Use of chlorisondamine to assess the neurogenic contribution to blood pressure in mice: An evaluation of method.

Chlorisondamine (CSD) has been used to assess the neurogenic contribution to blood pressure (BP) and vasomotor sympathetic tone in animal models. It is assumed that the reduction in BP following CSD administration is associated to decreases in cardiac output (CO) and peripheral resistance, reflecting cardiac and vasomotor sympathetic tone, respectively. Surprisingly, this has not been characterized experimentally in mice, despite the extensive use of this animal model in cardiovascular research. We hypothesize that a specific dose of CSD can selectively block the sympathetic vasomotor tone. To test this hypothesis, we evaluated the effects of different doses of CSD (intraperitoneal) on BP and heart rate (HR) using telemetry, and on CO using echocardiography. BP and HR in normotensive C57Bl/6J mice reduced to a similar extent by all CSD doses tested (1-6 mg/kg). CSD at 6 mg/kg also reduced CO without affecting left ventricular stroke volume or fractional shortening. On the other hand, lower doses of CSD (1 and 2 mg/kg) produced significantly larger BP and HR reductions in DOCA-salt-induced hypertensive mice, indicating a greater neurogenic BP response. In addition, all doses of CSD reduced CO in hypertensive mice. Our data suggest that the BP response to CSD in mice likely reflects reduced CO and vasomotor sympathetic tone. We conclude that CSD can be used to assess the neurogenic contribution to BP in mice but may not be appropriate for specifically estimating vasomotor sympathetic tone.

Soluble Prorenin Receptor Increases Blood Pressure in High Fat-Fed Male Mice.

Obesity-related hypertension is a major public health concern. We recently demonstrated that plasma levels of the soluble form of the prorenin receptor (sPRR) were elevated in obesity-associated hypertension. Therefore, in the present study, we investigated the contribution of sPRR to blood pressure (BP) elevation in the context of obesity. High fat-fed C57BL/6 male mice were infused with vehicle or sPRR (30 µg/kg per day) via subcutaneously implanted osmotic minipump for 4 weeks. BP parameters were recorded using radiotelemetry devices. Male mice infused with sPRR exhibited higher systolic BP and mean arterial pressure and lower spontaneous baroreflex sensitivity than mice infused with vehicle. To define mechanisms involved in systolic BP elevation, mice were injected with an AT1R (Ang II [angiotensin II] type 1 receptor) antagonist (losartan), a muscarinic receptor antagonist (atropine), a ß-adrenergic antagonist (propranolol), and a ganglionic blocker (chlorisondamine). Losartan did not blunt sPRR-induced elevation in systolic BP. Chlorisondamine treatment exacerbated the decrease in mean arterial pressure in male mice infused with sPRR. These results demonstrated that sPRR induced autonomic nervous dysfunction. Interestingly, plasma leptin levels were increased in high fat-fed C57BL/6 male mice infused with sPRR. Overall, our results indicated that sPRR increased systolic BP through an impairment of the baroreflex sensitivity and an increase in the sympathetic tone potentially mediated by leptin in high fat-fed C57BL/6 male mice.

Role of α7-Nicotinic Acetylcholine Receptors of B Cells in the Immunotoxic Effect of Organophosphorus Compounds.

Experiments on white non-inbred rats demonstrated that treatment with organophosphorus compound dimethyl dichlorovinyl phosphate (DDVP) decreased T cell-independent antibody production by B cells and blood levels of IL-10 and IL-12; a similar effect was produced by GTS-21, a selective agonist of α7-nicotinic acetylcholine receptor. N-nicotinic receptor antagonist chlorisondamine in combination with DDVP partially prevented suppression of antibody production in comparison with the effect observed during intoxication with DDVP.

Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.

Cerebrospinal Fluid Hypernatremia Elevates Sympathetic Nerve Activity and Blood Pressure via the Rostral Ventrolateral Medulla.

