Biomedical subjects

Maria Christina W Avellar

Publications and source records attributed to Maria Christina W Avellar.

11 recordsLinked to original sources

PubMed2006

Serotonin concentration, synthesis, cell origin, and targets in the rat caput epididymis during sexual maturation and variations associated with adult mating status: morphological and biochemical studies.

The caput epididymis of some mammals contains large quantities of serotonin whose origin, targets, and physiological variations have been poorly studied. We combined morphological and biochemical techniques to begin approaching these aspects of serotonin in the rat caput epididymis. Serotonin immunostaining was detected in mast, epithelial, and neuroendocrine cells. Epithelial cells displayed immunoreactivity to 5HT(1A), 5HT(2A,) and 5HT(3) serotonin receptors. Endothelial and mast cells labeled positive for 5HT(1B) serotonin receptors and spermatozoa displayed 5HT(2A) and 5HT(3) serotonin receptor immunoreactivity. Epithelial, endothelial, and mast cells stained positive for serotonin transporters. Only epithelial cells showed tryptophan hydroxylase immunoreactivity; this enzyme catalyzes the limiting step in the serotonin synthetic pathway. In addition, Western blot analyses of caput homogenates documented the presence of 2 protein bands ( approximately 51 kd and approximately 48 kd) that were immunoreactive for tryptophan hydroxylase. Chromatographic analyses documented the presence of tryptophan hydroxylase in the caput, and showed that both its activity and serotonin availability increased with sexual maturation and decreased following p-chlorophenylalanine treatment, an inhibitor of tryptophan hydroxylase activity. Interestingly, serotonin concentration and tryptophan hydroxylase activity tended to be higher in breeding males than in those with no mating experience. We think that these results support the existence of a local serotoninergic system in the rat caput epididymis that might regulate some aspects of male reproductive function.

Animals↗

PubMed2006

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.

Acetyl-CoA C-Acyltransferase↗

PubMed2005

Expression and localization of muscarinic acetylcholine receptor subtypes in rat efferent ductules and epididymis.

The expression of muscarinic acetylcholine receptor (mAChR) subtypes (M(1)-M(5)) was studied in the rat efferent ductules and epididymis at the mRNA and protein levels. The relative abundance of each mAChR transcript subtype differed depending on the tissue and the epididymal region analyzed. The M(1) mAChR mRNA level was more abundant in the efferent ductules than in the caput and cauda of the epididymis. The M(2) mAChR mRNA level was similar between the efferent ductules and caput of the epididymis and higher in the cauda region. The M(3) mAChR mRNA level was low in the efferent ductules and caput of the epididymis, but high levels were detected in the cauda region. mRNAs for M(4) and M(5) mAChRs were not detected in these tissues. Our studies indicated a variable degree of immunostaining for each mAChR subtype in a cell-type and tissue-specific pattern. M(1) mAChR was detected over the efferent ductule epithelium. M(2) and M(3) mAChRs were observed in the apical region of the ciliated cells. Apical and narrow cells of the initial segment showed distinct staining by M(1) antibody, whereas a supranuclear reaction was noted in the principal cells of the caput of the epididymis. In addition, staining for M(1) and M(2) mAChRs was visible in the apical membrane of some epithelial cells of the cauda region. M(3) mAChR was detected in the peritubular smooth muscle of the efferent ductules and epididymis. Functional studies suggested the involvement of this subtype in epididymal tubule contraction. Thus, the cell-specific expression of the various mAChR subtypes in the efferent ductules and epididymis suggests that these receptors play a role in the modulation of luminal fluid composition and smooth muscle contraction.

Animals↗

PubMed2005

Effects of testosterone on muscarinic acetylcholine receptors in the rat epididymis.

