Edilson Dantas da Silva Júnior

Increase of angiotensin-converting enzyme activity and peripheral sympathetic dysfunction could contribute to hypertension development in streptozotocin-induced diabetic rats

Diabetes augments the risk of hypertension. Although several factors have been implicated in the development of such hypertensive state, we designed this study to investigate blood pressure development, the activity of angiotensin-converting enzyme (ACE) in blood as well as sympathetic neurotransmission in the vas deferens of diabetic rats. We used streptozotocin (STZ)-induced diabetic rats (60 mg/kg) in order to evaluate the systolic blood pressure (SBP), ACE activity and peripheral sympathetic neurotransmission. We observed the following changes of parameters: increase of SBP, decrease of heart rate, augmentation of plasma ACE activity, enhancement of phasic and tonic vas deferens contractions elicited by electrical stimulation at 5 Hz, increase of maximal response to noradrenaline (NA) and decrease of adenosine triphosphate (ATP)-elicited contraction of vasa deferentia. The results reveal that in the development of hypertension in diabetic rats, augmentation of circulating ACE activity precedes the sympathetic dysfunction. Additionally, it seems that the purinergic and noradrenergic neurotransmission is compromised.

Changes of cytosolic calcium and contractility of young rat vas deferens by acute treatment with amphetamine, fluoxetine or sibutramine

Previous studies conducted in our laboratory indicated that administration of amphetamine, fluoxetine or sibutramine affects the sympathetic nervous system of the rat vas deferens. Therefore, our goal was to verify the role of calcium in vasa deferentia from young rats pretreated with a single dose of these drugs. Young 40-day-old male Wistar rats were pretreated with amphetamine 3 mg/kg, fluoxetine 10 mg/kg or sibutramine 6 mg/kg for 4 h before the experiments. CaCl(2) (10 mM) was used to induce contraction through time-effect curves in calcium-free solution to measure phasic and tonic components. We also evaluated the calcium-induced fluorescence of vas deferens cut into thin slices. In rats pretreated with amphetamine, we found an increase of the tonic contraction component which was reduced by verapamil. The phasic and tonic responses were increased in the group treated with fluoxetine, but only the tonic response was more sensitive to the antagonism by verapamil. The group treated with sibutramine showed an increase of phasic response whereas the tonic component was decreased. In this group an increase of the affinity for verapamil antagonism was found. In the calcium fluorescence study it was observed that the group treated with amphetamine, fluoxetine or sibutramine showed higher basal Ca(2+) fluorescence after stimulus with KCl (70 mM), noradrenaline (10(-4)M) or acetylcholine (10(-4)M). In all pretreated groups the calcium fluorescence was diminished by nifedipine 10(-7)M. Therefore, the pretreatment with amphetamine, fluoxetine or sibutramine seems to affect the calcium contractility and homeostasis in young rat vas deferens.

Functional antagonism of amphetamine versus ethanol on adrenergic neurotransmission in vas deferens of adolescent rats

Because of the few studies that emphasize the in vivo use of amphetamine and ethanol, and their consequences on autonomic neurotransmission, we decided to study the effect of these drugs on peripheral noradrenergic neurotransmission of young animals. We used contractions of the vas deferens of adolescent rats as a model for the study of pre-treatment with both agents. The 30 to 40 day old adolescent rats were pre-treated with amphetamine, at doses of 3mg/kg, or ethanol at doses of 1.2 g/kg. Both agents were also used simultaneously to investigate possible interactions. The group treated with amphetamine showed a potentiation of the vas deferens contractions evoked by noradrenaline and barium (about 20%), as well as time-response contractions of calcium (about 20%). However, the response to electrical field stimulation (EFS) was not significantly changed, but the content of noradrenaline was reduced by about 50%. The group treated with ethanol showed a decrease in vas deferens contractility to noradrenaline, phenylephrine, and barium, by less than 20%. In this group, contraction by EFS was reduced by about 40% (Tonic, 2 Hz) and 20% (Phasic, 5 Hz), but the response to calcium was not changed. As after amphetamine, the content of noradrenaline was reduced by about 50%. In the group treated with amphetamine+ethanol all the changes described after the single treatments with amphetamine or ethanol were neutralized. It is concluded that a functional antagonism was shown between amphetamine and ethanol when administered simultaneously on peripheral sympathetic neurotransmission in vas deferens of adolescent animals.

