Hydrogen sulfide ameliorates hypertension and vascular dysfunction induced by insulin resistance in rats by reducing oxidative stress and activating eNOS.

Hydrogen sulfide (H2S) is a gasotransmitter implied in metabolic diseases, insulin resistance, obesity, and type 2 Diabetes Mellitus. This study aimed to determine the effect of chronic administration of sodium hydrosulfide (NaHS; inorganic H2S donor), L-Cysteine (L-Cys; substrate of H2S producing enzymes) and DL-Propargylglycine (DL-PAG; cystathionine-gamma-lyase inhibitor) on the vascular dysfunction induced by insulin resistance in rat thoracic aorta. For this purpose, 72 animals were divided into two main sets that received: 1) tap water (control group; n = 12); and 2) fructose 15% w/v in drinking water [insulin resistance group (IR); n = 60] for 20 weeks. After 16 weeks, the group 2 was divided into five subgroups (n = 12 each), which received daily i. p. injections during 4 weeks of: 1) non-treatment (control); 2) vehicle (phosphate buffer saline; PBS, 1 ml/kg); 3) NaHS (5.6 mg/kg); 4) L-Cys (300 mg/kg); and (5) DL-PAG (10 mg/kg). Hemodynamic variables, metabolic variables, vascular function, ROS levels and the expression of p-eNOS and eNOS were determined. IR induced: 1) hyperinsulinemia; 2) increased HOMA-index; 3) decreased Matsuda index; 4) hypertension, vascular dysfunction, increased ROS levels; 5) increased iNOS, and 6) decreased CSE, p-eNOS and eNOS expression. Furthermore, IR did not affect contractile responses to norepinephrine. Interestingly, NaHS and L-Cys treatment, reversed IR-induced impairments and DL-PAG treatment decreased and increased the HOMA and Matsuda index, respectively. Taken together, these results suggest that NaHS and L-Cys decrease the metabolic and vascular alterations induced by insulin resistance by reducing oxidative stress and activating eNOS. Thus, hydrogen sulfide may have a therapeutic application.

NaHS restores the vascular alterations in the renin-angiotensin system induced by hyperglycemia in rats.

Hyperglycemia (HG) impairs the renin-angiotensin system (RAS), which may contribute to vascular dysfunction. Besides, hydrogen sulfide (H2S) exerts beneficial cardiovascular effects in metabolic diseases. Therefore, our study aimed to determine the effects of chronic administration of sodium hydrosulfide (NaHS; inorganic H2S donor) and DL-Propargylglycine [DL-PAG; cystathionine-×¥-lyase (CSE) inhibitor] on the RAS-mediated vascular responses impairments observed in thoracic aortas from male diabetic Wistar rats. For that purpose, neonatal rats were divided into two groups that received: 1) citrate buffer (n = 12) or 2) streptozotocin (STZ, 70 mg/kg; n = 48) on the third postnatal day. After 12 weeks, diabetic animals were divided into 4 subgroups (n = 12 each) that received daily i.p. injections during 4 weeks of: 1) non-treatment; 2) vehicle (PBS, 1 mL/kg); 3) NaHS (5.6 mg/kg); and 4) DL-PAG (10 mg/kg). After treatments (16 weeks), blood glucose, angiotensin-(1-7) [Ang-(1-7)], and angiotensin II (Ang II) levels, vascular responses to Ang-(1-7) and Ang II, and the expression of angiotensin AT1, AT2, and Mas receptors, angiotensin converting enzyme (ACE) and ACE type 2 (ACE2) were determined. HG induced: 1) increased blood glucose levels and expression of angiotensin II AT1 receptor; 2) impaired Ang-(1-7) and Ang II mediated vascular responses; 3) decreased angiotensin levels and expression of angiotensin II AT2 and angiotensin-(1-7) Mas receptors, and ACE2; and 4) no changes in ACE expression. Interestingly, NaHS, but not DL-PAG, reversed HG-induced impairments, except for blood glucose level changes. These results suggest that NaHS restores vascular function in streptozotocin-induced HG through RAS modulation.

Hydrogen sulfide prevents the vascular dysfunction induced by severe traumatic brain injury in rats by reducing reactive oxygen species and modulating eNOS and H2S-synthesizing enzyme expression.