Elevated NaCl concentrations of the cerebrospinal fluid increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. Neurons of the rostral ventrolateral medulla (RVLM) play a pivotal role in the regulation of SNA and receive mono- or polysynaptic inputs from several hypothalamic structures responsive to hypernatremia. Therefore, the present study investigated the contribution of RVLM neurons to the SNA and pressor response to cerebrospinal fluid hypernatremia. Lateral ventricle infusion of 0.15 mol/L, 0.6 mol/L, and 1.0 mol/L NaCl (5 µL/10 minutes) produced concentration-dependent increases in lumbar SNA, adrenal SNA, and arterial blood pressure, despite no change in splanchnic SNA and a decrease in renal SNA. Ganglionic blockade with chlorisondamine or acute lesion of the lamina terminalis blocked or significantly attenuated these responses, respectively. RVLM microinjection of the gamma-aminobutyric acid (GABAA) agonist muscimol abolished the sympathoexcitatory response to intracerebroventricular infusion of 1 mol/L NaCl. Furthermore, blockade of ionotropic glutamate, but not angiotensin II type 1, receptors significantly attenuated the increase in lumbar SNA, adrenal SNA, and arterial blood pressure. Finally, single-unit recordings of spinally projecting RVLM neurons revealed 3 distinct populations based on discharge responses to intracerebroventricular infusion of 1 mol/L NaCl: type I excited (46%; 11/24), type II inhibited (37%; 9/24), and type III no change (17%; 4/24). All neurons with slow conduction velocities were type I cells. Collectively, these findings suggest that acute increases in cerebrospinal fluid NaCl concentrations selectively activate a discrete population of RVLM neurons through glutamate receptor activation to increase SNA and arterial blood pressure.

Chlorisondamine, a sympathetic ganglionic blocker, moderates the effects of whole-body irradiation (WBI) on early host defense to a live bacterial challenge.

There is a growing consensus that long-term deficits in the brain are due to dynamic interactions between multiple neural and immune cell types. Specifically, radiation induces an inflammatory response, including changes in neuromodulatory pro- and anti-inflammatory cytokine secretion. The purpose of this study was to establish that there is sympathetic involvement in radiation-induced decrements early in in vivo immune function host defense. Female, 8-9 week-old C57BL/6J mice were exposed to whole-body irradiation (WBI). There were 8 groups with radiation (0 vs. 3 Gy protons), immune challenge (Escherichia coli) and exposure to the sympathetic ganglionic blocker, chlorisondamine (1 mg/kg weight, i.p.), as independent variables. Ten days post-irradiation, mice were inoculated with E. coli intraperitoneally and sacrificed 90-120 min later. The data suggest that radiation-induced changes in immune function may in part be mediated by the sympathetic nervous system. Briefly, we found that radiation augments the bacteria-induced inflammatory cytokine response, particularly those cytokines involved in innate immunity. However, this augmentation can be reduced by the ganglionic blockade.

Prevention of hemolysis-induced organ damage by nutritional activation of the vagal anti-inflammatory reflex*.

