Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor.

BACKGROUND AND PURPOSE: Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH: The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS: Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS: Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin.

Blunted sympathoinhibitory responses in obesity-related hypertension are due to aberrant central but not peripheral signalling mechanisms.

The gut hormone cholecystokinin (CCK) acts at subdiaphragmatic vagal afferents to induce renal and splanchnic sympathoinhibition and vasodilatation, via reflex inhibition of a subclass of cardiovascular-controlling neurons in the rostroventrolateral medulla (RVLM). These sympathoinhibitory and vasodilator responses are blunted in obese, hypertensive rats and our aim in the present study was to determine whether this is attributable to (i) altered sensitivity of presympathetic vasomotor RVLM neurons, and (ii) aberrant peripheral or central signalling mechanisms. Using a diet-induced obesity model, male Sprague-Dawley rats exhibited either an obesity-prone (OP) or obesity-resistant (OR) phenotype when placed on a medium high fat diet for 13-15 weeks; control animals were placed on a low fat diet. OP animals had elevated resting arterial pressure compared to OR/control animals (P < 0.05). Barosensitivity of RVLM neurons was significantly attenuated in OP animals (P < 0.05), suggesting altered baroreflex gain. CCK induced inhibitory responses in RVLM neurons of OR/control animals but not OP animals. Subdiaphragmatic vagal nerve responsiveness to CCK and CCK1 receptor mRNA expression in nodose ganglia did not differ between the groups, but CCK induced significantly less Fos-like immunoreactivity in both the nucleus of the solitary tract and the caudal ventrolateral medulla of OP animals compared to controls (P < 0.05). These results suggest that blunted sympathoinhibitory and vasodilator responses in obesity-related hypertension are due to alterations in RVLM neuronal responses, resulting from aberrant central but not peripheral signalling mechanisms. In obesity, blunted sympathoinhibitory mechanisms may lead to increased regional vascular resistance and contribute to the development of hypertension.

The circulatory and renal sympathoinhibitory effects of gastric leptin are altered by a high fat diet and obesity.

Gastric leptin elicits its cardiovascular and splanchnic sympathoinhibitory responses via a vagal afferent mechanism, however the latter are blunted/abolished in animals fed a medium high fat diet (MHFD). In a diet-induced obesity model we sought to determine whether the renal sympathetic nerve discharge (RSND) and regional vasodilator responses to gastric leptin are also affected by diet and/or obesity. The diet induced obesity model was used in 2 separate studies. After 13 weeks on a MHFD the animals were classified as either obesity prone (OP) or obesity resistant (OR) depending on their weight gain. Control animals were fed a low fat diet for an equivalent period. Arterial pressure (AP) and heart rate (HR) were monitored in isoflurane-anaesthetised, artificially ventilated animals and RSND or regional vascular responses to leptin (15 µg/kg) administered close to the coeliac artery were evaluated. OP rats had higher baseline AP compared to control/OR rats (P<0.05). Close arterial leptin inhibited RSND in control animals but this response was abolished in OR and OP animals (P<0.01 for both). Leptin administration increased renal vascular conductance in control animals but this response was significantly attenuated only in OP animals (P<0.05). The vasodilator response in the superior mesenteric artery was not significantly different in any of the groups (P>0.05). Together these results suggest that, while the renal sympathoinhibitory responses to gastric leptin are affected by diet, the vasodilator responses to leptin in the renal vascular bed are only affected in OP animals. These changes may impact on cardiovascular homeostatic mechanisms in obesity.

Renal sympathoinhibitory and regional vasodilator responses to cholecystokinin are altered in obesity-related hypertension.