AIM: To assess the effects of subchronic administration with NaHS, an exogenous H2S donor, on TBI-induced hypertension and vascular impairments. MAIN METHODS: Animals underweministration does not prevent the body weight loss but slightly imnt a lateral fluid percussion injury, and the hemodynamic variables were measured in vivo by plethysmograph method. The vascular function in vitro, the ROS levels by the DCFH-DA method and the expression of H2S-synthesizing enzymes and eNOS by Western blot were measured in isolated thoracic aortas at day 7 post-TBI. The effect of L-NAME on NaHS-induced effects in vascular function was evaluated. Brain water content was determined 7 days after trauma induction. Body weight was recorded throughout the experimental protocol, whereas the sensorimotor function was evaluated using the neuroscore test at days -1 (basal), 2, and 7 after the TBI induction. KEY FINDINGS: TBI animals showed: 1) an increase in hemodynamic variables and ROS levels in aortas; 2) vascular dysfunction; 3) sensorimotor dysfunction; and 4) a decrease in body weight, the expression of H2S-synthesizing enzymes, and eNOS phosphorylation. Interestingly, NaHS subchronic administration (3.1 mg/kg; i.p.; every 24 h for six days) prevented the development of hypertension, vascular dysfunction, and oxidative stress. L-NAME abolished NaHS-induced effects. Furthermore, NaHS treatment restored H2S-synthesizing enzymes and eNOS phosphorylation with no effect on body weight, sensorimotor impairments, or brain water content. SIGNIFICANCE: Taken together, these results demonstrate that H2S prevents TBI-induced hypertension by restoring vascular function and modulating ROS levels, H2S-synthesizing enzymes expression, and eNOS phosphorylation.

Pharmacological evidence that potassium channels mediate hydrogen sulfide-induced inhibition of the vasopressor sympathetic outflow in pithed rats.

Hydrogen sulfide (H2S) is a gasotransmitter that modulates neurotransmission. Indeed, it has been recently demonstrated that H2S inhibits the sympathetic outflow in male rats, although the mechanisms remain elusive. Thus, this study evaluated the role of potassium channels on NaHS-induced sympathoinhibition. For this purpose, male and female Wistar rats were anesthetized, pithed, and cannulated. After that, animals received selective electrical stimulation of the vasopressor sympathetic outflow (T7-T9). Prior to 310 µg/kg·min NaHS i.v. continuous infusion animals received: (1) bidistilled water (tetraethylammonium, TEA; 4-aminopyridine, 4-AP; and barium chloride, BaCl2; vehicle; 1 ml/kg); (2) TEA (non-selective K+ channels blocker; 16.5 mg/kg); (3) 4-AP (non-selective voltage-dependent K+ channels blocker; 5 mg/kg); (4) BaCl2 (inward rectifier K+ channels blocker; 65 µg/kg); (5) DMF 5%, glucose 10% and NaOH 0.1 N (glibenclamide vehicle; 1 ml/kg); (6) glibenclamide (ATP-dependent K+ channels blocker; 10 mg/kg); (7) DMSO 4% (paxilline vehicle; 1 ml/kg); and (8) paxilline (large-conductance voltage- and Ca2+-activated K+ channel blocker; 90 µg/kg). The NaHS-induced sympathoinhibition was: (1) equally observed in male and female rats; (2) unaffected by vehicles; (3) reversed by the potassium channel blockers. Taken together, our results suggest that NaHS-induced sympathoinhibition does not depend on sex and it is mediated by the activation of several potassium channels.

Exogenous hydrogen sulfide restores CSE and CBS but no 3-MST protein expression in the hypothalamus and brainstem after severe traumatic brain injury.

Hydrogen sulfide (H2S) is a gasotransmitter endogenously synthesized by cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), and 3-mercaptopiruvate sulfurtransferase (3-MST) enzymes. H2S exogenous administration prevents the development of hemodynamic impairments after traumatic brain injury (TBI). Since the hypothalamus and the brainstem highly regulate the cardiovascular system, this study aimed to evaluate the effect of NaHS subchronic treatment on the changes of H2S-sythesizing enzymes in those brain areas after TBI and in physiological conditions. For that purpose, animals were submitted to a lateral fluid percussion injury, and the changes in CBS, CSE, and 3-MST protein expression were measured by western blot at days 1, 2, 3, 7, and 28 in the vehicle group, and 7 and 28 days after NaHS treatment. After severe TBI induction, we found a decrease in CBS and CSE protein expression in the hypothalamus and brainstem; meanwhile, 3-MST protein expression diminished only in the hypothalamus compared to the Sham group. Remarkably, i.p. daily injections of NaHS, an H2S donor, (3.1 mg/kg) during seven days: (1) restored CBS and CSE but no 3-MST protein expression in the hypothalamus at day 28 post-TBI; (2) reestablished only CSE in brainstem 7 and 28 days after TBI; and (3) did not modify H2S-sythesizing enzymes protein expression in uninjured animals. Mainly, our results show that the NaHS effect on CBS and CSE protein expression is observed in a time- and tissue-dependent manner with no effect on 3-MST expression, which may suggest a potential role of H2S synthesis in hypothalamus and brainstem impairments observed after TBI.