OBJECTIVES: Acute hemolysis is associated with organ damage, inflammation, and impaired vascular function. Stimulation of the cholecystokinin-1 receptor-dependent vagal anti-inflammatory reflex with lipid-rich enteral nutrition was demonstrated to prevent tissue damage and attenuate inflammation. This study investigates the effects of nutritional activation of the vagal anti-inflammatory reflex on organ integrity, systemic inflammation, and microcirculation during hemolysis. DESIGN: Prospective randomized controlled study. SETTING: University research unit. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Intravascular hemolysis was simulated by infusion of prelysed erythrocytes. Animals were fasted or received lipid-rich enteral nutrition. Pegylated (PEG)-CCK9A, A70104 (a cholecystokinin-1 receptor antagonist), and chlorisondamine (a nicotinic acetylcholine receptor antagonist) were applied to investigate involvement of the vagal reflex. MEASUREMENTS AND MAIN RESULTS: Nutritional intervention reduced hemolysis-related renal tubular cell damage, hepatocyte damage, ileal leakage of horseradish peroxidase, and bacterial translocation compared with food deprivation (all p < 0.05). Also circulating interleukin (IL)-6 levels were decreased by enteral nutrition (p < 0.05). Blockage of the cholecystokinin-1 receptor or the nicotinic acetylcholine receptor reversed the protective nutritional effects compared with vehicle (p < 0.05), whereas PEG-CCK9 mimicked the impact of enteral feeding in fasted animals (p < 0.05). Furthermore, nutritional intervention increased renal, hepatic, and intestinal blood flow compared with fasting (all p < 0.05), as evaluated using fluorescent microspheres. CONCLUSIONS: Nutritional activation of the vagal anti-inflammatory reflex preserves tissue integrity and attenuates systemic inflammation in a rodent model of acute hemolysis. In addition, lipid-rich nutrition improves renal, hepatic, and intestinal microcirculation. These findings implicate stimulation of the autonomic nervous system by nutritional means as a potential therapy to prevent complications of acute hemolysis. (Crit Care Med 2013; 41:e361-e367).

Neural control of arterial pressure variability in the neuromuscularly blocked rat.

The baroreflexes stabilize moment-to-moment arterial pressure. Sinoaortic denervation (SAD) of the baroreflexes results in a large increase in arterial pressure variability (APV) across various species. Due to an incomplete understanding of the nonlinear interactions between central and peripheral systems, the major source of APV remains controversial. While some studies suggested that the variability is endogenous to the central nervous system (CNS), others argued that peripheral influences may be the main source. For decades, abnormal cardiovascular variability has been associated with a number of cardiovascular diseases including hypertension, heart failure, and stroke. Delineating mechanisms of the APV is critical for the improvement of current strategies that use APV as a clinical tool for the diagnosis and prognosis of cardiovascular diseases. In this study, with a unique chronic neuromuscularly blocked (NMB) rat preparation that largely constrains peripheral influences, we determined the CNS contribution to the post-SAD APV. First, we confirmed that SAD significantly increased APV in the NMB rat, then demonstrated that post-SAD ganglionic blockade substantially reduced APV, and subsequent intravenous infusions of phenylephrine and epinephrine (in presence of ganglionic blockade) only slightly increased APV. These data suggest that the CNS is an important source, and skeletal activity, thermal challenges or other forms of peripherally generated cardiovascular stress are not required for the post-SAD APV. In addition, we showed that bilateral aortic denervation produced a larger increase in APV than bilateral carotid sinus denervation, suggesting that the aortic baroreflex plays a more dominant role in the control of APV than the carotid sinus.

Drug metabolism within the brain changes drug response: selective manipulation of brain CYP2B alters propofol effects.

Drug-metabolizing cytochrome P450 (CYPs) enzymes are expressed in the liver, as well as in extrahepatic tissues such as the brain. Here we show for the first time that drug metabolism by a CYP within the brain, illustrated using CYP2B and the anesthetic propofol (2, 6-diisopropylphenol, Diprivan), can meaningfully alter the pharmacological response to a CNS acting drug. CYP2B is expressed in the brains of animals and humans, and this CYP isoform is able to metabolize centrally acting substrates such as propofol, ecstasy, and serotonin. Rats were given intracerebroventricularly (i.c.v.) injections of vehicle, C8-xanthate, or 8-methoxypsoralen (CYP2B mechanism-based inhibitors) and then tested for sleep time following propofol (80 mg/kg intraperitoneally). Both inhibitors significantly increased sleep-time (1.8- to 2-fold) and brain propofol levels, while having no effect on plasma propofol levels. Seven days of nicotine treatment can induce the expression of brain, but not hepatic, CYP2B, and this induction reduced propofol sleep times by 2.5-fold. This reduction was reversed in a dose-dependent manner by i.c.v. injections of inhibitor. Sleep times correlated with brain (r=0.76, P=0.0009), but not plasma (r=0.24, P=0.39) propofol concentrations. Inhibitor treatments increased brain, but not plasma, propofol levels, and had no effect on hepatic enzyme activity. These data indicate that brain CYP2B can metabolize neuroactive substrates (eg, propofol) and can alter their pharmacological response. This has wider implications for localized CYP-mediated metabolism of drugs, neurotransmitters, and neurotoxins within the brain by this highly variable enzyme family and other CYP subfamilies expressed in the brain.

