Leonardo M. Cardoso

Cardiovascular responses to hydrogen peroxide into the nucleus tractus solitarius

The nucleus tractus solitarius (NTS), a major hindbrain area involved in cardiovascular regulation, receives primary afferent fibers from peripheral baroreceptors and chemoreceptors. Hydrogen peroxide (H(2)O(2)) is a relatively stable and diffusible reactive oxygen species (ROS), which acting centrally, may affect neural mechanisms. In the present study, we investigated effects of H(2)O(2) alone or combined with the glutamatergic antagonist kynurenate into the NTS on mean arterial pressure (MAP) and heart rate (HR). Conscious or anesthetized (urethane and alpha-chloralose) male Holtzman rats (280-320 g) were used. Injections of H(2)O(2) (125 to 1500 pmol/40 nl) into the intermediate NTS of anesthetized rats evoked dose-dependent and transient hypotension (-18 +/- 3 to -55 +/- 11 mmHg) and bradycardia (-16 +/- 5 to -116 +/- 40 bpm). Injection of the catalase inhibitor 3-amino-1,2,4-triazole (100 nmol/40 nl) into the NTS also produced hypotension and bradycardia. Previous injection of the ionotropic L-glutamate receptor antagonist kynurenate (7 nmol/40 nl) attenuated by 48% the bradycardic response, without changing the hypotension evoked by H(2)O(2) (500 pmol/40 nl) in anesthetized rats. The antioxidant L-ascorbate (600 pmol/80 nl) injected into the NTS attenuated the bradycardic (42%) and hypotensive (67%) responses to H(2)O(2) (500 pmol/40 nl) into the NTS. In conscious rats, injection of H(2)O(2) (50 nmol/100 nl) into the NTS also evoked intense bradycardia (-207 +/- 8 bpm) and hypotension (-54 +/- 6 mmHg) that were abolished by prior injection of kynurenate (7 nmol/100 nl). The results show that H(2)O(2) into the NTS induces hypotension and bradycardia probably due to activation of glutamatergic mechanisms.

A Low Protein Diet Causes an Increase in the Basal Levels and Variability of Mean Arterial Pressure and Heart Rate in Fisher Rats

Abstract The correlation between nutrition and cardiovascular related disorders is a well-established fact. Previous work from our Laboratory has suggested a significant compromise of cardiovascular reflexes in conscious rats submitted to a low-protein (LP) diet. Our working hypothesis is that the basal level of mean arterial pressure (MAP), variability of the mean arterial pressure (VMAP), heart rate (HR) and variability of heart rate (VHR) are altered in rats submitted to a protein restricted diet. Two experimental groups were used: control group (normal protein 15%, NP) and malnourished group (low-protein 6%, LP). In order to verify the efficiency of the dietary restriction we measured body weight, total blood protein, plasma albumin, urea and glucose. Our experiments demonstrated that the malnourished rats presented augment levels of basal MAP (LP 122±2 mmHg vs. NP 113±1 mmHg) and of VMAP (LP 12.8±1.5 mmHg vs. NP 9±1 mmHg) when compared to the control group. We observed similar increased levels, in the malnourished group, for both HR (LP 429±8 bpm vs. NP 381±7 bpm) and VHR (LP 67.6±8.3 bpm vs. NP 44.4±4.9 bpm). Our results suggest a correlation between the LP diet in Fisher rats and the increased basal levels of mean arterial pressure, HR and their respective variability.

Effects of low-protein diet on the baroreflex and Bezold-Jarisch reflex in conscious rats.

The present study evaluated the effects of a low-protein diet (LP, 6% protein) on cardiovascular reflexes of Male Fisher rats. Three experimental groups, and their respective controls (15% protein), were used. (1) Baroreceptor reflex (BAR); (2) Bezold-Jarisch reflex (BJR); and (3) Prazosin treated. Dietary restriction began after weaning (three weeks) and lasted for a period of five weeks, after which animals were subjected to the experimental protocols. The BAR group was evaluated through injections of phenylephrine (0.55.0 μg/Kg, i.v.) and sodium nitroprusside (0.77.0 μg/Kg, i.v.) while the BJR was evaluated through injections of serotonin (2.510μg/Kg, i.v.). Our results showed an increased baroreflex gain bradycardia for the LP group (-0.96 ± 0.34 vs. -2.12 ± 1.06 bpm/mmHg) and a larger bradycardia for the BJR the LP group (160 ± 18% greater than controls). Basal cardiovascular parameters were not different between LP and control rats, however LP animals treated with prazosin resulted in a larger fall of blood pressure (-19±3 vs. -28±5 mmHg). In conclusion, LP rats present an increased responsiveness of BAR and BJR, which could contribute to their normal levels of cardiovascular parameters, in spite of the possible increase in the sympathetic vasomotor tonus observed with the prazosin protocol.

