Proteogenomics Reveals Orthologous Alternatively Spliced Proteoforms in the Same Human and Mouse Brain Regions with Differential Abundance in an Alzheimer's Disease Mouse Model.

Alternative splicing (AS) may increase the number of proteoforms produced by a gene. Alzheimer's disease (AD) is a neurodegenerative disease with well-characterized AS proteoforms. In this study, we used a proteogenomics strategy to build a customized protein sequence database and identify orthologous AS proteoforms between humans and mice on publicly available shotgun proteomics (MS/MS) data of the corpus callosum (CC) and olfactory bulb (OB). Identical proteotypic peptides of six orthologous AS proteoforms were found in both species: PKM1 (gene PKM/Pkm), STXBP1a (gene STXBP1/Stxbp1), Isoform 3 (gene HNRNPK/Hnrnpk), LCRMP-1 (gene CRMP1/Crmp1), SP3 (gene CADM1/Cadm1), and PKCßII (gene PRKCB/Prkcb). These AS variants were also detected at the transcript level by publicly available RNA-Seq data and experimentally validated by RT-qPCR. Additionally, PKM1 and STXBP1a were detected at higher abundances in a publicly available MS/MS dataset of the AD mouse model APP/PS1 than its wild type. These data corroborate other reports, which suggest that PKM1 and STXBP1a AS proteoforms might play a role in amyloid-like aggregate formation. To the best of our knowledge, this report is the first to describe PKM1 and STXBP1a overexpression in the OB of an AD mouse model. We hope that our strategy may be of use in future human neurodegenerative studies using mouse models.

Intravenous doxapram administration as a potential model of panic attacks in rats.

Panic disorder can be categorized into the nonrespiratory or the respiratory subtypes, the latter comprising dyspnea, shortness of breath, chest pain, feelings of suffocation, and paresthesias. Doxapram is an analeptic capable of inducing panic attacks with respiratory symptoms in individuals diagnosed with the disorder; however, its neuroanatomical targets and its effects on experimental animals remain uncharacterized. One of the brain regions proposed to trigger panic attacks is the midbrain periaqueductal gray (PAG). Therefore, in this study, we evaluated the effects of doxapram in Fos (c-Fos) protein expression in the PAG and characterized its cardiorespiratory and behavioral effects on the elevated T maze and in the conditioned place aversion (CPA) paradigms. Doxapram increased Fos expression in different columns of the PAG, increased respiratory frequency, decreased heart rate, and increased arterial pressure when injected via intravenous route. Alprazolam, a panicolytic benzodiazepine, injected via intraperitoneal route, decreased respiratory frequency, whereas URB597, an anandamide hydrolysis inhibitor injected via intraperitoneal route, was ineffective. Doxapram injected via intraperitoneal route induced an anxiogenic-like effect in the elevated T-maze model; however, it failed to induce CPA. This study suggests that the cardiorespiratory and behavioral effects of doxapram in rodents serve as an experimental model that can provide insights into the neurobiology of panic attacks.

Central Ang II (Angiotensin II)-Mediated Sympathoexcitation: Role for HIF-1α (Hypoxia-Inducible Factor-1α) Facilitated Glutamatergic Tone in the Paraventricular Nucleus of the Hypothalamus.