Lipid-enriched enteral nutrition controls the inflammatory response in murine Gram-negative sepsis.

OBJECTIVES: Controlling the inflammatory cascade during sepsis remains a major clinical challenge. Recently, it has become evident that the autonomic nervous system reduces inflammation through the vagus nerve. The current study investigates whether nutritional stimulation of the autonomic nervous system effectively attenuates the inflammatory response in murine Gram-negative sepsis. DESIGN: Controlled in vivo and ex vivo experimental study. SETTINGS: Research laboratory of a university hospital. SUBJECTS: Male C57bl6 mice. INTERVENTIONS: Mice were intraperitoneally challenged with lipopolysaccharide derived from Escherichia coli. Before lipopolysaccharide administration, mice were fasted or enterally fed either lipid-rich nutrition or low-lipid nutrition. Antagonists to cholecystokinin receptors or nicotinic receptors were administered before lipopolysaccharide administration. Blood and tissue samples were collected at 90 mins. Mesenteric afferent discharge was determined in ex vivo preparations in response to both nutritional compositions. MEASUREMENTS AND MAIN RESULTS: Both lipid-rich and low-lipid nutrition dose-dependently reduced lipopolysaccharide-induced tumor necrosis factor-α release (high dose: both 1.4 ± 0.4 ng/mL) compared with fasted mice (3.7 ± 0.8 ng/mL; p < .01). The anti-inflammatory effect of both nutritional compositions was mediated through cholecystokinin receptors (p < .01), activation of mesenteric vagal afferents (p < .05), and peripheral nicotinic receptors (p < .05). Lipid-rich nutrition attenuated the inflammatory response at lower dosages than low-lipid nutrition, indicating that enrichment of enteral nutrition with lipid augments the anti-inflammatory potential. Administration of lipid-rich nutrition prevented endotoxin-induced small intestinal epithelium damage and reduced inflammation in the liver and spleen compared with fasted (all p < .01) and low-lipid nutrition controls (all p < .05). CONCLUSIONS: The current study demonstrates that lipid-rich nutrition attenuates intestinal damage and systemic as well as organ-specific inflammation in murine Gram-negative sepsis through the nutritional vagal anti-inflammatory pathway. These findings implicate enteral administration of lipid-enriched nutrition as a promising intervention to modulate the inflammatory response during septic conditions.

Rat brain CYP2B induction by nicotine is persistent and does not involve nicotinic acetylcholine receptors.

CYP2B is a drug-metabolizing enzyme expressed in the liver and brain that metabolizes a variety of centrally acting drugs (e.g. propofol, bupropion and nicotine), endogenous neurochemicals (e.g. serotonin and testosterone) and toxins (e.g. chlorpyrifos). Human CYP2B6 is found at higher levels in the brains of smokers, and 7-day nicotine treatment induces rat brain CYP2B while not altering hepatic CYP2B. We characterized the time course of rat brain CYP2B induction by nicotine and determined if nicotinic acetylcholine receptors (nAChRs) mediated this induction. Rats were treated once daily with 1mg/kg nicotine base or saline s.c. for 1 or 7days and sacrificed from 30minutes to 7days after the last injection. One-day nicotine treatment did not induce brain CYP2B, whereas 7-day nicotine treatment significantly increased CYP2B expression for up to 24hours in the frontal cortex and brainstem; these levels returned to baseline by 7days post-treatment. CYP2B activity was also higher at 24hours in these regions. No change was seen in the cerebellar CYP2B levels or in vivo activity following nicotine treatment. Time of day of treatment and sacrifice altered the magnitude of brain CYP2B induction while chlorisondamine, an irreversible nAChR blocker, pre-treatment did not block CYP2B induction. Seven-day nicotine treatment resulted in an induction of rat brain CYP2B protein and in vivo activity for up to 24hours, which would suggest greater local drug metabolism by brain CYP2B. Humans or animals exposed to nicotine may have altered therapeutic drug response, brain levels of neurotransmitters and/or neurotoxicity.