The gut and kidney command >50% of cardiac output postprandially, highlighting the importance of these vascular beds in cardiovascular homeostasis. The gastrointestinal peptide cholecystokinin (CCK) induces vagally mediated splanchnic sympathoinhibition that is attenuated in animals fed a medium high-fat diet (MHFD); therefore, our aim was to determine whether renal sympathetic nerve discharge (RSND) responses to CCK are also affected by this diet, and whether these changes are associated with obesity and hypertension. Another aim was to determine whether regional vasodilator responses to CCK are affected in obesity-related hypertension. In two separate studies, Sprague-Dawley rats were fed either a low-fat diet (LFD; control) or a MHFD for 13 weeks, after which MHFD animals were classified as obesity prone (OP) or obesity resistant (OR) based on their weight gain falling into the upper or lower tertile, respectively. Arterial pressure and heart rate were monitored in isoflurane-anaesthetized, artificially ventilated animals, and either RSND or regional vascular responses to CCK (0.1-8 µg kg(-1)) were evaluated. The OP rats had higher baseline arterial pressure compared with control/OR rats (P < 0.05). Administration of CCK inhibited RSND and increased renal vascular conductance in control/OR rats, and these responses were significantly blunted in OP rats (P < 0.05 for all). Baseline arterial pressure was positively correlated with weight gain and inversely correlated with CCK-induced vasodilatation (P < 0.05 for both). We hypothesize that in obesity-related hypertension, disruption of the sympathoinhibitory signals elicited by CCK reduces vasodilatation in the splanchnic/renal regions, leading to increased postprandial vascular resistance.

Sympathoinhibitory signals from the gut and obesity-related hypertension.

Several gastrointestinal hormones are commonly associated with satiety and digestion, but recent studies suggest they are also involved in regulating hemodynamic demand after a meal. These hormones released from the gut postprandially play a role in short-term cardiovascular regulation via a vagally mediated sympathoinhibitory reflex mechanism, similar to that of the arterial baroreflex. It has been hypothesized that activation of this reflex may promote greater blood flow to the splanchnic and renal vasculature that have increased haemodynamic demand after a meal, while simultaneously inducing vasoconstriction to the skeletomuscular vasculature where it is needed less. Together, the renal and splanchnic circulations can command over 50 % of cardiac output so that the role of gut hormones in controlling sympathetic vasomotor tone to these vascular beds may be more important in cardiovascular regulation than previously thought. The exact aetiology of obesity-related hypertension remains to be determined and is likely to be multifactorial, although the involvement of gut hormone signalling in the development of this disease has not previously been considered. Diets rich in fats and increased food intake are amongst the leading causes of obesity and precipitate significant changes such as inflammation in the gastrointestinal environment that can lead to blunted vagal afferent signalling. In obesity, these changes may disrupt sympathoinhibitory mechanisms and subsequently lead to increased vascular resistance in the gastrointestinal and renal vascular beds, contributing to the development of hypertension.

Sites of action of ghrelin receptor ligands in cardiovascular control.

Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.

Effects of nitric oxide synthase blockade on dorsal vagal stimulation-induced pancreatic insulin secretion.

We and others have previously shown that the dorsal motor nucleus of the vagus (DMV) is involved in regulation of pancreatic exocrine secretion. Many pancreatic preganglionic neurons within the DMV are inhibited by pancreatic secretagogues suggesting that an inhibitory pathway may participate in the control of pancreatic exocrine secretion. Accordingly, the present study examined whether chemical stimulation of the DMV activates the endocrine pancreas and whether an inhibitory pathway is involved in this response. All experiments were conducted in overnight fasted isoflurane/urethane-anesthetized Sprague Dawley rats. Activation of the DMV by bilateral microinjection of bicuculline methiodide (BIM, GABA(A) receptor antagonist, 100 pmol/25 nl; 4 mM) resulted in a significant and rapid increase in glucose-induced insulin secretion (9.2±0.1 ng/ml peak response) compared to control microinjection (4.0±0.6 ng/ml). Activation of glucose-induced insulin secretion by chemical stimulation of the DMV was inhibited (2.1±1.1 ng/ml and 1.6±0.1 ng/ml 5 min later) in the presence of the muscarinic receptor antagonist atropine methonitrate (100 µg/kg/min, i.v.). On the other hand, the nitric oxide (NO) synthesis inhibitor l-nitroarginine methyl ester (30 mg/kg, i.v.) significantly increased the excitatory effect of DMV stimulation on glucose-induced insulin secretion to 15.3±3.0 ng/ml and 16.1±3.1 ng/ml 5 min later. These findings suggest that NO may play an inhibitory role in the central regulation of insulin secretion.

High-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and cholecystokinin: implications for circulatory control.

