson's disease (PD) is characterized by progressive neurodegeneration of nigrastriatal dopaminergic neurons leading to clinical motor dysfunctions. Many animal models of PD have been developed using exogenous neurotoxins and pesticides. Evidence strongly indicates that the dopaminergic neurons of the substantia nigra pars compacta (SNpc) are highly susceptible to neurodegeneration due to a number of factors including oxidative stress and mitochondrial dysfunction. Oxidation of DA to a potential endogenous neurotoxin, dopaminochrome (DAC), may be a potential contributor to the vulnerability of the nigrostriatal tract to oxidative insult. In this study, we show that DAC causes slow and progressive degeneration of dopaminergic neurons in contrast to 1-methyl-4-phenylpyridinium (MPP(+)), which induces rapid lesions of the region. The DAC model may be more reflective of early stresses that initiate the progressive neurodegenerative process of PD, and may prove a useful model for future neurodegenerative studies.

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.

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.

Age-related increases in monoamine oxidase B (MAO-B) may contribute to neurodegeneration associated with Parkinson's disease (PD). The MAO-B inhibitor deprenyl, a long-standing antiparkinsonian therapy, is currently used clinically in concert with the dopamine precursor L-DOPA. Clinical studies suggesting that deprenyl treatment alone is not protective against PD associated mortality were targeted to symptomatic patients. However, dopamine loss is at least 60% by the time PD is symptomatically detectable, therefore lack of effect of MAO-B inhibition in these patients does not negate a role for MAO-B in pre-symptomatic dopaminergic loss. In order to directly evaluate the role of age-related elevations in astroglial MAO-B in the early initiation or progression of PD, we created genetically engineered transgenic mice in which MAO-B levels could be specifically induced within astroglia in adult animals. Elevated astrocytic MAO-B mimicking age related increase resulted in specific, selective and progressive loss of dopaminergic neurons in the substantia nigra (SN), the same subset of neurons primarily impacted in the human condition. This was accompanied by other PD-related alterations including selective decreases in mitochondrial complex I activity and increased mitochondrial oxidative stress. Along with a global astrogliosis, we observed local microglial activation within the SN. These pathologies correlated with decreased locomotor activity. Importantly, these events occurred even in the absence of the PD-inducing neurotoxin MPTP. Our data demonstrates that elevation of murine astrocytic MAO-B by itself can induce several phenotypes of PD, signifying that MAO-B could be directly involved in multiple aspects of disease neuropathology. Mechanistically this may involve increases in membrane permeant H(2)O(2) which can oxidize dopamine within dopaminergic neurons to dopaminochrome which, via interaction with mitochondrial complex I, can result in increased mitochondrial superoxide. Our inducible astrocytic MAO-B transgenic provides a novel model for exploring pathways involved in initiation and progression of several key features associated with PD pathology and for therapeutic drug testing.

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)

Parkinson's disease is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis-inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3 h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.

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)

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.