Prenatal high salt programs enhanced sympathoadrenal activation of the cardiovascular response to restraint.

We recently reported that feeding Sprague Dawley rats a high-salt diet during pregnancy programmed an exaggerated pressor and tachycardic response to restraint in adult female offspring. In the present investigation, a pharmacologic approach was used to determine the contribution of the sympathoadrenal system to the exaggerated response. Injection of a cocktail containing a ganglionic blocker (chlorisondamine) and a beta-adrenoceptor antagonist (propranolol) prevented the stress-induced tachycardia and increase in blood pressure and abolished the difference between high-salt and normal-salt offspring. These data suggest that the prenatal high salt programmed a sympathoadrenal hyperresponsiveness to restraint stress.

Effects of dipeptidyl peptidase iv inhibition on arterial blood pressure.

1. The aim of the present study was to determine whether inhibition of dipeptidyl peptidase IV (DPP IV) elevates arterial blood pressure and whether any such effect is dependent on genetic background, the sympathetic nervous system and Y(1) receptors. The rationale behind this study was that: (i) neuropeptide (NP) Y(1-36) and peptide YY(1-36) (PYY(1-36)) are endogenous Y(1) receptor agonists and are metabolised by DPP IV to NPY(3-36) and PYY(3-36), which are not Y(1) but rather selective Y(2) receptor agonists; (ii) Y(1) receptors mediate vasoconstriction, whereas Y(2) receptors have little effect on vascular tone; (iii) vaso-constrictor effect of the Y(1) receptor is enhanced in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) rats; and (iv) NPY(1-36) is released from sympathetic nerve terminals. 2. We examined the effects of acute administration of 3-N-[(2S,3S)-2-amino-3-methylpentanoyl]-1,3-thiazolidine (P32/98; a DPP IV inhibitor) on arterial blood pressure in anaesthetized adult SHR and WKY rats in the absence and presence of either captopril, hydralazine or chlorisondamine to lower basal mean arterial blood pressure (MABP) by different mechanisms (inhibition of angiotensin-converting enzyme, direct vasodilation and ganglionic blockade, respectively). 3. In naïve SHR with severely elevated basal blood pressures (MABP = 176 +/- 3 mmHg; n = 4), i.v. boluses (1, 3 and 10 mg/kg) of P32/98 did not affect blood pressure. 4. When basal blood pressure was reduced by pretreatment of SHR with either captopril (30 mg/kg, i.v.; MABP = 116 +/- 3 mmHg; n = 9) or hydralazine (5 mg/kg, i.p.; MABP = 84 +/- 3 mmHg; n = 7), P32/98 (1, 3 and 10 mg/kg) caused significant dose-related increases in arterial blood pressure (4 +/- 2, 10 +/- 2 and 12 +/- 3 mmHg in the captopril-pretreated group, respectively (P < 0.01); 5 +/- 2, 8 +/- 3 and 11 +/- 4 mmHg in the hydralazine-pretreated group, respectively (P < 0.01)). 5. The increases in arterial blood pressure induced by P32/98 in captopril- or hydralazine-pretreated SHR were entirely blocked by pretreatment with the selective Y(1) receptor antagonist N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine amide (BIBP 3226; 6 mg/kg per h). 6. When basal blood pressure was reduced in SHR by pretreatment with chlorisondamine (10 mg/kg, s.c.; MABP = 108 +/- 4 mmHg; n = 7), inhibition of DPP IV with P32/98 did not affect arterial blood pressure. Basal heart rate in chlorisondamine-treated SHR was significantly reduced compared with naïve SHR, captopril-pretreated SHR and hydralazine-pretreated SHR, indicating effectiveness of ganglionic blockade. 7. Unlike the results in genetically hypertensive animals, in normotensive WKY rats pretreated with captopril (30 mg/kg, i.v.; MABP = 81 +/- 4 mmHg; n = 6), or hydralazine (5 mg/kg, i.p.; MABP = 63 +/- 4 mmHg; n = 4) or chlorisondamine (10 mg/kg, s.c.; MABP = 63 +/- 4 mmHg; n = 5), P32/98 did not affect arterial blood pressure. 8. We conclude that, in genetically susceptible animals, inhibition of DPP IV increases arterial blood pressure via Y(1) receptors when elevated blood pressure is reduced with antihypertensive drugs provided that the sympathetic nervous system is functional. The results suggest vigilance because DPP IV inhibitors are used more widely in hypertensive patients treated with antihypertensive drugs.