Gastric leptin and cholecystokinin (CCK) act on vagal afferents to induce cardiovascular effects and reflex inhibition of splanchnic sympathetic nerve discharge (SSND) and may act cooperatively in these responses. We sought to determine whether these effects are altered in animals that developed obesity in response to a medium high-fat diet (MHFD). Male Sprague-Dawley rats were placed on a low-fat diet (LFD; n = 8) or a MHFD (n = 24) for 13 wk, after which the animals were anesthetized and artificially ventilated. Arterial pressure was monitored and blood was collected for the determination of plasma leptin and CCK. SSND responses to leptin (15 µg/kg) and CCK (2 µg/kg) administered close to the coeliac artery were evaluated. Collectively, MHFD animals had significantly higher plasma leptin but lower plasma CCK levels than LFD rats (P < 0.05), and this corresponded to attenuated or reversed SSND responses to CCK (LFD, -21 ± 2%; and MHFD, -12 ± 2%; P < 0.05) and leptin (LFD, -6 ± 2%; and MHFD, 4 ± 1%; P < 0.001). Alternatively, animals on the MHFD were stratified into obesity-prone (OP; n = 8) or obesity-resistant (OR; n = 8) groups according to their weight gain falling within the upper or lower tertile, respectively. OP rats had significantly higher resting arterial pressure, adiposity, and plasma leptin but lower plasma CCK compared with LFD rats (P < 0.05). The SSND responses to CCK or leptin were not significantly different between OP and OR animals. These results demonstrate that a high-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and CCK and may impact on sympathetic vasomotor mechanisms involved in circulatory control.

Dorsal vagal preganglionic neurons: differential responses to CCK1 and 5-HT3 receptor stimulation.

The dorsal motor nucleus of the vagus (DMV) is the main source of the vagal innervation of the pancreas. Several studies in vitro have demonstrated that the DMV consists of a heterogeneous population of preganglionic neurons but little is known about their electrophysiological characteristics in vivo. The aims of this study were to (i) identify DMV preganglionic neurons in vivo with axons in the pancreatic vagus and (ii) characterize their responses to stimulation of cholecystokinin (CCK(1)) and serotonin (5-HT(3)) receptors which are major regulators of pancreatic secretion. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O(2)) were used throughout. Dorsal vagal preganglionic neurons were identified by antidromic activation in response to stimulation of the pancreatic vagus. Dorsal vagal preganglionic neurons had axonal conduction velocities in the C-fibre range (0.7+/-0.03 m/s). Forty-four neurons were identified within the rostral, intermediate and caudal DMV and thirty-eight were tested for responsiveness to CCK-8S (CCK(1) agonist) and phenylbiguanide (PBG; 5-HT(3) receptor agonist). CCK-8S and PBG (0.1-10 microg/kg, i.v.) produced three types of response: (i) preganglionic neurons in the intermediate DMV were inhibited by CCK-8S (n=18) and PBG (n=10), (ii) neurons in the caudal DMV were activated by CCK (n=5) and PBG (n=2) and (iii) CCK-8S (n=9) and PBG (n=7) had no effect on preganglionic neurons in the rostral DMV. CCK-8S and PBG have complex actions on preganglionic neurons in the DMV that may be related to their effects on pancreatic secretion.

Activation of cholecystokinin (CCK 1) and serotonin (5-HT 3) receptors increases the discharge of pancreatic vagal afferents.