Cardiovascular responses produced by central injection of hydrogen peroxide in conscious rats

Reactive oxygen species (ROS) have been shown to modulate neuronal synaptic transmission and may play a role on the autonomic control of the cardiovascular system. In this study we investigated the effects produced by hydrogen peroxide (H(2)O(2)) injected alone or combined with the anti-oxidant agent N-acetil-l-cysteine (NAC) or catalase into the fourth brain ventricle (4th V) on mean arterial pressure and heart rate of conscious rats. Moreover the involvement of the autonomic nervous system on the cardiovascular responses to H(2)O(2) into the 4th V was also investigated. Male Holtzman rats (280-320 g) with a stainless steel cannula implanted into the 4th V and polyethylene cannulas inserted into the femoral artery and vein were used. Injections of H(2)O(2) (0.5, 1.0 and 1.5 micromol/0.2 microL, n=6) into the 4th V produced transient (for 10 min) dose-dependent pressor responses. The 1.0 and 1.5 micromol doses of H(2)O(2) also produced a long lasting bradycardia (at least 24 h with the high dose of H(2)O(2)). Prior injection of N-acetyl-l-cysteine (250 nmol/1 microL/rat) into the 4th V blockade the pressor response and attenuated the bradycardic response to H(2)O(2) (1 micromol/0.5 microL/rat, n=7) into the 4th V. Intravenous (i.v.) atropine methyl bromide (1.0 mg/kg, n=11) abolished the bradycardia but did not affect the pressor response to H(2)O(2). Prazosin hydrochloride (1.0 mg/kg, n=6) i.v. abolished the pressor response but did not affect the bradycardia. The increase in the catalase activity (500 UEA/1 microL/rat injected into the 4th V) also abolished both, pressor and bradycardic responses to H(2)O(2). The results suggest that increased ROS availability into 4th V simultaneously activate sympathetic and parasympathetic outflow inducing pressor and bradycardic responses.

Central antioxidant therapy inhibits parasympathetic baroreflex control in conscious rats

Baroreceptor reflex is an important system for neural control of blood pressure. Recently, reactive oxygen species (ROS) have been shown to play an important role in neuronal activity of central areas related to blood pressure control. The aim of this study was to investigate the effects elicited by ascorbic acid (AAC) and N-acetylcysteine (NAC) injections into the 4thV on the parasympathetic component of the baroreflex. Male Wistar rats were implanted with a stainless steel guide cannula into the 4thV. One day prior to the experiments, the femoral artery and vein were cannulated for pulsatile arterial pressure, mean arterial pressure and heart rate measurements and drug administration, respectively. After baseline recordings, the baroreflex was tested with a pressor dose of phenylephrine (PHE, 3 μg/kg, i.v.) and a depressor dose of sodium nitroprusside (SNP, 30 μg/kg, i.v.) before (control) and 5, 15, 30 and 60 min after AAC or NAC into the 4thV. Control PHE injection induced baroreflex-mediated bradycardia (-93 ± 13 bpm, n=7). Interestingly, after AAC injection into the 4thV, PHE injection produced a transient tachycardia at 5 (40 ± 23 bpm), 15 (26 ± 22 bpm) and 30 min (59 ± 21 bpm). No changes were observed in baroreflex-mediated tachycardia evoked by SNP after AAC injection on 4thV (control: 151 ± 23bpm vs. 135 ± 18 bpm at 5 min after AAC, n=7). In the NAC treated group, PHE induced a reduction in reflex bradycardia at 5 min when compared to control (-11 ± 17 bpm vs. -83 ± 15 bpm, n=7). No changes were observed in baroreflex-mediated tachycardia evoked by SNP after NAC injection on 4thV. The antioxidants AAC and NAC may act in the central nervous system affecting the parasympathetic component of the cardiac baroreflex.

Increased activity of the renin-angiotensin and sympathetic nervous systems is required for regulation of the blood pressure in rats fed a low-protein diet.

•  What is the central question of this study? Is the increase in blood pressure observed in rats fed a low protein diet due to overactivation of sympathetic and renin angiontensin systems? •  What is the main finding and its importance? The data show an increase in the expression of angiotensin II type 1 receptors and an incrased sympathetic activity in this experimental model, suggesting that both systems are contributing to the high blood pressure observed in these animals.