Hydrogen Sulfide Subchronic Treatment Improves Hypertension Induced by Traumatic Brain Injury in Rats through Vasopressor Sympathetic Outflow Inhibition.

Traumatic brain injury (TBI) represents a critical public health problem around the world. To date, there are no accurate therapeutic approaches for the management of cardiovascular impairments induce by TBI. In this regard, hydrogen sulfide (H2S), a novel gasotransmitter, has been proposed as a neuro- and cardioprotective molecule. This study was designed to determine the effect of subchronic management with sodium hydrosulfide (NaHS) on hemodynamic, vasopressor sympathetic outflow and sensorimotor alterations produced by TBI. Animals underwent a lateral fluid percussion injury, and changes in hemodynamic variables were measured by pletismographic methods. In addition, vasopressor sympathetic outflow was assessed by a pithed rat model. Last, sensorimotor impairments were evaluated by neuroscore test and beam-walking test. At seven, 14, 21, and 28 days after moderate-severe TBI, the animals showed: (1) a decrease on sensorimotor function in the neuroscore test and beam-walking test; (2) an increase in heart rate, systolic, diastolic, and mean blood pressure; (3) progressive sympathetic hyperactivity; and (4) a decrease in vasopressor responses induced by noradrenaline (α1/2-adrenoceptors agonist) and UK 14,304 (selective α2-adrenoceptor agonist). Interestingly, intraperitoneal daily injections of NaHS, an H2S donor (3.1 and 5.6 mg/kg), during seven days after TBI prevented the development of the impairments in hemodynamic variables, which were similar to those obtained in sham animals. Moreover, NaHS treatment prevented the sympathetic hyperactivity and decreased noradrenaline-induced vasopressor responses. No effects on sensorimotor dysfunction were observed, however. Taken together, our results suggest that H2S ameliorates the hemodynamic and sympathetic system impairments observed after TBI.

Fenofibrate Protects Cardiomyocytes from Hypoxia/Reperfusion- and High Glucose-Induced Detrimental Effects.

Lesions caused by high glucose (HG), hypoxia/reperfusion (H/R), and the coexistence of both conditions in cardiomyocytes are linked to an overproduction of reactive oxygen species (ROS), causing irreversible damage to macromolecules in the cardiomyocyte as well as its ultrastructure. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, promotes beneficial activities counteracting cardiac injury. Therefore, the objective of this work was to determine the potential protective effect of fenofibrate in cardiomyocytes exposed to HG, H/R, and HG+H/R. Cardiomyocyte cultures were divided into four main groups: (1) control (CT), (2) HG (25 mM), (3) H/R, and (4) HG+H/R. Our results indicate that cell viability decreases in cardiomyocytes undergoing HG, H/R, and both conditions, while fenofibrate improves cell viability in every case. Fenofibrate also decreases ROS production as well as nicotinamide adenine dinucleotide phosphate oxidase (NADPH) subunit expression. Regarding the antioxidant defense, superoxide dismutase (SOD Cu2+/Zn2+ and SOD Mn2+), catalase, and the antioxidant capacity were decreased in HG, H/R, and HG+H/R-exposed cardiomyocytes, while fenofibrate increased those parameters. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) increased significantly in treated cells, while pathologies increased the expression of its inhibitor Keap1. Oxidative stress-induced mitochondrial damage was lower in fenofibrate-exposed cardiomyocytes. Endothelial nitric oxide synthase was also favored in cardiomyocytes treated with fenofibrate. Our results suggest that fenofibrate preserves the antioxidant status and the ultrastructure in cardiomyocytes undergoing HG, H/R, and HG+H/R preventing damage to essential macromolecules involved in the proper functioning of the cardiomyocyte.

