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.

Functional effects of alcohol withdrawal syndrome on peripheral sympathetic neurotransmission in vas deferens of adult rats.

AIMS: Alcohol withdrawal syndrome (AWS) is characterized by a set of physiological modifications triggered by abrupt withdrawal and/or decreasing consumption of ethanol (EtOH), which may manifest 16-48 h after ceasing consumption. The relationship between the effects of AWS and central and peripheral sympathetic neurotransmission is unknown. This study investigates the possible mechanisms on the sympathetic system during periods of AWS. MAIN METHODS: Male Wistar rats were treated with EtOH (6-10 g/kg/day/v.o. 5 days). Subsequently, 1h, 24h, 48 h and 120 h after administration of the last dose of EtOH, the animals were sacrificed, and their vas deferens (VD) were removed to perform the following evaluations: (a) concentration-effect curves of sympathetic agonist; (b) activity of α2-adrenoreceptor; (c) function of voltage-dependent calcium channels (Cav); and (d) release of endogenous catecholamines measured in real time coupled to HPLC. KEY FINDINGS: The results showed that the maximum effects of contraction were increased by agonists tested in at 24h and 48 h EtOH withdrawal. The inhibitory affinity (pIC50) of guanfacine was decreased 24h EtOH withdrawal. The function of Cav was also decreased as pIC50 values dropped 24h and 48 h EtOH withdrawal. The release of catecholamines increased 48 h after EtOH withdrawal. It is suggested that AWS triggers hyperactivity in peripheral sympathetic neurotransmission. SIGNIFICANCE: The mechanisms underlying hyperactivity are possibly explained by a failure of autoregulation from catecholamines released by α2-adrenoreceptors and/or an increase of Cav function, which may be potential targets to attenuate the symptoms of AWS at the peripheral level.

Time-dependent up-regulation of Ca(2+) channels in vas deferens of newborn rats fed with breast milk of mothers under treatment with nifedipine.

Our aim was to check for calcium channel maturation and regulation on newborn rats during breastfeeding by mothers treated with the L-type calcium channel blocker nifedipine. Contractions by KCl and radioligand binding techniques were used to verify if Ca(2+) channels are modified in rat vas deferens of 40-day old litters that were breastfed by mothers injected daily with nifedipine during nursery. Injections were applied in the beginning (1st until 8th day), middle (9th until 16th day), or end (17th until 24th day) of nursery, to verify the period of highest susceptibility of newborn to nifedipine receptor regulation. Contractile responses revealed that only after the middle period of treatment of mothers the maximal effects (E(max)) induced in pups by KCl were increased by about 35%, without changes of apparent affinity (pD(2)). Additionally, binding studies with [(3)H] Isradipine in cell membrane preparations showed a greater density (B(max)) of Ca(2+) channels by about 55%, without changes of affinity (K(d)). Changes were not detected after treatment of mothers in the beginning or end of breastfeeding. In addition, in vas deferens of 60-day old litters, the E(max) returned to control values, showing that changes were not persistent. Moreover, body and vas deferens weights and blood testosterone of newborn were never changed. The histology of mammary gland was similar for treated and control mothers, suggesting a stable milk production. It is concluded that nifedipine treatment of mothers, if made during the 9th to 16th day of lactation, produced a short lasting reversible up-regulation of L-type Ca(2+) channels.

Sympathetic neurotransmission in the rat testicular capsule: functional characterization and identification of mRNA encoding alpha1-adrenoceptor subtypes.

The rat testicular capsule is a thin tissue surrounding the testis, whose precise function is still unknown. We have studied the contractile effects of electrical field stimulation, noradrenaline, and the blockade by antagonists of adrenergic receptors, in order to characterize sympathetic neurotransmission, and adrenoceptor subtypes. In addition, reverse transcription polymerase chain reaction (RT-PCR) assays were made to check for the expression of the three known subtypes of alpha(1)-adrenoceptors. The effects of electrical field stimulation (2 to 20 Hz, 1 ms, 60 V) were almost totally abolished by depletion of neuronal noradrenaline storage with reserpine (10 mg/Kg), but not by the purinergic receptor antagonist suramin (10(-5) M), indicating that noradrenaline, but not ATP, was involved in contractions. The selective alpha(1)-adrenoceptor antagonist prazosin (10(-7) M) was more effective than the selective alpha(2)-adrenoceptor antagonist idazoxan (10(-7) M) to inhibit contractions induced by electrical field stimulation, pointing out a major involvement of alpha(1)-adrenoceptor. When noradrenaline was used instead of electrical field stimulation, it showed a high potency (pD(2)=7.9). Noradrenaline-induced contractions were competitively blocked by the selective alpha(1A)-adrenoceptor antagonists WB 4101 (pA(2)=8.88), phentolamine (pA(2)=8.39) and by the alpha(1B)-adrenoceptor antagonist spiperone (pA(2)=8.57), indicating the presence of functional alpha(1A)- and alpha(1B)-adrenoceptors. In addition, contractions were not blocked by the selective alpha(1D)-adrenoceptor antagonist BMY 7378 (up to 10(-6) M), while selective alpha(2)-adrenoceptor antagonists showed low pA(2) values (yohimbine, 7.25 and idazoxan, 7.49), suggesting a minor role, if any, for alpha(1D)- and alpha(2)-adrenoceptors. To check the proportionate role of alpha(1A)- and alpha(1B)-adrenoceptors, we blocked alpha(1B)-adrenoceptors with chloroethylclonidine (CEC, 30 microM, 45 min), that reduced the maximal effect of noradrenaline by about 60%. The remnant CEC-insensitive noradrenaline contraction was assumed to be unrelated to alpha(1B)-adrenoceptor, and was inhibited by 5-methyl-urapidil (pA(2)=8.94) and by the Ca(2+) channel blocker nifedipine (3 microM), confirming the involvement of alpha(1A)-adrenoceptors. The presence of mRNA encoding alpha(1A)- and alpha(1B)-adrenoceptor was also shown on RT-PCR assays. Unexpectedly, alpha(1D)-transcripts were also detected in these assays. Taken together, our results show that ATP co-transmission could not be detected, and that neurotransmission involves the interaction of noradrenaline with both alpha(1A)- and alpha(1B)-, but not with alpha(1D)- or alpha(2)-adrenoceptor. The fact that the functional alpha(1D)-adrenoceptor could not be detected in spite of the presence of the corresponding mRNA, remains to be investigated.

Up-regulation of Ca(2+) channels in vas deferens after chronic treatment of newborn rats with nifedipine.

Radioligand binding and contraction techniques were used to verify if L-type Ca(2+) channels are modified in rat vas deferens after treatment with the blocker nifedipine (15 microg), injected at 7, 14, 21 and 28 days after birth. Vas deferens tissue was used 10, 30 and 90 days after the last injection, to verify if modifications are persistent. Binding studies with cell membranes, using [(3)H]isradipine, showed an increase of the density (B(max)) of Ca(2+) channels by more than 60%, after 10 and 30 days, without changes of affinity (K(d)). Maximal contractions (E(max)) of KCl, were increased by 106% and 37%, respectively, after 10 and 30 days, without changes of apparent affinity (pD(2)). After 90 days, the values of B(max), K(d), E(max) and pD(2) were not different from the controls. Differences were also not found for rats injected when adult. It is concluded that treatment of newborn, but not of adult, rats with nifedipine produced a long-lasting, though reversible, up-regulation of L-type Ca(2+) channels.