The effect of the cholinergic anti-inflammatory pathway on experimental colitis.

Inflammatory bowel diseases (IBD) are characterized by proinflammatory cytokines, tissue damage and loss of neuron in inflamed mucosa, which implies the cholinergic anti-inflammatory pathway may be destroyed during the process of inflammatory response. In the study, we identified the effect of cholinergic agonist as anabaseine (AN) and nicotinic receptor antagonist as chlorisondamine diiodide (CHD) on trinitrobenzene sulfonic acid (TNBS)-induced colitis, to investigate the potential therapeutic effect of the cholinergic anti-inflammatory pathway on IBD. Experimental colitis was induced by TNBS at day 1, 10 mug AN or 1.5 mug CHD was injected i.p. to mouse right after the induction of colitis, and repeated on interval day till the mice were sacrificed at day 8. Colonic inflammation was examined by histological analysis, myeloperoxidase (MPO) activity, and the production of tumour necrosis factor (TNF)-alpha in tissue. Lamina propria mononuclear cells (LPMC) were isolated, and NF-kappaB activation was detected by western blot. The mice with colitis treated by AN showed less tissue damage, less MPO activity, less TNF-alpha production in colon, and inhibited NF-kappaB activation in LPMC, compared with those mice with colitis untreated, whereas the mice with colitis treated by CHD showed the worst tissue damage, the highest MPO activity, the highest TNF-alpha level, and enlarged NF-kappaB activation in LPMC. Agonist of the cholinergic anti-inflammatory pathway inhibits colonic inflammatory response by downregulating the production of TNF-alpha, and inhibiting NF-kappaB activation, which suggests that modulating the cholinergic anti-inflammatory pathway may be a new potential management for IBD.

Chlorisondamine inhibits the nicotine-induced stimulation of c-fos in the pigeon brain for up to 2 weeks.

Chlorisondamine is a charged molecule that acts as long-acting nicotinic antagonist in many species, including pigeon. Evidence indicates that, despite the charged nature of chlorisondamine, it blocks some central effects of nicotine. The present study examined the time course of chlorisondamine's blockade of nicotine-induced c-fos expression in the pigeon brain. Chlorisondamine's central blockade was examined from 1 hr to 28 days prior to nicotine administration. Nicotine stimulated increases in c-fos mRNA in the hippocampus, hyperstriatum accessorium, hyperstriatum ventrale, nucleus accumbens, bulbus olfactorius, paleostriatum augmentatum, and stratum griseum et fibrosum superficiale. Nicotinic receptors labeled by [(125)I]-epibatidine were not always found in the same regions as nicotine-induced increases in c-fos expression. Acute chlorisondamine increased the level of c-fos mRNA in the cerebellum, hippocampus, hyperstriatum accessorium, locus parolfactorius, nucleus accumbens, tectum opticum, paleostriatum augmentatum, and stratum griseum et fibrosum superficiale but had no effect on its own 24 hr after administration. Chlorisondamine blocked nicotine-induced increases in c-fos RNA for 4 days in the nucleus accumbens, a week in the bulbus olfactorius, and 2 weeks in the stratum griseum et fibrosum superficiale. The time course of chlorisondamine's blockade of nicotine-induced c-fos expression is consistent with the time course of the ability of chlorisondamine to block behavioral and physiological responses to nicotine.