Chronic administration of NaHS and L-Cysteine restores cardiovascular changes induced by high-fat diet in rats.

Hydrogen sulfide plays an important role in the regulation of the cardiovascular system, insulin secretion, and glucose homeostasis. The aim of the present study was to examine the effects of chronic treatment with sodium hydrosulfide (NaHS), L-Cysteine (L-Cys) and DL-Propargylglycine (DL-PAG) on the changes induced by a high-fat diet (HFD) in zoometric and metabolic variables as well as cardiovascular changes such as hypertension and sympathetic hyperactivity. For this purpose, male Wistar rats were fed a normal fat diet (NFD) or HFD for 12 weeks. Next, the HFD rats were divided into 5 subgroups which received daily i.p. injections during 4 weeks of: (1) nothing (no injection, Control); (2) vehicle (PBS; 1ml/kg); (3) NaHS (5.6 mg/kg); (4) L-Cys (300mg/kg); or (5) DL-PAG (1mg/kg). Then, an oral glucose tolerance test, hormone serum levels and blood pressure were determined. The cardiovascular responses to stimulation of the vasopressor sympathetic tone or intravenous administration of the agonists noradrenaline (α1/2-adrenoceptors), methoxamine (α1-adrenoceptors) and UK 14,304 (α2-adrenoceptors) were determined in pithed rats. Lastly, the heart, liver and adipose tissue were weighted. HFD significantly increased: (1) zoometric variables, which were decreased by NaHS and L-Cys; (2) metabolic variables, ameliorated by DL-PAG; (3) haemodynamic variables, which were reversed by NaHS and L-Cys; and (4) the vasopressor responses induced by sympathetic stimulation, which were diminished by NaHS and L-Cys. In conclusion, chronic treatment with NaHS and L-Cys are effective in reducing adipose tissue and ameliorating the cardiovascular changes induced by obesity; meanwhile, DL-PAG ameliorates metabolic variables.

Therapeutic effect of treatment with metformin and/or 4-hydroxychalcone in male Wistar rats with nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. Despite the impact of this pathology in the population, nowadays there is no specific treatment for this disease, focusing its treatment on risks factors. However, it is imperative the existence of a specific treatment, due to this, the aim of this study was to determine the therapeutic effect of treatment with metformin, 4-hydroxychalcone or co-treatment on male Wistar rats with NAFLD. Wistar rats were divided into two groups with free access to either tap water or 50% sucrose (NAFLD) during 25 weeks. After 20 weeks of induction each were divided into four groups that received daily p.o. administration of: i) saline solution (1 ml); ii) metformin (200 mg/kg/day); iii) 4-hydroxychalcone (80 mg/kg/day) and i.v.) co-treatment (metformin plus 4-hydroxychalcone at the doses mentioned above), for 5 weeks. In healthy rats: metformin and co-treatment modified food and total caloric intake and induced diarrhea; but none of the treatments changed the other parameters evaluated. Meanwhile in rats with NAFLD: i) metformin inhibited hepatic total cholesterol and TGF-ß, increased diarrhea frequency, and slightly decreased liver steatosis, and fibrosis; ii) 4-hydroxychalcone decreased IL-6, TNF-α and TGF-ß, increased IL-10, and markedly decreased liver steatosis and fibrosis; and iii) co-treatment markedly decreased food intake, total caloric intake, and body weight, increased diarrhea; increased IL-10, showing and intermediate effect on decrease TNF-α, TGF-ß, liver steatosis and fibrosis. Our results showed that 4-hydroxychalcone treatment was the most effective among the treatments tested against NAFLD.

Potential vascular α1-adrenoceptor blocking properties of metformin in rat aorta and tail artery.

Metformin is a widely used drug for the treatment of type 2 Diabetes Mellitus. Several studies have also suggested that metformin decreases blood pressure; although an interaction with α-adrenoceptors has been proposed, this mechanism needs to be further investigated. Since α1-adrenoceptors play a significant role to regulate vascular tone, this study has analysed the potential ability of metformin to block α1-adrenoceptors in rat aorta and tail artery. For this purpose, the contractile responses induced by noradrenaline, methoxamine, and phenylephrine were determined in the absence or presence of metformin in rat aorta and tail artery rings. In both arteries, noradrenaline, methoxamine, and phenylephrine produced concentration-dependent contractile responses. Interestingly, the contractile responses to noradrenaline, methoxamine, and phenylephrine were significantly and differentially blocked by metformin (1, 3.1 and/or 10 mM) but not by vehicle. These results suggest that metformin is capable to block α1-adrenoceptors and may explain, at least in part, the anti-hypertensive effect observed in several clinical trials.