Central infusion of Ang II (angiotensin II) has been associated with increased sympathetic outflow resulting in neurogenic hypertension. In the present study, we appraised whether the chronic increase in central Ang II activates the paraventricular nucleus of the hypothalamus (PVN) resulting in elevated sympathetic tone and altered baro- and chemoreflexes. Further, we evaluated the contribution of HIF-1α (hypoxia-inducible factor-1α), a transcription factor involved in enhancing the expression of N-methyl-D-aspartate receptors and thus glutamatergic-mediated sympathetic tone from the PVN. Ang II infusion (20 ng/minute, intracerebroventricular, 14 days) increased mean arterial pressure (126±9 versus 84±4 mm Hg), cardiac sympathetic tone (96±7 versus 75±6 bpm), and decreased cardiac parasympathetic tone (16±2 versus 36±3 versus bpm) compared with saline-infused controls in conscious rats. The Ang II-infused group also showed an impaired baroreflex control of heart rate (-1.50±0.1 versus -2.50±0.3 bpm/mm Hg), potentiation of the chemoreflex pressor response (53±7 versus 30±7 mm Hg) and increased number of FosB-labeled cells (53±3 versus 19±4) in the PVN. Concomitant with the activation of the PVN, there was an increased expression of HIF-1α and N-Methyl-D-Aspartate-type1 receptors in the PVN. Further, Ang II-infusion showed increased renal sympathetic nerve activity (20.5±2.3% versus 6.4±1.9% of Max) and 3-fold enhanced renal sympathetic nerve activity responses to microinjection of N-methyl-D-aspartate (200 pmol) into the PVN of anesthetized rats. Further, silencing of HIF-1α in NG108 cells abrogated the expression of N-methyl-D-aspartate-N-methyl-D-aspartate-type1 induced by Ang II. Taken together, our studies suggest a novel Ang II-HIF-1α-N-methyl-D-aspartate receptor-mediated activation of preautonomic neurons in the PVN, resulting in increased sympathetic outflow and alterations in baro- and chemoreflexes.

Central angiotensin II-Protein inhibitor of neuronal nitric oxide synthase (PIN) axis contribute to neurogenic hypertension.

Activation of renin-angiotensin- system, nitric oxide (NO•) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases, including hypertension. Previously we have shown increased protein expression of PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with heart failure. To elucidate the molecular mechanism by which Angiotensin II (Ang II) increases PIN expression, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 0.5 µl/h) or saline as vehicle (Veh) for 14 days through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline renal sympathetic nerve activity and mean arterial pressure. Ang II infusion increased the expression of PIN (1.24 ± 0.04* Ang II vs. 0.65 ± 0.07 Veh) with a concomitant 50% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1.00 ± 0.03 Veh) in the PVN. Substrate-dependent ligase assay in cells transfected with pCMV-(HA-Ub)8 vector revealed a reduction of HA-Ub-PIN conjugates after Ang II and a proteasome inhibitor, Lactacystin (LC), treatment (4.5 ± 0.7* LC Ang II vs. 9.2 ± 2.5 LC). TUBE (Tandem Ubiquitin-Binding Entities) assay showed decrease PIN-Ub conjugates in Ang II-treated cells (0.82 ± 0.12* LC Ang II vs. 1.21 ± 0.06 LC) while AT1R blocker, Losartan (Los) treatment diminished the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.16 ± 0.04* LC Ang II Los). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO• on pre-autonomic neurons in the PVN resulting in increased sympathetic outflow.

Effects of alprazolam and cannabinoid-related compounds in an animal model of panic attack.

Selective stimulation of carotid chemoreceptors by intravenous infusion of low doses of potassium cyanide (KCN) produces short-lasting escape responses that have been proposed as a model of panic attack. In turn, preclinical studies suggest that facilitation of the endocannabinoid system attenuate panic-like responses. Here, we compared the effects of cannabinoid-related compounds to those of alprazolam, a clinically effective panicolytic, on the duration of the escape reaction induced by intravenous infusion of KCN (80µg) in rats. Alprazolam (1, 2, 4mg/kg) decreased escape duration at doses that did not alter basal locomotor activity. URB597 (0.1, 0.3, 1mg/kg; inhibitor of anandamide hydrolysis), WIN55,212-2 (0.1, 0.3, 1mg/kg; synthetic cannabinoid), arachidonoyl-serotonin (1, 2.5, 5mg/kg; dual TRPV1 and anandamide hydrolysis inhibitor), and cannabidiol (5, 10, 20, 40mg/kg; a phytocannabinoid) did not decrease escape duration. Alprazolam also prevented the increase in arterial pressure evoked by KCN, while bradycardia was unchanged. This study reinforces the validity of the KCN-evoked escape as a model of panic attack. However, it does not support a role for the endocannabinoid system in this behavioral response. These results might have implications for the screening of novel treatments for panic disorder.