Cholecystokinin and serotonin are released from the gastrointestinal tract in response to the products of digestion and play critical roles in mediating pancreatic secretion via vago-vagal reflex pathways. This study was designed to investigate the effects of activation of cholecystokinin CCK(1) and serotonin (5-hydroxytryptamine, 5-HT) 5-HT(3) receptors on pancreatic vagal afferent discharge and to determine whether there is an interaction between these receptors. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O(2)) were used in all experiments. The effects of systemic administration of cholecystokinin and the serotonin 5-HT(3) receptor agonist phenylbiguanide on pancreatic vagal afferent discharge were recorded before and after administration of cholecystokinin CCK(1) and serotonin 5-HT(3) receptor antagonists. Cholecystokinin (0.1-10 microg/kg, i.v.) and phenylbiguanide (1 and 10 microg/kg, i.v.) increased pancreatic vagal afferent discharge dose-dependently. Cholecystokinin CCK(1) receptor antagonists, lorglumide (10 mg/kg, i.v.) and devazepide (0.5 mg/kg, i.v.), reduced cholecystokinin- and phenylbiguanide-induced increases in pancreatic vagal afferent discharge significantly (n=5, P<0.05). On the other hand, serotonin 5-HT(3) receptor blockade with granisetron (1 mg/kg, i.v.) or MDL72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate; 0.1 mg/kg, i.v.) inhibited the pancreatic vagal afferent discharge responses to phenylbiguanide but not those to cholecystokinin. This study has confirmed that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via activation of cholecystokinin CCK(1) and serotonin 5-HT(3) receptors, respectively. In addition, it has demonstrated that (i) the serotonin 5-HT(3) agonist phenylbiguanide acts partly via an interaction with cholecystokinin CCK(1) receptors, and (ii) the actions of cholecystokinin are not dependent on serotonin 5-HT(3) receptor activation.

Abdominal vagal signalling: a novel role for cholecystokinin in circulatory control?

It is generally accepted that the gastrointestinal circulation is primarily under the control of the enteric nervous system. However, recent studies have demonstrated that the sympathetic nervous system may play a greater role in postprandial gastrointestinal circulatory function than was thought previously. Cholecystokinin (CCK) is a gastrointestinal hormone released from enteroendocrine cells lining the intestinal mucosa in response to feeding. Systemic administration of CCK induces gastrointestinal vasodilation mediated by withdrawal of sympathetic vasomotor drive. CCK differentially influences the discharge rate of presympathetic vasomotor neurons in the rostral ventrolateral medulla and this response is mirrored by differential responses in the gastrointestinal and skeletal muscle sympathetic vasomotor outflows. CCK1 receptors located on abdominal vagal afferent neurons are activated by CCK which, in turn, activates an intramedullary circuit in a manner analogous to that of other sympathetic cardiovascular reflexes. Evidently, abdominal vagal afferent neurons influence sympathetic vasomotor discharge in a fashion that contrasts markedly with changes in sympathetic vasomotor outflow and regional circulatory function produced by activation of vagal cardiopulmonary reflexes. The clinical implications of this mechanism may extend to the treatment of disorders such as postprandial hypotension and gastrointestinal diseases that are contingent on local blood flow.

The role of NMDA and non-NMDA receptors in the NTS in mediating three distinct sympathoinhibitory reflexes.

Cholecystokinin (CCK) elicits a sympathetic vasomotor reflex that is implicated in gastrointestinal circulatory control. We sought to determine (1) the site in the solitary tract nucleus (NTS) responsible for mediating this reflex and (2) the possible involvement of excitatory amino acid (EAA) receptors. In addition, we sought to determine whether the NTS site responsible for mediating the baroreflex (phenylephrine, PE, 10 microg/kg i.v.) and the von Bezold-Jarisch reflex (phenylbiguanide, PBG, 10 microg/kg i.v) overlap with that involved in the CCK-induced reflex (CCK, 4 microg/kg, i.v.), and to compare the relative importance of NMDA and non-NDMA receptors in these reflexes. In separate experiments, the effects of PE, PBG, and CCK on mean arterial blood pressure, heart rate, and splanchnic sympathetic nerve discharge were tested before and after bilateral microinjection into the NTS of the gamma-aminobutyric acid(A) (GABA(A)) agonist muscimol, the EAA antagonist kynurenate, the NMDA receptor antagonist D: (-)-2-amino-5-phosphopentanoic acid (AP-5), the non-NMDA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), AP-5 + NBQX, or vehicle. While all treatments (except vehicle) significantly attenuated/abolished/reversed the splanchnic sympathoinhibitory responses to PE, PBG, and CCK, the extent of blockade varied between the different treatment groups. Both NMDA and non-NMDA receptors were essential to the baroreflex and the von Bezold-Jarisch reflex, whereas the CCK reflex was more dependent on non-NMDA receptors. Muscimol, kynurenate, and AP-5 + NBQX significantly attenuated the bradycardic responses to PE and PBG (P < 0.05), whereas AP-5, NBQX, or vehicle did not. The bradycardic responses to CCK remained intact after all treatments. These results suggest that while there is overlap in the area of the NTS responsible for eliciting all three reflexes, NMDA and non-NMDA receptors are recruited differentially for the full expression of these reflexes. The CCK-induced sympathoinhibitory reflex is unique in that it relies predominantly on non-NMDA receptors in the NTS and elicits bradycardic effects that are independent of the NTS.