Histopathological and biochemical changes in the development of nonalcoholic fatty liver disease induced by high-sucrose diet at different times.

The high intake of sweetened drinks is associated with obesity and insulin resistance. These pathologies are directly related to the development of nonalcoholic fatty liver disease (NAFLD), considered a condition of metabolic syndrome (MS). Due to their increasing worldwide prevalence, experimental animal models have been developed to gain a better understanding of its physiopathology; notwithstanding, few studies have evaluated its progression in association with MS and ingestion of sweetened drinks. Therefore, the aim of this study was to understand the pathophysiologic characteristics of NAFLD related to sucrose concentration and time of ingestion in rats. Wistar rats were divided into 2 groups with free access to either tap water or 30% sucrose, and euthanized at 12, 16, or 20 weeks; and 2 additional groups were given free access to either 40% or 50% sucrose and were euthanized at 20 weeks. Biochemical parameters and levels of serum cytokines were measured, and histology was performed. Ingestion of 30% sucrose induced liver steatosis until 16 weeks (grade 2) and 20 weeks (grade 3). Meanwhile, during 20 weeks, 40% sucrose induced grade 5 of nonalcoholic steatohepatitis (NASH) and 50% sucrose induced grade 6 of NASH and fibrosis. This study demonstrated that increasing time of induction and concentration of sucrose ingestion resulted in a higher grade of NAFLD.

Pharmacological evidence that metformin blocks the vasopressor responses mediated by stimulation of α1- and α2-adrenoceptors in pithed rats.

It has been reported that metformin reduces blood pressure although the mechanisms have not been described. Indeed, several mechanisms could be implicated including the interaction with α-adrenoceptors or inhibition of sympathetic outflow. Therefore, this study was designed to determine the capability of metformin to block the vasopressor responses induced by α1/2-adrenoceptor agonists or selective electrical stimulation of sympathetic outflow. For this purpose, Wistar male rats were anesthetized, pithed and cannulated for selective preganglionic stimulation of the vasopressor sympathetic outflow or drugs administration. The effect of i.v. bolus injection of metformin (180 and 310mg/kg) or its vehicle (bidistilled water) was studied on the vasopressor responses induced by: (1) selective sympathetic stimulation (0.03-3Hz); (2) exogenous noradrenaline (0.03-3µg/kg); (3) methoxamine (1-100µg/kg); and (4) UK 14,304 (0.1-30µg/kg). The tachycardic responses to noradrenaline were also investigated in presence of metformin. The vasopressor responses induced by selective electrical stimulation of sympathetic outflow were diminished by metformin (180 and 310mg/kg) and remained unchanged in presence of vehicle. Moreover, the vasopressor responses induced by exogenous noradrenaline, methoxamine and UK 14,304 were dose-dependently inhibited by i.v. bolus injections of metformin (180 and 310mg/kg) and were not affected by vehicle. Metformin practically did not block the tachycardic responses to noradrenaline except at the dose of 3µg/kg. Taken together, these results demonstrate that metformin is capable to block vascular α1/2-adrenoceptors but not cardiac ß-adrenoceptors. Thus, this mechanism could contribute, at least in part, on the hypotensive responses induced by metformin.

Pharmacological evaluation of metformin and N-benzylbiguanide, a novel analogue of metformin, on the vasopressor responses to adrenergic system stimulation in pithed rats with fructose-induced insulin resistance.