Changes in cardiovascular responses to chemoreflex activation of rats recovered from protein restriction are not related to AT1 receptors.

NEW FINDINGS: What is the central question of this study? In this study, we sought to investigate whether cardiovascular responses to peripheral chemoreflex activation of rats recovered from protein restriction are related to activation of AT1 receptors. What is the main finding and its importance? This study highlights the fact that angiotensinergic mechanisms activated by AT1 receptors do not support increased responses to peripheral chemoreflex activation by KCN in rats recovered from protein restriction. Also, we found that protein restriction led to increased resting ventilation in adult rats, even after recovery. The effects of a low-protein diet followed by recovery on cardiorespiratory responses to peripheral chemoreflex activation were tested before and after systemic angiotensin II type 1 (AT1 ) receptor antagonism. Male Fischer rats were divided into control and recovered (R-PR) groups after weaning. The R-PR rats were fed a low-protein (8%) diet for 35 days and recovered with a normal protein (20%) diet for 70 days. Control rats received a normal protein diet for 105 days (CG105 ). After cannulation surgery, mean arterial pressure, heart rate, respiratory frequency, tidal volume and minute ventilation were acquired using a digital recording system in freely moving rats. The role of angintensin II was evaluated by systemic antagonism of AT1 receptors with losartan (20 mg kg-1 i.v.). The peripheral chemoreflex was elicited by increasing doses of KCN (20-160 µg kg min-1 , i.v.). At baseline, R-PR rats presented increased heart rate and minute ventilation (372 ± 34 beats min-1 and 1.274 ± 377 ml kg-1  min-1 ) compared with CG105 animals (332 ± 22 beats min-1 and 856 ± 112 ml kg-1  min-1 ). Mean arterial pressure was not different between the groups. Pressor and bradycardic responses evoked by KCN (60 µg kg-1 ) were increased in R-PR (+45 ± 13 mmHg and -77 ± 47 beats min-1 ) compared with CG105 rats (+25 ± 17 mmHg and -27 ± 28 beats min-1 ), but no difference was found in the tachypnoeic response. These differences were preserved after losartan. The data suggest that angiotensin II acting on AT1 receptors may not be associated with the increased heart rate, increased minute ventilation and acute cardiovascular responses to peripheral chemoreflex activation in rats that underwent postweaning protein restriction followed by recovery.

Increased vascular sympathetic modulation in mice with Mas receptor deficiency.

INTRODUCTION: The angiotensin-converting enzyme 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas axis could modulate the heart rate (HR) and blood pressure variabilities (BPV) which are important predictors of cardiovascular risk and provide information about the autonomic modulation of the cardiovascular system. Therefore we investigated the effect of Mas deficiency on autonomic modulation in wild type and Mas-knockout (KO) mice. METHODS: Blood pressure was recorded at high sample rate (4000 Hz). Stationary sequences of 200-250 beats were randomly chosen. Frequency domain analysis of HR and BPV was performed with an autoregressive algorithm on the pulse interval sequences and on respective systolic sequences. RESULTS: The KO group presented an increase of systolic arterial pressure (SAP; 127.26±11.20 vs 135.07±6.98 mmHg), BPV (3.54±1.54 vs 5.87±2.12 mmHg(2)), and low-frequency component of systolic BPV (0.12±0.11 vs 0.47±0.34 mmHg(2)). CONCLUSIONS: The deletion of Mas receptor is associated with an increase of SAP and with an increased BPV, indicating alterations in autonomic control. Increase of sympathetic vascular modulation in absence of Mas evidences the important role of Ang-(1-7)/Mas on cardiovascular regulation. Moreover, the absence of significant changes in HR and HRV can indicate an adaptation of autonomic cardiac balance. Our results suggest that the Ang-(1-7)/Mas axis seems more important in autonomic modulation of arterial pressure than HR.

Excitatory amino acid receptors in the dorsomedial hypothalamic area contribute to the chemoreflex tachypneic response.