Functional characterization of acetylcholine receptors and calcium signaling in rat testicular capsule contraction

The motor activity of mammalian testicular capsule (TC) contributes to male fertility and infertility, but the acetylcholine receptors related to the contractions induced by cholinergic drugs are poorly known. Indeed to characterize the acetylcholine receptors and cellular signaling by Ca(2+) involved in TC motor activity of rats, the potency of agonists (pD₂) and antagonists (pA₂) of acetylcholine receptors, and effects of Ca(2+) cellular transport blockers on the cholinergic contractions were evaluated. pD₂ values of acetylcholine (5.98) were ten-fold higher than that of carbachol (4.99). Efficacy (Emax) of acetylcholine and carbachol to induce contractions corresponded to 95% and 97% of Emax for KCl, but Emax for nicotine was very low (8% of Emax for KCl). Further, physostigmine did not affect the acetylcholine potency. Contractions induced by acetylcholine or carbachol were antagonized by muscarinic but not nicotinic antagonist. The order of pA₂ values obtained for muscarinic antagonists, namely atropine>4-DAMP>AF-DX116>pirenzepine, corresponded to a typical profile of M3 receptors. Contractions induced by acetylcholine or carbachol were inhibited by blockers of Ca(2+) influx through voltage-dependent calcium channels (nifedipine and Ni(2+)), Ca(2+) reuptake by sarco-endoplasmic reticulum (cyclopiazonic acid) and mitochondria (FCCP). The protein kinase C (PKC) inhibitor chelerythrine only affected the acetylcholine-induced contraction. These results suggest that TC motor activity of rats are mediated mainly by M₃ receptors followed by the increase of cytosolic Ca(2+) concentration regulated by voltage-dependent calcium channels, sarco-endoplasmic reticulum and mitochondria. Furthermore, the differential effects of chelerythrine in the acetylcholine or carbachol-induced contractions are discussed.

Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats

Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration-effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000μM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1-300µM and cardiac arrest at 1000μM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels.

Influence of acute treatment with sibutramine on the sympathetic neurotransmission of the young rat vas deferens.

The effects of acute treatment with sibutramine on the peripheral sympathetic neurotransmission in vas deferens of young rats were still not evaluated. Therefore, we carried out this study in order to verify the effects of acute sibutramine treatment on the neuronal- and exogenous agonist-induced contractions of the young rat vas deferens. Young 45-day-old male Wistar rats were pretreated with sibutramine 6 mg/kg and after 4h the vas deferens was used for experiment. The acute treatment with sibutramine was able to increase the potency (pD2) of noradrenaline and phenylephrine. Moreover, the efficacy (Emax) of noradrenaline was increased while the efficacy of serotonin and nicotine were decreased. The maximum effect induced by a single concentration of tyramine was diminished in the vas deferens from treated group. Moreover, the leftward shift of the noradrenaline curves promoted by uptake blockers (cocaine and corticosterone) and β-adrenoceptor antagonist (propranolol) was reduced in the vas deferens of treated group. The initial phasic and secondary tonic components of the neuronal-evoked contractions of vas deferens from treated group at the frequencies of 2 Hz were decreased. Moreover, only the initial phasic component at 5 Hz was diminished by the acute treatment with sibutramine. In conclusion, we showed that the acute treatment with sibutramine in young rats was able to affect the peripheral sympathetic nervous system by inhibition of noradrenaline uptake and reduction of the neuronal content of this neurotransmitter, leading to an enhancement of vas deferens sensitivity to noradrenaline.

Cardiac arrest induced by muscarinic or adenosine receptors agonists is reversed by DPCPX through double mechanism