Metformin has been associated with cardioprotection, vasorelaxation and normalization of endothelial function during type 2 Diabetes Mellitus. However, few studies have analysed its effects on vascular adrenergic system. Our study has evaluated the vasopressor responses induced by sympathetic stimulation or by i.v. bolus injections of the agonists noradrenaline (α1/2), methoxamine (α1) and UK 14,304 (α2) in rats with fructose-induced insulin resistance chronically pretreated with either metformin or EGL-6M (N-benzylbiguanide), a novel analogue of metformin. Rats were treated with fructose (15%) or tap water (control) during 16 weeks. Next, both groups were treated daily during 4 weeks with: (1) vehicle; (2) metformin (50mg/kg); or (3) EGL-6M (50mg/kg). Blood glucose and plasma insulin were determined before and after administration of glucose during oral glucose tolerance test. Animals treated with fructose showed hyperinsulinemia and insulin resistance, which were decreased by metformin and EGL-6M. In animals treated with fructose, the vasopressor responses induced by: (1) sympathetic stimulation were decreased; (2) noradrenaline were increased; and (3) methoxamine and UK 14,304 remained unaffected compared with control group. In control animals, metformin failed to modify the vasopressor responses analysed, while EGL-6M increased the vasopressor responses to sympathetic stimulation. In rats treated with fructose, metformin decreased vasopressor response to noradrenaline but did not modify the sympathetic stimulation responses. EGL-6M increased the vasopressor responses to sympathetic stimulation without modifying those to noradrenaline, methoxamine or UK 14,304. Collectively, these data suggest that EGL-6M is capable to increase insulin sensitivity and the vasopressor sympathetic outflow in rats.

Pharmacological analysis of the cardiac sympatho-inhibitory actions of moxonidine and agmatine in pithed spontaneously hypertensive rats.

This study shows that in spontaneously hypertensive rats (SHR) of 14-weeks-old, the sympathetically-induced, but not noradrenaline-induced tachycardic response are higher than age-matched Wistar normotensive rats. Furthermore, in SHR the sympathetically-induced tachycardic response was: (1) unaffected by moxonidine (3µg/kgmin); (2) partially inhibited by B-HT 933 (30µg/kgmin), both at the lowest doses; and (3) completely inhibited by the highest doses of B-HT 933 (100µg/kgmin), moxonidine (10µg/kgmin) or agmatine (1000 and 3000µg/kgmin) while the noradrenaline-induced tachycardic responses remained unaffected by the above compounds, except by 3000µg/kgmin agmatine. In SHR, 300µg/kg rauwolscine failed to block the sympatho-inhibition to 100µg/kgmin B-HT 933 or 10µg/kgmin moxonidine, but 1000µg/kg rauwolscine abolished, partially antagonized, and did not modify the sympatho-inhibition to the highest doses of B-HT 933, moxonidine, and agmatine, respectively, 3000µg/kg AGN 192403 or 300µg/kg BU224 given alone had no effect in the moxonidine- or agmatine-induced sympatho-inhibition, and the combination rauwolscine plus AGN 192403 but not plus BU224, abolished the sympatho-inhibition to the highest doses of moxonidine and agmatine. In conclusion, the sympathetically-induced tachycardic responses in SHR are inhibited by moxonidine and agmatine. The inhibition of moxonidine is mainly mediated by prejunctional α2-adrenoceptors and to a lesser extent by I1-imidazoline receptors, while the inhibition of agmatine is mediated by prejunctional α2-adrenoceptors and I1-imidazoline receptors at the same extent. Notwithstanding, the inhibitory function of α2-adrenoceptors seems to be altered in SHR compared with Wistar normotensive rats.

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17ß-estradiol on isolated canine basilar and internal carotid arteries.

Progesterone and 17ß-estradiol induce vasorelaxation through non-genomic mechanisms in several isolated blood vessels; however, no study has systematically evaluated the mechanisms involved in the relaxation induced by 17ß-estradiol and progesterone in the canine basilar and internal carotid arteries that play a key role in cerebral circulation. Thus, relaxant effects of progesterone and 17ß-estradiol on KCl- and/or PGF2α-pre-contracted arterial rings were investigated in absence or presence of several antagonists/inhibitors/blockers; the effect on the contractile responses to CaCl2 was also determined. In both arteries progesterone (5.6-180 µM) and 17ß-estradiol (1.8-180 µM): (1) produced concentration-dependent relaxations of KCl- or PGF2α-pre-contracted arterial rings; (2) the relaxations were unaffected by actinomycin D (10 µM), cycloheximide (10 µM), SQ 22,536 (100 µM) or ODQ (30 µM), potassium channel blockers and ICI 182,780 (only for 17ß-estradiol). In the basilar artery the vasorelaxation induced by 17ß-estradiol was slightly blocked by tetraethylammonium (10mM) and glibenclamide (KATP; 10 µM). In both arteries, progesterone (10-100 µM), 17ß-estradiol (3.1-31 µM) and nifedipine (0.01-1 µM) produced a concentration-dependent blockade of the contraction to CaCl2 (10 µM-10mM). These results suggest that progesterone and 17ß-estradiol produced relaxation in the basilar and internal carotid arteries by blockade of L-type voltage dependent Ca(2+) channel but not by genomic mechanisms or production of cAMP/cGMP. Potassium channels did not play a role in the relaxation to progesterone in both arteries or in the effect of 17ß-estradiol in the internal carotid artery; meanwhile KATP channels play a minor role on the effect of 17ß-estradiol in the basilar artery.