This study evaluated the effect of blockade of the excitatory amino acid (EAA) receptors in the dorsomedial hypothalamic (DMH) area on the ventilatory and cardiovascular responses of the chemoreflex activation in conscious rats. Bilateral microinjection of kynurenic acid (2.7 nmol, n = 6) into the DMH area reduced the tachypneic (+ 264 ± 13 versus + 204 ± 14 cpm, P < 0.05) and pressor (+ 52 ± 5 versus + 31 ± 6 mmHg, P < 0.05) components of chemoreflex but had no effect on the bradycardic component (-214 ± 7 versus -244 ± 17 bpm) of the chemoreflex. The magnitudes of the reduction in pressor and tachypneic chemoreflex responses were not significantly correlated (r = 0.308, P = 0.330). These data indicate that neurons located in the DMH area are activated by chemoreflex; that this activation is mediated via EAA receptors; and that it is essential for the full expression of the respiratory component of the chemoreflex.

Exercise training starting at weaning age preserves cardiac pacemaker function in adulthood of diet-induced obese rats.

Peripheral sympathetic overdrive in young obese subjects contributes to further aggravation of insulin resistance, diabetes, and hypertension, thus inducing worsening clinical conditions in adulthood. Exercise training has been considered a strategy to repair obesity autonomic dysfunction, thereby reducing the cardiometabolic risk. Therefore, the aim of this study was to assess the effect of early exercise training, starting immediately after weaning, on cardiac autonomic control in diet-induced obese rats. Male Wistar rats (weaning) were divided into four groups: (i) a control group (n = 6); (ii) an exercise-trained control group (n = 6); (iii) a diet-induced obesity group (n = 6); and (iv) an exercise-trained diet-induced obesity group (n = 6). The development of obesity was induced by 9 weeks of palatable diet intake, and the training program was implemented in a motor-driven treadmill (5 times per week) during the same period. After this period, animals were submitted to vein and artery catheter implantation to assess cardiac autonomic balance by methylatropine (3 mg/kg) and propranolol (4 mg/kg) administration. Exercise training increased running performance in both groups (p < 0.05). Exercise training also prevented the increased resting heart rate in obese rats, which seemed to be related to cardiac pacemaker activity preservation (p < 0.05). Additionally, the training program preserved the pressure and bradycardia responses to autonomic blockade in obese rats (p < 0.05). An exercise program beginning at weaning age prevents cardiovascular dysfunction in obese rats, indicating that exercise training may be used as a nonpharmacological therapeutic strategy for the treatment of cardiometabolic diseases.

Physical exercise performance in temperate and warm environments is decreased by an impaired arterial baroreflex.

The present study aimed to investigate whether running performance in different environments is dependent on intact arterial baroreceptor reflexes. We also assessed the exercise-induced cardiovascular and thermoregulatory responses in animals lacking arterial baroafferent signals. To accomplish these goals, male Wistar rats were subjected to sinoaortic denervation (SAD) or sham surgery (SHAM) and had a catheter implanted into the ascending aorta to record arterial pressure and a telemetry sensor implanted in the abdominal cavity to record core temperature. After recovering from these surgeries, the animals were subjected to constant- or incremental-speed exercises performed until the voluntary interruption of effort under temperate (25° C) and warm (35° C) conditions. During the constant-speed exercises, the running time until the rats were fatigued was shorter in SAD rats in both environments. Although the core temperature was not significantly different between the groups, tail skin temperature was higher in SAD rats under temperate conditions. The denervated rats also displayed exaggerated increases in blood pressure and double product compared with the SHAM rats; in particular, in the warm environment, these exaggerated cardiovascular responses in the SAD rats persisted until they were fatigued. These SAD-mediated changes occurred in parallel with increased variability in the very low and low components of the systolic arterial pressure power spectrum. The running performance was also affected by SAD during the incremental-speed exercises, with the maximal speed attained being decreased by approximately 20% in both environments. Furthermore, at the maximal power output tolerated during the incremental exercises, the mean arterial pressure, heart rate and double product were exaggerated in the SAD relative to SHAM rats. In conclusion, the chronic absence of the arterial baroafferents accelerates exercise fatigue in temperate and warm environments. Our findings also suggest that an augmented cardiovascular strain accounted for the early interruption of exercise in the SAD rats.