Baroreflex function in conscious rats submitted to iron overload

Our hypothesis is that iron accumulated in tissue, rather than in serum, may compromise cardiovascular control. Male Fischer 344 rats weighing 180 to 220 g were divided into 2 groups. In the serum iron overload group (SIO, N = 12), 20 mg elemental iron was injected ip daily for 7 days. In the tissue iron overload group (TIO, N = 19), a smaller amount of elemental iron was injected (10 mg, daily) for 5 days followed by a resting period of 7 days. Reflex heart rate responses were elicited by iv injections of either phenylephrine (0.5 to 5.0 microg/kg) or sodium nitroprusside (1.0 to 10.0 microg/kg). Baroreflex curves were determined and fitted to sigmoidal equations and the baroreflex gain coefficient was evaluated. To evaluate the role of other than a direct effect of iron on tissue, acute treatment with the iron chelator deferoxamine (20 mg/kg, iv) was performed on the TIO group and the baroreflex was re-evaluated. At the end of the experiments, evaluation of iron levels in serum confirmed a pronounced overload for the SIO group (30-fold), in contrast to the TIO group (2-fold). Tissue levels of iron, however, were higher in the TIO group. The SIO protocol did not produce significant alterations in the baroreflex curve response, while the TIO protocol produced a nearly 2-fold increase in baroreflex gain (-4.34 +/- 0.74 and -7.93 +/- 1.08 bpm/mmHg, respectively). The TIO protocol animals treated with deferoxamine returned to sham levels of baroreflex gain (-3.7 +/- 0.3 sham vs -3.6 +/- 0.2 bpm/mmHg) 30 min after the injection. Our results indicate an effect of tissue iron overload on the enhancement of baroreflex sensitivity.

Oxidative stress causes hypertension and activation of nuclear factor-κB after high-fructose and salt treatments

There is evidence that diets rich in salt or simple sugars as fructose are associated with abnormalities in blood pressure regulation. However, the mechanisms underlying pathogenesis of salt- and fructose-induced kidney damage and/or consequent hypertension yet remain largely unexplored. Here, we tested the role of oxidative state as an essential factor along with high salt and fructose treatment in causing hypertension. Fischer male rats were supplemented with a high-fructose diet (20% in water) for 20 weeks and maintained on high-salt diet (8%) associate in the last 10 weeks. Fructose-fed rats exhibited a salt-dependent hypertension accompanied by decrease in renal superoxide dismutase activity, which is the first footprint of antioxidant inactivation by reactive oxygen species (ROS). Metabolic changes and the hypertensive effect of the combined fructose-salt diet (20 weeks) were markedly reversed by a superoxide scavenger, Tempol (10 mg/kg, gavage); moreover, Tempol (50 mM) potentially reduced ROS production and abolished nuclear factor-kappa B (NF-κB) activation in human embryonic kidney HEK293 cells incubated with L-fructose (30 mM) and NaCl (500 mosmol/kg added). Taken together, our data suggested a possible role of oxygen radicals and ROS-induced activation of NF-κB in the fructose- and salt-induced hypertension associated with the progression of the renal disease.

Bezold–Jarisch reflex in sino-aortic denervated malnourished rats

In this study we assessed the role of Bezold-Jarisch reflex (BJR) in the regulation of blood pressure (BP) of malnourished (MN) and control rats (CN) with sino-aortic denervation (SAD). Fischer rats were fed diets containing either 6% (MN) or 15% (CN) protein for 35 days after weaning. These rats underwent sham or SAD and catheterization of femoral artery and vein for BP measurements and drug injection. Phenylbiguanide (PBG 5 μg/kg, i.v.) for activation BJR, produced bradycardia (-317±22 bpm for CN vs. -372±16 bpm for MN) and hypotension (-57±4 mm Hg for CN vs. -54±6 mm Hg for MN. After SAD, MN rats had reduced hypotensive (-37±7 mm Hg for MN vs. -82±6 mm Hg for CN) and bradycardic (-124±17 for MN vs. -414±20 bpm CN) responses to BJR activation. To evaluate the contribution of the parasympathetic component due to BJR for the fall in BP, methyl atropine bromide, was given between two injections of PBG (5 μg/kg) separated by 10 min each other. Both bradycardic (-216±21 bpm before and -4±3 bpm after for CN -226±43 bpm before and -9±20 bpm after for MN) and hypotensive (-42±4 mm Hg before and -6±1 mm Hg after for CN -33±9 mm Hg before and -5±2 mm Hg after for MN) responses were abolished in CN and MN groups. These data indicate that dietary protein malnutrition changes the relation between baroreflex and BJR required for maintenance of the BP during malnourishment.

Sympathoinhibition to Bezold–Jarisch reflex is attenuated in protein malnourished rats

Malnutrition affects cardiovascular reflexes, including chemoreflex and baroreflex. In this study we assessed the hypothesis that malnourishment changes the responses in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) evoked from Bezold-Jarisch reflex (BJR). Fischer rats were fed diets containing either (6% malnourished or 14% control) protein for 35 days after weaning. There were no differences in baseline MAP (102 ± 4 vs. 95 ± 3 mmHg) whereas higher baseline HR (478 ± 18 vs. 360 ± 11 bpm; P<0.05,) and reduced sympathoinhibition (ΔRSNA=-54 ± 9 vs. -84 ± 7%; P=0.0208) to BJR activation were found in malnourished rats. We conclude that malnutrition affects the sympathetic control of BJR.