Long- and short-term changes in the neuroimmune-endocrine parameters following inhalation exposures of F344 rats to low-dose sarin.

Inhalation of subclinical doses of sarin suppresses the antibody-forming cell (AFC) response, T-cell mitogenesis, and serum corticosterone (CORT) levels, and high doses of sarin cause lung inflammation. However, the duration of these changes is not known. In these studies, rats were exposed to a subclinical dose of sarin (0.4 mg/m3/h/day) for 1 or 5 days, and immune and inflammatory parameters were assayed up to 8 weeks before sarin exposure. Our results showed that the effects of a 5-day sarin exposure on the AFC response and T-cell receptor (TCR)-mediated Ca2+ response disappeared within 2-4 weeks after sarin exposure, whereas the CORT and adrenocorticotropin hormone (ACTH) levels remained significantly decreased. Pretreatment of rats with chlorisondamine attenuated the effects of sarin on the AFC and the TCR-mediated Ca2+ response, implicating the autonomic nervous system (ANS) in the sarin-induced changes in T-cell function. Moreover, exposure to a single or five repeated subclinical doses of sarin upregulated the mRNA expression of proinflammatory cytokines in the lung, which is associated with the activation of NFkappaB in bronchoalveolar lavage cells. These effects were lost within 2 weeks of sarin inhalation. Our results suggest that while sarin-induced changes in T cells and cytokine gene expression were short lived, suppression of CORT and ACTH levels were relatively long lived and might represent biomarkers of sarin exposure. Moreover, while the effects of sarin on T-cell function were regulated by the ANS, the decreased CORT levels by sarin might result from its effects on the hypothalamus-pituitary-adrenal axis.

Effect of anabasine on catecholamine secretion from the perfused rat adrenal medulla.

OBJECTIVES: The present study was designed to investigate the characteristic effects of anabasine on secretion of catecholamines (CA) from the isolated perfused rat adrenal gland and to establish its mechanism of adrenomedullary secretion. METHODS: The adrenal gland was isolated by a modification of the Wakade method, and perfused with normal Krebs-bicarbonate solution. The content of CA was measured using fluorometry. RESULTS: The perfusion of anabasine(30-300 microM) into an adrenal vein for 60 min resulted in great increases in CA secretions in a dose-dependent fashion. Upon repeated injection of anabasine (100 microM) at 120 min-intervals, CA secretion was rapidly decreased after the third injection of anabasine. However, there was no statistical difference between the CA secretory responses of both 1st and 2nd treated groups by the successive administration of anabasine at 120 min-intervals. Tachyphylaxis to the releasing effects of CA evoked by anabasine was observed by repeated administration. Therefore, in all subsequent experiments, anabasine was not administered successively more than twice at only 120 min-intervals. The CA-releasing effects of anabasine were depressed by pretreatment with chlorisondamine (selective neuronal nicotinic receptor antagonist, 1 microM), atropine (muscarinic receptor antagonist, 2 microM), nicardipine (L-type dihydropyridine Ca2+ channel blocker, 1 microM), TMB-8 (anti-releaser of intracellular Ca2 +, 30 microM), and perfusion of EGTA (Ca2+ chelator, 5 mM) plus Ca2+ -free medium. In the presence of anabasine (100 microM), the CA secretory responses induced by acetylcholine (5.32 mM), high K+ (direct membrane-depolarizer, 56 mM), DMPP(selective neuronal nicotinic receptor agonist, 10(-4) M), and McN-A-343 (selective muscarinic M1 receptor agonist, 10(-4) M) were maximally enhanced in the first 4 min. However, as time elapsed, these responses became more inhibited at later periods. Furthermore, the perfusion of nicotine (30 microM) into an adrenal vein for 60 min also caused a great increase in CA secretion, leading to peak response in the first 0-5 min period. In the presence of nicotine (30 microM), the CA secretory responses induced by acetylcholine, high K+, DMPP and McN-A-343 were also enhanced for the first 4min, but later reduced to less than the control release. CONCLUSIONS: Taken together, these experimental results indicate that anabasine affects rat adrenomedullary CA secretion in a calcium-dependent fashion. This facilitatory effect of anabasine may be mediated by activation of both cholinergic nicotinic and muscarinic receptors, which is relevant to both stimulation of Ca2+ influx into adrenomedullary chromaffin cells and Ca2+ release from cytoplasmic Ca2+ Anabasine may be less potent than nicotine in rat adrenomedullary CA secretion. Anabasine, in addition to nicotine, alkaloids present in tobacco smoke may be a risk factor in causing cardiovascular diseases.