ABSTRACT In the right atrium (RA), adenosine and acetylcholine inhibit the pacemaker function of the sinoatrial node and induce cardiac arrest. Pre‐incubation of receptor antagonists is known to inhibit the cardiac arrest induced by these agonists; however, the effect of antagonist administration after established cardiac arrest has not been described. Therefore, we assessed whether specific receptor antagonists could revert cardiac arrest induced by adenosine and muscarinic receptors activation. RA isolated from adults Wistar rats were mounted in an organ bath containing Krebs solution. Cardiac arrest was induced by adenosine or ATP (1 mM), the A1 adenosine receptor agonist CPA (0.1–1 &mgr;M), and muscarinic receptor agonists, carbachol (0.3–1 &mgr;M) and acetylcholine (1 mM). After establishing the cardiac arrest, the A1 adenosine receptor antagonist DPCPX (0.3–30 &mgr;M), the muscarinic receptor antagonist atropine (10 nM to 100 &mgr;M) or the phosphodiesterase inhibitor IBMX (10–300 &mgr;M) were incubated in order to check for the return of spontaneous contractions. DPCPX reversed the cardiac arrest induced by adenosine, ATP and CPA. In addition, atropine reversed the cardiac arrest induced by carbachol. Unexpectedly, DPCPX also reversed the cardiac arrest induced by carbachol. Similarly to DPCPX, the phosphodiesterase inhibitor IBMX reversed the cardiac arrest induced by adenosine, CPA and carbachol. The antagonism of adenosine and acetylcholine receptors activation, as well as phosphodiesterase inhibition, are able to revert cardiac arrest. DPCPX restore spontaneous contractions via the selective antagonism of A1 adenosine receptor and through a secondary mechanism likely related to phosphodiesterase inhibition.

Could α1-adrenoceptors and androgen receptors be modified by sexual maturation and testosterone in the rat testicular capsule?

AIMS Testicular capsule contractile dysfunctions are recognized to contribute to male infertility, but the influence of sexual maturation and exogenous testosterone on the expression and function of androgen receptor and α1-adrenoceptors on rat testicular capsule is unclear. Here, these two biological parameters were evaluated on testicular capsule from sexually immature and young adult rats treated or not with exogenous testosterone. MAIN METHODS Male Wistar rats (45- and 60-day-old) were assigned into groups: control (saline 0.9%) or testosterone-treated (propionate testosterone). Testicular capsule was isolated and processed for functional studies, immunohistochemistry, Western blot and RT-PCR studies. KEY FINDINGS Relative testicular capsule wet weight was not affected by sexual maturation or exogenous testosterone treatment. The expression and immunolocalization of androgen receptor (mRNA and protein) was identified in testicular capsule. Androgen receptor and α1-adrenoceptor (Adra1a, Adra1b, and Adra1d) mRNA levels were similar in testicular capsule from all experimental groups. Functional studies indicated that contractions produced by noradrenaline in testicular capsule from 45- and 60-day-old rats treated or not with testosterone were mainly mediated by α1A- and α1B-adrenoceptors. The L-type Ca(2+) channel blocker nifedipine induced a higher inhibitory effect on noradrenaline induced contractions in testicular capsule from 45- than 60-day-old rats treated with testosterone. SIGNIFICANCE Molecular studies, immunohistochemistry and pharmacological functional assays used in this study provide evidences of the androgen receptor expression in testicular capsule and that function, and not mRNA and protein expression levels of the α1-adrenoceptor subtypes in this tissue, is differentially influenced by the rat androgen status.

Effects of in vitro, acute and chronic treatment with fluoxetine on the sympathetic neurotransmission of rat vas deferens

It is described that fluoxetine treatment is able to induce ejaculatory disorders. However, the exact mechanism is still not fully understood. Therefore, this study was carried out to further evaluate the anti-ejaculatory effects of fluoxetine, using different approaches (in vitro or in vivo treatments), on the sympathetic neurotransmission of the rat vas deferens. Vas deferens from male Wistar rats were used to check the in vitro effects of fluoxetine 10-6M, 3.10-6M or 10-5M. Animals were also acutely (20mg/kg, i.p. 4h or 24h) or chronically (10mg/kg, i.p., 30days) treated with fluoxetine or drug-free vehicle. The vas deferens from non-treated and treated animals were isolated and mounted in an isolated organ bath for the study of the contractions induced by adrenergic agonists, tyramine, 5-HT, Ca2+ or electrical field stimulation. In vitro or acute treatment with fluoxetine decreased the contraction induced by agonists, Ca2+ or electrical field stimulation. The chronic treatment with fluoxetine decreased the contractions induced agonists, tyramine or Ca2+, but did not modify the contractions induced by electrical field stimulation. We have shown that in vitro or in vivo fluoxetine treatment is able to alter the sympathetic neurotransmission of the rat vas deferens which could be related to alterations in the calcium signalling.