Excitatory amino acid receptors in the dorsomedial hypothalamus are involved in the cardiovascular and behavioural chemoreflex responses.

The present study investigated the role of the dorsomedial hypothalamus (DMH) on cardiovascular and behavioural responses of chemoreflex activation in conscious rats. The arterial chemoreflex was activated by potassium cyanide (KCN, 40 µg, i.v.) before and after bilateral microinjection of lidocaine (2%) or kynurenic acid (2.7 nmol) into the DMH. Locomotor activity was measured to assess the chemoreflex behavioural response. Bilateral microinjection of lidocaine into the DMH produced a significant reduction in the pressor response induced by chemoreflex activation (+51 ± 4 versus +34 ± 5 mmHg, n = 5, P < 0.05). A similar reduction in the pressor chemoreflex response was also observed after microinjection of kynurenic acid into the DMH (+50 ± 3 versus +22 ± 5 mmHg, n = 6, P < 0.05). Strikingly, the behaviour/locomotor activity induced by chemoreflex activation was virtually abolished after blockade of excitatory amino acid receptors in the DMH with kynurenic acid (44 ± 6 versus 5 ± 4 cm, n = 6, P < 0.05). There was no correlation between the reduction in pressor and behavioural chemoreflex responses (r = -0.186, P > 0.05). The bradycardic response of the chemoreflex was not altered by lidocaine or kynurenic acid microinjected into the DMH. These results strongly suggest that the excitatory amino acid receptors in the DMH are essential for full expression of the behavioural response of the chemoreflex and participate, at least in part, in the integration of the pressor response of the chemoreflex in conscious rats.

Sinoaortic denervation prevents enhanced heat loss induced by central cholinergic stimulation during physical exercise.

The present study investigated whether the effects of central cholinergic stimulation on thermoregulation during exercise are modulated by arterial baroreceptors. Wistar rats were submitted to sinoaortic denervation (SAD) or sham denervation (SHAM) and then fitted with a chronic guide cannula into the lateral cerebral ventricle. After 2 weeks, a catheter was implanted into the ascending aorta, and a temperature sensor was implanted into the peritoneal cavity. Two days later, the rats were submitted to exercise on a treadmill at 18 m/min until fatigued. Thermoregulatory and cardiovascular responses were measured after injection of 2 µL of 10mM physostigmine (Phy) or 0.15M NaCl solution (Sal) into the cerebral ventricle. In SHAM rats, Phy injection induced a greater exercise-induced increase in blood pressure and lower increase in heart rate than Sal treatment. In the SAD group, the attenuation of heart rate in response to Phy was blocked despite an exaggerated increase in blood pressure. SHAM rats treated with Phy had a higher increase in tail skin temperature compared to Sal injection (31.9 ± 0.4 °C Phy-SHAM vs. 30.1 ± 0.6 °C Sal-SHAM, 5 min after injection; p<0.05), resulting in a lower exercise-induced increase in core temperature. In contrast, SAD blocked the Phy injection effects in thermoregulatory responses during exercise (tail temperature: 30.1 ± 1.2 °C Phy-SAD vs. 29.5 ± 1.2 °C Sal-SAD, 5 min, p = 0.65). Therefore, we conclude that the enhancement of cutaneous heat loss induced by central cholinergic stimulation during exercise is mediated primarily by arterial baroreceptors.

Effect of L-NAME microinjected into the nucleus tractus solitarius on jejunal glucose and electrolyte absorption in anesthetized rats.