Role of beta-endorphin, corticotropin-releasing hormone, and autonomic nervous system in mediation of the effect of chronic ethanol on natural killer cell cytolytic activity.

BACKGROUND: We have recently shown that alcohol feeding suppresses natural killer (NK) cell cytolytic activity partly by decreasing the function of hypothalamic beta-endorphin (beta-EP) neurons. The neuronal mechanism by which hypothalamic beta-EP communicates with the spleen to regulate the action of ethanol on NK cells is not known. In the present study, we evaluated the roles of beta-EP neurons, corticotropin releasing hormone (CRH) neurons, and the autonomic nervous system (ANS) in regulation of the ethanol effect on splenic NK cell cytolytic function. METHODS: Male rats were fed an ethanol-containing liquid diet or control diets. These rats were used to determine the hormone release from the paraventricular nuclei (PVN) of the hypothalamus or used to determine the splenic NK cell cytolytic function after PVN administration of CRH or intraperitoneal (i.p.) administration of a ganglionic blocker chlorisondamine. The release of hormones from the PVN was measured using the push-pull perfusion method. Splenic cytolytic activity was determined using the 4-hour (51)Cr release assay against YAC-1 lymphoma target cells. RESULTS: Alcohol feeding decreased the amount of beta-EP but increased the amount of CRH in the push-pull perfusate (PPP) samples collected from the PVN. When exogenous beta-EP was perfused into the PVN, it suppressed the release of endogenous CRH found in PPP samples of the PVN. Conversely, perfusion of an opiate antagonist naltrexone into the PVN increased the levels of endogenous CRH in PPP samples of the PVN. In addition, administration of exogenous beta-EP in the PVN stimulated the cytolytic function of NK cells, an action that was antagonized by CRH as well as by ethanol. Corticotropin-releasing hormone and ethanol alone also had an inhibitory action on NK cells. Finally, the ganglionic blocker used prevented the effect that ethanol, beta-EP, and CRH had on NK cells. These data suggest that ethanol inhibits the function of NK cells partly by suppressing the influence of the beta-EP-CRH-ANS signal to the spleen.

Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.

The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited IkappaBalpha liver loss, blunted the augmented nuclear factor-kappaB activity, decreased hepatic tumor necrosis factor (TNF)-alpha mRNA (VGX + SAO + Sham STIM = 1.0 +/- 1.9 TNF-alpha/glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 +/- 0.2 TNF-alpha/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-alpha (VGX + SAO + Sham STIM = 118 +/- 19 pg/mL; VGX + SAO + STIM = 39 +/- 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 +/- 1 U/g tissue; VGX + SAO + STIM = 3.1 +/- 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 +/- 1.0 U/g tissue; VGX + SAO + STIM = 3.2 +/- 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-kappaB and TNF-alpha in SAO shock.