Autonomic Mechanisms Of Blood Pressure Alterations During Sleep In Orexin/Hypocretin-Deficient Narcoleptic Mice

Study Objectives increases in arterial pressure (AP) during sleep and smaller differences in AP between sleep and wakefulness have been reported in orexin (hypocretin)-deficient mouse models of narcolepsy type 1 (NT1) and confirmed in NT1 patients. We tested whether these alterations are mediated by parasympathetic or sympathetic control of the heart and/or resistance vessels in an orexin-deficient mouse model of NT1. Methods 13 orexin knock-out (ORX-KO) mice were compared with 12 congenic wild-type (WT) mice. The electroencephalogram, electromyogram, and AP of the mice were recorded in the light (rest) period during intraperitoneal infusion of atropine methyl nitrate, atenolol, or prazosin to block muscarinic cholinergic, β1-adrenergic, or α1-adrenergic receptors, respectively, while saline was infused as control. Results AP significantly depended on a 3-way interaction among the mouse group (ORX-KO vs WT), the wake-sleep state, and the drug or vehicle infused. During the control vehicle infusion, ORX-KO had significantly higher AP values during REM sleep, smaller decreases in AP from wakefulness to either non-rapid-eye-movement (non-REM) sleep or REM sleep, and greater increases in AP from non-REM sleep to REM sleep compared to WT. These differences remained significant with atropine methyl nitrate, whereas they were abolished by prazosin and, except for the smaller AP decrease from wakefulness to REM sleep in ORX-KO, also by atenolol. Conclusions sleep-related alterations of AP due to orexin deficiency significantly depend on alterations in cardiovascular sympathetic control in a mouse model of NT1.

Cephalic phase insulin secretion is KATP channel independent

Glucose-induced insulin secretion from pancreatic β-cells critically depends on the activity of ATP-sensitive K+channels (KATPchannel). We previously generated mice lackingKir6.2, the pore subunit of the β-cell KATPchannel (Kir6.2−/−), that show almost no insulin secretion in response to glucosein vitro. In this study, we compared insulin secretion by voluntary feeding (self-motivated, oral nutrient ingestion) and by forced feeding (intra-gastric nutrient injection via gavage) in wild-type (Kir6.2+/+) andKir6.2−/−mice. Underad libitumfeeding or during voluntary feeding of standard chow, blood glucose levels and plasma insulin levels were similar inKir6.2+/+andKir6.2−/−mice. By voluntary feeding of carbohydrate alone, insulin secretion was induced significantly inKir6.2−/−mice but was markedly attenuated compared with that inKir6.2+/+mice. On forced feeding of standard chow or carbohydrate alone, the insulin secretory response was markedly impaired or completely absent inKir6.2−/−mice. Pretreatment with a muscarine receptor antagonist, atropine methyl nitrate, which does not cross the blood–brain barrier, almost completely blocked insulin secretion induced by voluntary feeding of standard chow or carbohydrate inKir6.2−/−mice. Substantial glucose-induced insulin secretion was induced in the pancreas perfusion study ofKir6.2−/−mice only in the presence of carbamylcholine. These results suggest that a KATPchannel-independent mechanism mediated by the vagal nerve plays a critical role in insulin secretion in response to nutrientsin vivo.

Heterogeneity of muscarinic receptor-mediated Ca2+ responses in cultured urothelial cells from rat

Muscarinic receptors (mAChRs) have been identified in the urothelium, a tissue that may be involved in bladder sensory mechanisms. This study investigates the expression and function of mAChRs using cultured urothelial cells from the rat. RT-PCR established the expression of all five mAChR subtypes. Muscarinic agonists acetylcholine (ACh; 10 μM), muscarine (Musc; 20 μM), and oxotremorine methiodide (OxoM; 0.001–20 μM) elicited transient repeatable increases in the intracellular calcium concentration ([Ca2+]i) in ∼50% of cells. These effects were blocked by the mAChR antagonist atropine methyl nitrate (10 μM). The sources of [Ca2+]i changes included influx from external milieu in 63% of cells and influx from external milieu plus release from internal stores in 27% of cells. The use of specific agonists and antagonists (10 μM M1 agonist McN-A-343; 10 μM M2, M3 antagonists AF-DX 116, 4-DAMP) revealed that M1, M2, M3 subtypes were involved in [Ca2+]i changes. The PLC inhibitor U-73122 (10 μM) abolished OxoM-elicited Ca2+ responses in the presence of the M2 antagonist AF-DX 116, suggesting that M1, M3, or M5 mediates [Ca2+]i increases via PLC pathway. ACh (0.1 μM), Musc (10 μM), oxotremorine sesquifumarate (20 μM), and McN-A-343 (1 μM) acting on M1, M2, and M3 mAChR subtypes stimulated ATP release from cultured urothelial cells. In summary, cultured urothelial cells express functional M1, M2, and M3 mAChR subtypes whose activation results in ATP release, possibly through mechanisms involving [Ca2+]i changes.