The aim of the present study was to investigate the effects of N(G)-nitro-L-arginine-methyl-ester (L-NAME) microinjected into the rostral nucleus tractus solitarius (NTS) on jejunal glucose, sodium and potassium absorption. Male Wistar rats (210-250 g, n=6-12) were anesthetized and submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and perform a subdiaphragmatic truncal vagotomy or sympathectomy. Either 0.9% NaCl or L-NAME (10 nmol 100 nl⁻¹) was microinjected into the rostral NTS using a stereotaxic instrument. Tyrode solution (pH 8) containing twice the usual concentrations of glucose, sodium and potassium was infused (0.5 ml min⁻¹) into the jejunal loop and samples were taken at 10-min intervals during the 40-min experiment. Results were expressed by the difference between influx and efflux. L-NAME into the NTS increased glucose absorption and decreased potassium absorption when compared to the saline group (38.8 ± 3.8 vs. 50.3 ± 3.3 mg/dl and 0.6±0.01 vs. 0.4 ± 0.03 mM, respectively; p<0.05). Sympathectomy inhibited the glucose absorption caused by L-NAME alone (50.3 ± 3.3 vs. 30.7 ± 4.6 mg/dl; p<0.05), whereas vagotomy inhibited the L-NAME effect on potassium absorption (0.40 ± 0.02 vs. 0.70 ± 0.05; p<0.05). Moreover, increased sodium absorption was observed only in the group that received 30 nmol of L-NAME into NTS (33.0 ± 4.2 vs. 48.4 ± 3.9). In conclusion, the results suggest the participation of endogenous nitric oxide (NO) in the NTS in modulating intestinal glucose and potassium absorption mediated by the autonomic nervous system.

Altered cardiovascular reflexes responses in conscious Angiotensin-(1-7) receptor Mas-knockout mice.

This study evaluated the physiological importance of Angiotensin-(1-7) receptor Mas on reflex control of circulation. Experiments were performed in male Mas-knockout (Mas-KO) and Wild Type (WT) conscious mice (12-20 wk of age). Baroreceptor reflex was evaluated by the bradycardic response induced by phenylephrine (0.25 µg/5 µl, i.v.). Bezold-Jarisch reflex was evaluated by phenylbiguanide (0.5 µg/5 µl, i.v.) and chemoreflex by potassium cyanide (2.5 µg/5 µl, i.v.). Baseline mean arterial pressure was higher in Mas-KO (n=14) as compared with WT mice (n=18) (118±1 mmHg vs. 109±2 mmHg); however, heart rate was similar in both strains (615±30 bpm vs. 648±13 bpm). Baroreflex bradycardia was lower (0.78±0.44 ms/mmHg vs. 1.30±0.14 ms/mmHg) in Mas-KO compared with WT mice. The depressor (-17±5 mmHg vs. -45±6 mmHg) and bradycardic (-212±36 bpm vs. -391±29 bpm) components of the Bezold-Jarisch reflex were also lower in Mas-KO mice. In addition, chemoreflex pressor response (+20±3 mmHg vs. +12±0.8 mmHg) and bradycardic response (-250±74 bpm vs. -52±26 bpm) were significantly higher in Mas-KO. These results further advances previous studies by showing that the lack of Mas receptor induced important imbalance in the neural control of blood pressure, altering not only the baroreflex but also the chemo- and Bezold-Jarisch reflexes.

The enhanced hyperglycemic response to hemorrhage hypotension in obese rats is related to an impaired baroreflex.