Chemical stimulation of visceral afferents activates medullary neurones projecting to the central amygdala and periaqueductal grey.

Cholecystokinin (CCK) stimulates gastrointestinal vagal afferent neurones that signal visceral sensations. We wished to determine whether neurones of the nucleus of the solitary tract (NTS) or ventrolateral medulla (VLM) convey visceral afferent information to the central nucleus of the amygdala (CeA) or periaqueductal grey region (PAG), structures that play a key role in adaptive autonomic responses triggered by stress or fear. Male Sprague-Dawley rats received a unilateral microinjection of the tracer cholera toxin subunit B (CTB, 1%) into the CeA or PAG followed, 7 days later, by an injection of CCK (100 microg/kg, i.p.) or saline. Brains were processed for detection of Fos protein (Fos-IR) and CTB. CCK induced increased expression of Fos-IR in the NTS and the VLM, relative to control. When CTB was injected into the CeA, CTB-immunoreactive (CTB-IR) neurones were more numerous in the rostral NTS ipsilateral to the injection site, whereas they were homogeneously distributed throughout the VLM. Double-labelled neurones (Fos-IR+CTB-IR) were most numerous in the ipsilateral NTS and caudal VLM. The NTS contained the higher percentage of CTB-IR neurones activated by CCK. When CTB was injected into the PAG, CTB-IR neurones were more numerous in the ipsilateral NTS whereas they were distributed relatively evenly bilaterally in the rostral VLM. Double-labelled neurones were not differentially distributed along the rostrocaudal axis of the NTS but were more numerous in this structure when compared with the VLM. NTS and VLM neurones may convey visceral afferent information to the CeA and the PAG.

An enteric signal regulates putative gastrointestinal presympathetic vasomotor neurons in rats.

Ingestion of a meal results in gastrointestinal (GI) hyperemia and is associated with systemic and paracrine release of a number of peptide hormones, including cholecystokinin (CCK) and 5-hydroxytryptamine (5-HT). Systemic administration of CCK octapeptide inhibits a subset of presympathetic neurons of the rostroventrolateral medulla (RVLM) that may be responsible for driving the sympathetic vasomotor tone to the GI viscera. The aim of this study was to determine whether endogenous release of CCK and/or 5-HT also inhibits CCK-sensitive RVLM neurons. The effects of intraduodenal administration of the secretagogues sodium oleate (SO) and soybean trypsin inhibitor (SBTI) on circulating levels of CCK and 5-HT were examined. In separate experiments, the discharge rates of barosensitive, medullospinal, CCK-sensitive RVLM presympathetic vasomotor neurons were recorded after rapid intraduodenal infusion of SO-SBTI or water. Alternatively, animals were pretreated with the CCK1 receptor antagonists devazepide and lorglumide or the 5-HT3 antagonist MDL-72222 before SO-SBTI administration. Secretagogue infusion significantly increased the level of circulating CCK, but not 5-HT. SO-SBTI significantly decreased (58%) the neuronal firing rate of CCK-sensitive RVLM neurons compared with water (5%). CCK1 receptor antagonists did not reverse SO-SBTI-induced neuronal inhibition (58%), whereas the 5-HT3 antagonist significantly attenuated the effect (22%). This study demonstrates a functional relation between a subset of RVLM presympathetic vasomotor neurons and meal-related signals arising from the GI tract. It is likely that endogenously released 5-HT acts in a paracrine fashion on GI 5-HT3 receptors to initiate reflex inhibition of these neurons, resulting in GI vasodilatation by withdrawal of sympathetic tone.

Cocaine- and amphetamine-regulated transcript in catecholamine and noncatecholamine presympathetic vasomotor neurons of rat rostral ventrolateral medulla.