Evidence that chronic administration of 17ß-oestradiol decreases the vasopressor responses to adrenergic system stimulation in streptozotocin-diabetic female rats.

In vitro studies have indicated that 17ß-oestradiol exerts beneficial effects on the cardiovascular system by activating the nitric oxide pathway. However, these effects have not been demonstrated in vivo in the systemic vasculature of rats made diabetic through streptozotocin induction. Therefore, the goal of this study was to determine the effect of 17ß-oestradiol on vasopressor responses induced by sympathetic stimulation or i.v. injections of noradrenaline, methoxamine and B-HT 933 in sham-operated or ovariectomised, diabetic or non-diabetic female rats. Thus, rats were ovariectomised or sham-operated for this experiment. One week later, the animals were treated with streptozotocin (60mg/kg, i.p.) or its vehicle. Two weeks later, these rats were treated daily with 17ß-oestradiol (10µg/kg, s.c.) or its vehicle for five weeks. Next, under anaesthesia, the animals were pithed and prepared for blood pressure and heart rate measurements. 17ß-oestradiol failed to modify the vasopressor responses to (i) sympathetic stimulation, noradrenaline, methoxamine or B-HT 933 in sham-operated non-diabetic rats; (ii) sympathetic stimulation or B-HT 933 in sham-operated diabetic rats; (iii) noradrenaline or methoxamine in ovariectomised non-diabetic rats. In contrast, 17ß-oestradiol significantly decreased the vasopressor responses to (i) noradrenaline and methoxamine in sham-operated diabetic rats; (ii) sympathetic stimulation or B-HT 933 in ovariectomised non-diabetic rats; and (iii) sympathetic stimulation, noradrenaline, methoxamine or B-HT 933 in ovariectomised diabetic rats. These results suggest that chronic administration of 17ß-oestradiol decreases the vasopressor responses to adrenergic system stimulation in streptozotocin-induced diabetic rats. This report describes the first in vivo study reporting this effect of 17ß-oestradiol in diabetes.

Pharmacological evidence that dopamine inhibits the cardioaccelerator sympathetic outflow via D2-like receptors in pithed rats.

It has been suggested that N,N-di-n-propyl-dopamine (dopamine analogue) decreased heart rate in rats through stimulation of dopamine receptors. Nevertheless, the role of prejunctional dopamine D1/2-like receptors or even α2-adrenoceptors to mediate cardiac sympatho-inhibition induced by dopamine remains unclear. Hence, this study identified the pharmacological profile of the cardiac sympatho-inhibition to dopamine in pithed rats. Male Wistar rats were pithed and prepared to stimulate the cardiac sympathetic outflow or to receive i.v. bolus of exogenous noradrenaline. I.v. continuous infusions of dopamine (endogenous ligand) or quinpirole (D2-like agonist) dose-dependently inhibited the tachycardic responses to sympathetic stimulation, but not those to exogenous noradrenaline. In contrast, SKF-38393 (100 µg/kg∙min, D1-like agonist) failed to modify both of these responses. The sympatho-inhibition to dopamine (1.8 µg/kg∙min) or quinpirole (100 µg/kg∙min): i) remained unaltered after saline or the antagonists SCH-23390 (D1-like, 300 µg/kg) and rauwolscine (α2-adrenoceptors, 300 µg/kg); and ii) was significantly antagonized by raclopride (D2-like, 300 µg/kg). These antagonists, at the above doses, failed to modify the sympathetically-induced tachycardic responses. The above results suggest that the inhibition of the cardiac sympathetic outflow to dopamine and quinpirole is primarily mediated by prejunctional D2-like receptors but not D1-like receptors or α2-adrenoceptors.

The α2-adrenoceptors mediating inhibition of the vasopressor sympathetic outflow in pithed rats: pharmacological correlation with α2A, α2B and α2C subtypes.