Esophageal-gastric relaxation reflex in rat: dual control of peripheral nitrergic and cholinergic transmission

It has long been known that the esophageal distension produced by swallowing elicits a powerful proximal gastric relaxation. Gastroinhibitory control by the esophagus involves neural pathways from esophageal distension-sensitive neurons in the nucleus tractus solitarius centralis (cNTS) with connections to virtually all levels of the dorsal motor nucleus of the vagus (DMV). We have shown recently that cNTS responses are excitatory and primarily involve tyrosine hydroxylase-immunoreactive cells, whereas the DMV response involves both an α1 excitatory and an α2 inhibitory response. In the present study, using an esophageal balloon distension to evoke gastric relaxation (esophageal-gastric reflex, EGR), we investigated the peripheral pharmacological basis responsible for this reflex. Systemic administration of atropine methyl nitrate reduced the amplitude of the gastric relaxation to 52.0 ± 4.4% of the original EGR, whereas NG-nitro-l-arginine methyl ester (l-NAME) reduced it to 26.3 ± 7.2% of the original EGR. Concomitant administration of atropine methyl nitrate and l-NAME reduced the amplitude of the gastric relaxation to 4.0 ± 2.5% of control. This reduction in the amplitude of induced EGR is quite comparable (4.3 ± 2.6%) to that seen when the animal was pretreated with the nicotinic ganglionic blocker hexamethonium. In the presence of bethanechol, the amplitude of the esophageal distension-induced gastric relaxation was increased to 177.0 ± 10.0% of control; administration of l-NAME reduced this amplitude to 19.9 ± 9.5%. Our data provide a clear demonstration that the gastroinhibitory control by the esophagus is mediated via a dual vagal innervation consisting of inhibitory nitrergic and excitatory cholinergic transmission.

Central neuropeptide Y induces proximal stomach relaxation via Y1 receptors in the dorsal vagal complex of the rat

Effects of neuropeptide Y (NPY) on motility of the proximal stomach was examined in anesthetized rats. Intragastric pressure was measured using a balloon situated in the proximal part of the stomach. Administration of NPY into the fourth ventricle induced relaxation of the proximal stomach in a dose-dependent manner. Administration of an Y1 receptor (Y1R) agonist [Leu31, Pro34]NPY induced a larger relaxation than NPY. The administration of an Y2 receptor agonist (NPY 13-36) did not induce significant changes in motility. Microinjections of [Leu31, Pro34]NPY into the caudal part of the dorsal vagal complex (DVC) induced relaxation of the proximal stomach. In contrast, similar injections into the intermediate part of the DVC increased IGP of the proximal stomach. Administration of NPY into the fourth ventricle did not induce relaxation after bilateral injections of the Y1R antagonist (1229U91) into the caudal DVC. These results indicate that NPY induces relaxation in the proximal stomach via Y1Rs situated in the DVC. Because bilateral vagotomy below the diaphragm abolished the relaxation induced by the administration of NPY into the fourth ventricle, relaxation induced by NPY is probably mediated by vagal preganglionic neurons. Intravenous injection of atropine methyl nitrate reduced relaxation induced by administration of NPY. Therefore, relaxation induced by NPY is likely mediated by peripheral cholinergic neurons.

Effects of chronic bromocriptine (CB-154) treatment on the plasma glucose and insulin secretion response to neurocytoglucopenia in rats

Neurocytoglucopenia has been reported to increase both parasympathetic and sympathetic tone with a predominant effect on the latter, which accounts for the major effect of plasma hyperglycemia and the inhibition of insulin secretion. The aim of this study was to determine the effects of chronic treatment with bromocriptine (0.4 mg/100 g body wt per day), a potent sympatholytic D(2)-dopaminergic agonist, on hyperglycemia and insulin secretion in response to neurocytoglucopenia induced by 2-deoxy-d-glucose (2DG). After 2 weeks of bromocriptine treatment the animals, freely moving in their cages, were submitted to 2DG administration (50 mg/100 g body wt) via atrial catheter infusion. After 2DG infusion, the plasma prolactin of vehicle-treated (VEH) rats increased rapidly, reaching a peak at 10 min (34.3+/-7.6 ng/ml; P<0.01). In contrast, 2DG infusion failed to induce any significant change in the plasma prolactin levels of bromocriptine-treated (BR) rats. BR rats showed higher resting glucose levels than control rats (8.2+/-0.28 mM (BR) vs 6.0+/-0.18 mM (VEH); P<0.01). However, the hyperglycemic response of BR rats to 2DG injection was 30% lower than that of VEH rats (P<0.05). BR rats also showed a rapid rise in plasma insulin levels reaching a peak at 30 min after 2DG injection (243% higher than basal values; P<0.01). This increased rise in the insulin response to neurocytoglucopenia of BR rats was blocked by previous intravenous injection of atropine methyl nitrate (0.2 mg/100 g body wt). The present results suggest that chronic treatment with bromocriptine determines a strong increase in the parasympathetic tone response to neurocytoglucopenia, which is responsible for the higher stimulation of insulin secretion observed in BR rats. The data also provide further evidence that D(2)-dopaminergic agonist can block neurocytoglucopenia-induced prolactin release.