The aim of the present study was to assess the metabolic adjustments in adult rats with autonomic imbalance induced by hypercaloric diet. Male Wistar rats (4 weeks of age) were fed a chow diet (CD, n = 12) or hypercaloric diet (HD, n = 13) for 19 weeks. Body weight and dietary intake were measured every week and the basal metabolic rate was assessed. After 19 weeks of diet, six animals from each group were anesthetized with a lethal dose of barbital sodium (100 mg/Kg body weight, intraperitoneal; i.p.). Lee index was evaluated and adipose pads weighted. The remaining animals had a silastic cannula placed into the jugular vein for drug administration, blood collecting, and hemorrhage (1.2 mL/100 g bw/2 min). A polyethylene catheter (PE50) was inserted into the abdominal artery through the femoral artery for cardiovascular monitoring. The assessment of autonomic balance was done by evaluation of baroreflex sensitivity (intravenous (IV) injection of phenylephrine and sodium nitroprusside) and hemorrhage (1.2 mL/100 g bw/2 min). As expected, the HD induced obesity; increased weight gain (28%), adipose pads weight, and baroreflex dysfunction. The plasma level of free fatty acids and triacylglycerols were increased in HD rats by about 124% and 424%, respectively, as well as the basal metabolic rate measured at 19th weeks of diet (p < 0.01). We observed that baroreflex sensitivity to phenylephrine was reduced by about 50%, and the hyperglycemic response to hemorrhage hypotension was increased by 128% in HD rats. We found also a negative correlation between the alteration in baroreflex sensitivity and the increase in hyperglycemic response to hemorrhage in the obese rats (r = 0.72, p < 0.01) and a strong positive correlation between the increased Lee index and the hemorrhagic hyperglycemia (r = 0.93, p < 0.01). Our data demonstrate that obesity induced by hypercaloric diet in Wistar rats promotes an autonomic imbalance, which interferes with metabolic responses dependent on baroreflex sensitivity. In addition, we showed the existence of close correlation between the loss of baroreflex sensitivity and the degree of obesity.

Malnutrition enhances cardiovascular responses to chemoreflex activation in awake rats.

Several studies in the literature suggest that low-protein intake is associated with increases in sympathetic efferent activity and cardiovascular disease. Among the possible mechanisms, changes in the neurotransmission of cardiovascular reflexes have been implicated. Therefore, the present study comprised the evaluation of chemoreflex responsiveness in rats subjected to a low-protein diet during the 35 days after weaning. As a result, we observed that malnourished rats presented higher levels of baseline mean arterial pressure and heart rate and exhibited a mild increase in the pressor response to chemoreflex activation. They also exhibited a massive bradycardic response to chemoreflex activation. Interestingly, bilateral ligature of the carotid body arteries further increased baseline mean arterial pressure and heart rate in malnourished animals. The data suggest severe autonomic imbalance and/or change in the central interplay between neural and cardiovascular mechanisms.

Hyperglycemic response to hemorrhage is modulated by baroreceptors unloading but not by peripheral chemoreceptors activation.

The aim of this study was to assess the relative participation of carotid baro- and chemoreceptors on plasma glucose and lactate level in response to hemorrhagic hypotension. We also evaluated the effects of selective activation of carotid chemoreceptors. One week before the experiments, male Wistar rats (250-300 g) were submitted to bilateral total carotid denervation (BCD-group), or to bilateral ligature of the carotid body artery (ChD-group). During the same surgical procedure, a chronic jugular catheter for blood sampling and hemorrhage (1.2 mL/100 g/2 min) and polyethylene cannula was inserted into the left femoral artery for cardiovascular monitoring. One group submitted to fictitious surgery was used as a surgical control (Sham-group). Carotid chemoreceptors were selectively activated by sodium cyanide (NaCN, 40 microg/0.1 mL i.v.) in the Sham and ChD group. The results showed that hyperglycemic response to hemorrhage in the BCD-group was reduced whereas in the ChD-group there was no significant change in this parameter compared to the Sham group (8.6 +/- 0.5 mM, Sham-hemorrhaged, n = 8; 7.2 +/- 0.3 mM, BCD-hemorrhaged, n = 8 and 9.4 +/- 0.6 mM, ChD, n = 8, p < 0.05). Increased plasma lactate levels following hemorrhage were observed in all the three experimental groups throughout the experimental period and there were no differences between the groups. Chemoreceptor stimulation by NaCN also produced hyperglycemia, as well as an increase in blood pressure and bradycardia but did not affect plasma lactate concentration. Ligature of the carotid body artery annulled the cardiovascular responses induced by NaCN, but did not change the hyperglycemic response to hypoxia. In conclusion, our data indicate that carotid chemoreceptors do not play any major role in overall metabolic response to hypoxia or hemorrhagic hypotension. Furthermore, the results suggest that carotid baroreceptors unloading play a predominant role as main source of afferent impulses leading to the hyperglycemic response to hemorrhage. In addition our data shows that the metabolic response and cardiovascular adjustment to hypoxia can be dissociated by ligature of the carotid body artery.