α2-Adrenoceptors were first described as presynaptic receptors inhibiting the release of various transmitters from neurons in the central and peripheral nervous systems. In vitro studies have confirmed that α2A, α2B and α2C subtypes inhibited noradrenaline release from postganglionic sympathetic neurons but no study has been reported their involvement in the vasopressor sympathetic outflow in vivo. Thus, this study analysed the subtype(s) involved in the inhibition produced by the α2-adrenoceptor agonist, B-HT 933, on the vasopressor sympathetic outflow. Male Wistar pithed rats were pre-treated with i.v. bolus injections of gallamine (25mg/kg) and desipramine (50 µg/kg) and prepared to stimulate the vasopressor sympathetic outflow (T7-T9) or to receive i.v. bolus of exogenous noradrenaline. Sympathetic stimulation or exogenous noradrenaline produced, respectively, frequency-dependent and dose-dependent vasopressor responses. I.v. continuous infusion of B-HT 933 (30 µg/kg min) failed to modify the vasopressor responses to exogenous noradrenaline and inhibited those induced by preganglionic stimulation of the vasopressor sympathetic outflow at all frequencies of stimulation (0.03-3 Hz). The sympatho-inhibition elicited by B-HT 933 was: (i) unaffected by vehicles (1 ml/kg); (ii) partially antagonised by BRL44408 (300 µg/kg; α2A), imiloxan (3000 µg/kg; α2B) and/or JP-1302 (300 µg/kg; α2C) given separately; and (iii) completely blocked by rauwolscine (300 µg/kg) or the combination of BRL44408 (300 µg/kg)+imiloxan (3000 µg/kg)+JP-1302 (300 µg/kg). The above doses of antagonists did not modify per se the sympathetically-induced vasopressor responses. These results suggest that the vasopressor sympatho-inhibition to B-HT 933 is primarily mediated by activation of α2A/2B/2C-adrenoceptors in pithed rats.

Pharmacological evidence that Ca²+ channels and, to a lesser extent, K+ channels mediate the relaxation of testosterone in the canine basilar artery.

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF(2α)-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl2 was also determined. Testosterone (10-180 µM) produced concentration-dependent relaxations of KCl- or PGF(2α)-precontracted arterial rings which were: (i) unaffected by flutamide (10 µM), DL-aminoglutethimide (10 µM), actinomycin D (10 µM), cycloheximide (10 µM), SQ 22,536 (100 µM) or ODQ (30 µM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K(V); 1 mM), BaCl2 (K(IR); 30 µM), iberiotoxin (BK(Ca²+); 20 nM), but not by glybenclamide (K(ATP); 10 µM). In addition, testosterone (31, 56 and 180 µM) and nifedipine (0.01-1 µM) produced a concentration-dependent blockade of the contraction to CaCl2 (10 µM to 10 mM) in arterial rings depolarized by 60mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca²+ channels and, to a lesser extent, by activation of K+ channels (K(IR), K(V) and BK(Ca²+)). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17ß-estradiol.

Activation of 5-HT1B receptors inhibits the vasodepressor sensory CGRPergic outflow in pithed rats.

The importance of calcitonin gene-related peptide (CGRP) in the regulation of vascular tone has been widely documented. Indeed, stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses, which are mediated by CGRP release. These vasodepressor responses are inhibited by clonidine via prejunctional alpha(2A/2C)-adrenoceptors, but no study has yet reported the role of prejunctional 5-hydroxytryptamine (5-HT) receptors in this experimental model. Since activation of prejunctional 5-HT(1) receptors results in inhibition of neurotransmitter release, this study sets out to investigate as an initial approach the role of 5-HT(1B) receptors in the inhibition of the vasodepressor sensory outflow in pithed rats. Male Wistar pithed rats were pretreated with hexamethonium (2mg/kg.min) followed by i.v. continuous infusions of methoxamine (20 microg/kg min), and then by saline (0.02 ml/min) or CP-93,129 (a rodent 5-HT(1B) receptor agonist; 0.1, 1 and 10 microg/kg min). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T(9)-T(12)) resulted in frequency-dependent decreases in diastolic blood pressure. The infusions of CP-93,129, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous alpha-CGRP (0.1, 0.18, 0.31, 0.56 and 1 microg/kg). This inhibition by CP-93,129 was abolished by the antagonists GR127935 (5-HT(1B/1D)) or SB224289 (5-HT(1B)), but not by BRL15572 (5-HT(1D)). The above results suggest that CP-93,129-induced inhibition of the vasodepressor (perivascular) sensory outflow in pithed rats is mainly mediated by activation of prejunctional 5-HT(1B) receptors.