Lateral hypothalamic lesions cause gastric injury by stimulating gastric contractility

Changes in gastric contractility following lateral hypothalamic (LH) lesions with and without bilateral cervical vagotomy were measured in urethan-anesthetized rats. LH lesions were induced with direct current passed through stereotaxically placed electrodes. Gastric contractility was recorded continuously for 4 h with acutely implanted strain gauge force transducers and analyzed by computer. LH lesions consistently stimulated gastric contractility and caused more gastric mucosal injury than control conditions. Vagotomy blocked both gastric mucosal injury and high-amplitude gastric contractions. In rats with LH lesions and exogenously infused intragastric hydrochloric acid, atropine methyl nitrate inhibited high-amplitude gastric contractions and gastric erosions. These findings indicate that LH lesions stimulate vagally mediated high-amplitude gastric contractions, which, in the presence of hydrochloric acid, cause gastric mucosal erosions.

Influence of autonomic blockade on cardiovascular responses to exercise in rats

The purposes of this study were to determine the role of the sympathetic and parasympathetic nervous systems in producing the heart rate (HR) response to dynamic exercise in rats and to determine the effect of attenuation of the HR response to exercise on blood flow redistribution. Sprague-Dawley rats (n = 10) were instrumented with arterial and venous catheters and Doppler flow probes. Mean arterial pressure (MAP), HR, mesenteric blood flow (MBF), and iliac blood flow (IBF) were determined during four exercise tests. On 4 consecutive days, rats were treated with saline (SAL, 1 mg/kg iv), atropine methyl nitrate (ATR, 2 mg/kg), timolol maleate (TIM, 0.5 mg/kg), and combined timolol and atropine. One minute of mild exercise (10 m/min) produced an increase in HR of 90 +/- 6 beats/min after SAL treatment, which was significantly less than the increment after ATR (56 +/- 5 beats/min) or TIM (4 +/- 3 beats/min). For the remainder of graded exercise, ATR treatment produced a modest attenuation in the increment in HR and no effect on MAP, IBF, and MBF. At 30 m/min, TIM markedly blunted the exercise-induced increment in HR (SAL, 138 +/- 8 beats/min; TIM, 53 +/- 4 beats/min) and IBF (SAL, 324 +/- 33%; TIM, 197 +/- 33%) with no effect on MAP or MBF. The results suggest that 1) the sympathetic nervous system is an important mediator of exercise-induced tachycardia in rats and 2) exercised-induced hyperemia, but not MAP, is attenuated by nonselective beta-blockade during exercise in rats.

CRF in the paraventricular nucleus mediates gastric and colonic motor response to restraint stress

The role of corticotropin-releasing factor (CRF) in the paraventricular nucleus of the hypothalamus (PVN) in the control of gastric emptying of a nonnutrient meal and colonic transit was investigated in conscious fasted rats chronically implanted with hypothalamic cannulas and catheters in both the stomach and proximal colon. CRF (0.06-0.6 nmol) microinfused unilaterally into the PVN resulted in a dose-dependent inhibition of gastric emptying (0-51%) and stimulation of colonic transit (0-93%). CRF (0.6 nmol)-induced delay in gastric emptying was inhibited by 50% by subdiaphragmatic vagotomy or atropine methyl nitrate (1 mg/kg ip), whereas the stimulation of colonic transit was completely abolished by atropine methyl nitrate and reduced by 19% by vagotomy. Microinfusion of CRF (0.6 nmol) into the lateral hypothalamus or central amygdala had no effect. Restraint exposure for 1 h delayed gastric emptying and stimulated colonic transit by 28 and 78%, respectively. Bilateral microinfusion of the CRF antagonist alpha-helical CRF-(9-41) (13 nmol) into the PVN before restraint abolished stress-induced alterations of gastric and colonic transit. The CRF antagonist did not alter basal gastric and colonic transit under basal conditions. These data indicate that the PVN is a specific responsive site for central CRF-induced alterations of gastric and colonic transit and suggest that endogenous CRF in the PVN plays a role in mediating restraint stress-related alterations of gastrointestinal transit.