Effects of vasoactive intestinal polypeptide microinjected into the nucleus tractus solitarius on jejunal electrolytes absorption in rats.

It has been shown that vasoactive intestinal polypeptide (VIP) injected into the nucleus tractus solitarius inhibits alanine absorption across the jejunum. The aim of the present study was to investigate the effects of VIP injection into the nucleus tractus solitarius on jejunal absorption of electrolytes in the rat. Fifty-three Wistar rats were submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and to perform a subdiaphragmatic troncular vagotomy. Saline or VIP (10 pg 100 nl(-1)) was injected into the rostral nucleus tractus solitarius using a stereotaxic instrument. Tyrode solution, pH 8, containing twice glucose, sodium and potassium concentration was infused (0.5 ml min(-1)) into the jejunal loop. Samples were taken at 10-min intervals during the 40-min-experiment. Injection of VIP into the nucleus tractus solitarius increased jejunal potassium absorption. Moreover, VIP associated with vagotomy resulted in inhibition of jejunal potassium absorption by VIP alone at 40 min after perfusion (5.99 +/- 0.74 vs. 9.83 +/- 0.57 microM). There was no change in jejunal sodium absorption in any of the experimental groups. VIP had a modulatory action on jejunal potassium absorption when injected into the nucleus tractus solitarius.

The hypothalamic paraventricular nucleus and carotid receptors modulate hyperglycemia induced by hemorrhage.

The aim of this study was to assess the role of cholinergic transmission in the paraventricular nucleus of the hypothalamus (PVN) and carotid body receptors in mediating a rise in plasma glucose levels in response to hemorrhagic hypotension in rats. Methylatropine (1x10(-9) mol) or 0.15 M NaCl (0.2 microl) was injected into the PVN of Wistar rats weighing 250-300 g bearing a chronic jugular catheter for blood sampling and hemorrhage (1.2 ml/100 g/2 min). Polyethylene cannulae (PE-10) were inserted into the left femoral artery for cardiovascular monitoring. In the other experimental protocol, hemorrhage was performed on rats submitted to bilateral carotid receptor denervation (H-CD). The results show that the hyperglycemic response to hemorrhage was decreased by either methylatropine (H-MA) treatment or bilateral carotid receptor denervation (10.3+/-0.4 mM, control, n=15 vs. 7.7+/-0.2 mM, H-MA, n=12, and 7.6+/-0.3 mM, H-CD, n=5, p<0.01). Furthermore, methylatropine did not affect the recovery of blood pressure after hemorrhage-induced hypotension, suggesting that the metabolic and pressor adjustments have different efferent pathways. Our data demonstrate that cholinergic input from the PVN and carotid receptors (chemo- and/or baroreceptors) might participate in the same neural pathway activated by hemorrhage-induced hypotension that produces hyperglycemia.

Effects of vasoactive intestinal polypeptide microinjected into the nucleus tractus solitarius on jejunal glucose absorption in rats.

It has been shown that vasoactive intestinal polypeptide (VIP) injected into the nucleus tractus solitarius and into the dorsal motor nucleus of the vagus inhibits alanine absorption across the jejunum. The aim of the present study was to investigate the effects of VIP injection into the nucleus tractus solitarius on jejunal absorption of glucose in the rat. Forty Wistar rats were submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and to perform a subdiaphragmatic troncular vagotomy. Saline or VIP (10 pg 100 nl(-1)) was injected into the rostral nucleus tractus solitarius using a stereotaxic instrument. Tyrode solution, pH 8, containing twice glucose, sodium, and potassium concentrations was infused (0.5 ml min(-1)) into the jejunal loop. Samples were taken at 10-min intervals during the 40-min experiment. Injection of VIP into the nucleus tractus solitarius associated with vagotomy resulted in inhibition of jejunal glucose absorption by VIP alone at 10 and 40 min after perfusion (2.75+/-0.19 vs. 3.53+/-0.29 mg). The vagal outflow tract maintained jejunal glucose absorption even when VIP was microinjected into the nucleus tractus solitarius.