Presympathetic vasomotor adrenergic (C1) and nonadrenergic (non-C1) neurons in the rostral ventrolateral medulla (RVLM) provide the main excitatory drive to cardiovascular sympathetic preganglionic neurons in the spinal cord. C1 and non-C1 neurons contain cocaine- and amphetamine-regulated transcript (CART), suggesting that CART may be a common marker for RVLM presympathetic neurons. To test this hypothesis, we first used double-immunofluorescence staining for CART and tyrosine hydroxylase (TH) to quantify CART-immunoreactive (-IR) catecholamine and noncatecholamine neurons in the C1 region. Next, we quantified the proportion of CART-IR RVLM neurons that expressed Fos in response to a hypotensive stimulus, using peroxidase immunohistochemistry for Fos and dual immunofluorescence for CART and TH. Finally, we fluorescently detected CART immunoreactivity in electrophysiologically identified, juxtacellularly labeled RVLM presympathetic neurons. In the RVLM, 97% of TH-IR neurons were CART-IR, and 74% of CART-IR neurons were TH-IR. Nitroprusside infusion significantly increased the number of Fos-IR RVLM neurons compared with saline controls. In nitroprusside-treated rats, virtually all Fos/TH neurons in the RVLM were immunoreactive for CART (98% +/- 1.3%, SD; n = 7), whereas 29% +/- 8.3% of CART-positive, TH-negative neurons showed Fos immunoreactivity. Six fast (2.8-5.8 m/second, noncatecholamine)-, two intermediate (2.1 and 2.2 m/second)-, and five slow (<1 m/second, catecholamine)-conducting RVLM presympathetic vasomotor neurons were juxtacellularly labeled. After fluorescent detection of CART and biotinamide, all 13 neurons were found to be CART-IR. These results suggest that, in rat RVLM, all catecholamine and noncatecholamine presympathetic vasomotor neurons contain CART.

CCK-induced inhibition of presympathetic vasomotor neurons: dependence on subdiaphragmatic vagal afferents and central NMDA receptors in the rat.

Systemic administration of cholecystokinin (CCK) inhibits a subpopulation of rostral ventrolateral medulla (RVLM) presympathetic vasomotor neurons. This study was designed to determine whether this effect involved subdiaphragmatic vagal afferents and/or central N-methyl-d-aspartic acid (NMDA) receptors. Recordings were made from CCK-sensitive RVLM presympathetic vasomotor neurons in halothane-anesthetized, paralyzed male Sprague-Dawley rats. The responses of the neurons to CCK (2 and 4 microg/kg iv), phenylephrine (PE; 5 microg/kg iv), and phenylbiguanide (PBG; 5 microg/kg iv) were tested before and after application of the local anesthetic lidocaine (2% wt/vol gel; 1 ml) to the subdiaphragmatic vagi at the level of the esophagus. In seven separate experiments, lidocaine markedly reduced the inhibitory effects of CCK on RVLM presympathetic neuronal discharge rate. In other experiments, the effect of systemic administration of dizocilpine (1 mg/kg iv), a noncompetitive antagonist at NMDA receptor ion channels, on the RVLM presympathetic neuronal responses to CCK, PBG, and PE was tested. In all cases (n = 6 neurons in 6 individual rats), dizocilpine inhibited the effects of CCK, PBG, and PE on RVLM presympathetic neuronal discharge. These results suggest that the effects of systemic CCK on the discharge of RVLM presympathetic neurons is mediated via an action on receptors located on subdiaphragmatic vagal afferents. Furthermore, the data suggest that CCK activates a central pathway involving NMDA receptors to produce inhibition of RVLM presympathetic neuronal discharge.

Phenotypic identification of rat rostroventrolateral medullary presympathetic vasomotor neurons inhibited by exogenous cholecystokinin.

Systemic administration of the gastrointestinal hormone cholecystokinin (CCK) selectively inhibits splanchnic sympathetic vasomotor discharge and differentially affects presympathetic vasomotor neurons of the rostroventrolateral medulla (RVLM). Stimulation of the sympathoexcitatory region of the periaqueductal grey (PAG) produces profound mesenteric vasoconstriction. In this study, our aim was to identify phenotypically different populations of RVLM presympathetic vasomotor neurons using juxtacellular neuronal labelling and immunohistochemical detection of the adrenergic neuronal marker phenylethanolamine-N-methyl transferase (PNMT) and to determine whether the PAG provides functional excitatory input to these neurons. Fifty-eight percent (36/62) of RVLM presympathetic neurons were inhibited by systemic administration of CCK. These cells had conduction velocities (3.6 +/- 0.2 m/sec) in the non-C-fiber range consistent with neurons possessing lightly myelinated spinal axons. Of these, 79% (22/28) were excited by PAG stimulation, and 59% (10/17) were not immunoreactive for PNMT. Conversely, 42% (26/62) of RVLM presympathetic neurons were either unaffected or activated by CCK administration and had slower conduction velocities (1.4 +/- 0.3 m/sec) than cells inhibited by CCK. Fifty percent (11/22) of these cells were driven by PAG stimulation, and most (11/14 or 79%) were PNMT-positive. These results suggest that cardiovascular responses elicited by PAG stimulation occur via activation of non-C1 and C1 RVLM presympathetic neurons. RVLM neurons inhibited by CCK were more likely to be driven by PAG stimulation and may be a subset of neurons responsible for driving gastrointestinal sympathetic vasomotor tone. CCK-induced inhibition of a subpopulation of RVLM presympathetic neurons may be implicated in postprandial hyperemia and postprandial hypotension.

Chemical stimulation of vagal afferent neurons and sympathetic vasomotor tone.

Vagal afferents innervate a diverse range of structures of the thoracic and abdominal viscera. While a proportion of these afferents function as mechanoreceptors and respond to changes in intramural tension within the structures that they innervate, many also sense a broad range of chemical substances ranging from peptides, sugars and lipids present in the intraluminal contents of the gastrointestinal tract, as well as tissue prostanoids, cytokines and monoamines in the cardiopulmonary circulation. This review examines the effects of chemical stimulation of vagal afferents on circulatory and sympathetic vasomotor function. Notably, the von Bezold-Jarisch reflex is a cardiorespiratory reflex produced by chemical activation of cardiopulmonary vagal afferents. Classical stimulants of the von Bezold-Jarisch reflex include the Veratrum alkaloids and 5-HT(3) receptor agonists. Atrial natriuretic peptides are agents which also produce a von Bezold-Jarisch reflex-like response or a sensitisation of this reflex via an action on vagal afferents. Cholecystokinin (CCK) activates abdominal visceral vagal afferents, which apart from a clear role in mediation of satiety, also produces selective sympathetic vasomotor inhibition probably by inhibition of sub-groups of presympathetic vasomotor neurons of the rostral ventrolateral medulla. These actions of CCK may constitute a novel gastrointestinal-cardiovascular reflex. The afferent vagus transmits a diverse array of signals to the central nervous system, influencing sympathetic vasomotor and cardiomotor function, gastrointestinal function, neuroimmune function and endocrine function.

Cholecystokinin selectively affects presympathetic vasomotor neurons and sympathetic vasomotor outflow.

Cholecystokinin (CCK) is a potential mediator of gastrointestinal vasodilatation during digestion. To determine whether CCK influences sympathetic vasomotor function, we examined the effect of systemic CCK administration on mean arterial blood pressure (MAP), heart rate (HR), lumbar sympathetic nerve discharge (LSND), splanchnic sympathetic nerve discharge (SSND), and the discharge of presympathetic neurons of the rostral ventrolateral medulla (RVLM) in alpha-chloralose-anesthetized rats. CCK (1-8 microg/kg iv) reduced MAP, HR, and SSND and transiently increased LSND. Vagotomy abolished the effects of CCK on MAP and SSND as did the CCK-A receptor antagonist devazepide (0.5 mg/kg iv). The bradycardic effect of CCK was unaltered by vagotomy but abolished by devazepide. CCK increased superior mesenteric arterial conductance but did not alter iliac conductance. CCK inhibited a subpopulation (approximately 49%) of RVLM presympathetic neurons whereas approximately 28% of neurons tested were activated by CCK. The effects of CCK on RVLM neuronal discharge were blocked by devazepide. RVLM neurons inhibited by exogenous CCK acting via CCK-A receptors on vagal afferents may control sympathetic vasomotor outflow to the gastrointestinal tract vasculature.