The effect of testosterone on the expression of muscarinic acetylcholine receptor (mAChR) subtypes was studied in the rat epididymis, at mRNA and protein level. The rat androgen status was monitored by measuring plasma testosterone level and caput and cauda epididymis wet weight. Ribonuclease protection assay (RPA) and [3H]quinuclidinyl benzilate ([3H]QNB) binding assay were performed in the caput and cauda epididymis from control (50-day old), castrated, castrated and treated with testosterone and sexually immature (30-day old) rats. The expression of each mAChR transcript subtype differed depending on the epididymal region analyzed and rat testosterone and/or testicular factors status. In control rats, RPA showed the presence of mRNA for M1, M2 and M3 mAChR in the caput and cauda epididymis. The abundance of m2 and m3 transcripts in the cauda was higher than that in the caput epididymis. Low amount of m1 transcript was observed in both regions. Orchidectomy increased m1 mRNA amount in the caput and cauda epididymis when compared to control rats, an effect slightly modified by testosterone replacement. Although orchidectomy down-regulated the level of m2 transcript in both epididymal regions, castration significantly increased m3 mRNA amount in the caput region. These effects on m2 and m3 transcripts were prevented by testosterone replacement to castrated rats. Similar abundance of m3 transcript, however, was detected in the cauda epididymis of all experimental group tested. [3H]QNB binding studies revealed that orchidectomy down-regulated the number of mAChR detected in both epididymal regions, an effect also prevented by testosterone replacement. Thus, testosterone and/or testicular factors may play a role in the regulation of mAChR expression in the rat epididymis.

Animals↗

PubMed2004

Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study.

The aim of the present work was to study the effect of muscarinic agonist on cell proliferation and muscarinic acetylcholine receptors (mAChRs) regulation in rat Sertoli cells. Primary cultures of Sertoli cells were obtained from 8-day and 15-day old male Wistar rats. In proliferation assays, [methyl-3H]thymidine incorporation in Sertoli cells from 8-day and 15-day old rats reached a plateau after 60 min of carbachol incubation and decreased after 120 min of agonist incubation. Binding studies with [N-Methyl-3H]scopolamine ([3H]NMS) indicated a rapid loss of cell surface mAChRs when Sertoli cells from 15-day old rats were incubated with carbachol at 35 degrees C for 2 min. This effect was temperature-dependent. When the incubation of the cells was prolonged at 35 degrees C or at 4 degrees C, after the agonist had been washed away, 94% of mAChRs were present in the cell surface after 120 min incubation at 35 degrees C. At 4 degrees C, however, a low percentage of mAChRs was detected in the cell surface. In the presence of cycloheximide, the recycling of mAChRs to the cell surface was not changed, suggesting that the appearance of mAChRs on cell surface was not dependent on de novo receptor synthesis. In conclusion, our studies indicate that the activation of mAChRs may play a role in rat Sertoli cell proliferation. These receptors may be under regulation (internalization and recycling) when cells are exposed to muscarinic cholinergic agonist.

Analysis of Variance↗

PubMed2004

Effects of androgen manipulation on alpha1-adrenoceptor subtypes in the rat seminal vesicle.

This study analyses possible changes during surgical and chemical castration in the expression and pharmacological characteristics of alpha1-adrenoceptor in adult rat seminal vesicle. Ribonuclease protection assays indicated that alpha1a--was the predominant mRNA, while alpha1b-and alpha1d-adrenoceptor transcripts were detected in lower abundance in this tissue. alpha1a-adrenoceptor mRNA expression presented a complex dependency on androgens, while alpha1b- and alpha1d-adrenoceptor transcripts were both upregulated with surgical and chemical castration, suggesting a negative modulation by androgens. Testosterone treatment reversed the effects caused by surgical castration. Functional studies confirmed the involvement of alpha1A- and alpha1B-adrenoceptor in the seminal vesicle contractile responses, and suggested that alpha1B-induced contractile response was upregulated after castration. Taken together, the results suggest that alpha1-adrenoceptor expression in seminal vesicle is differentially regulated by the androgen status of the rat.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2004

Catecholamine effects on human melanoma cells evoked by alpha1-adrenoceptors.

The biological effects of catecholamines in mammalian pigment cells are poorly understood. Our previous results showed the presence of alpha(1)-adrenoceptors in SK-Mel 23 human melanoma cells. The aims of this work were to (1) characterize catecholamine effects on proliferation, tyrosinase activity and expression, (2) identify the alpha(1)-adrenoceptor subtypes, and (3) verify whether chronic norepinephrine (NE) treatment modified the types and/or pharmacological characteristics of adrenoceptors present in SK-Mel 23 human melanoma cells. Cells treated with the alpha(1)-adrenergic agonist, phenylephrine (PHE, 10(-5) or 10(-4) M), for 24-72 h, exhibited decreased cell proliferation and enhanced tyrosinase activity, but unaltered tyrosinase expression as compared with the control. The proliferation and tyrosinase activity responses were inhibited by the alpha(1)-adrenergic antagonist prazosin, suggesting they were evoked by alpha(1)-adrenoceptors. The presence of actinomycin D, a transcription inhibitor, did not diminish PHE-induced effects. RT-PCR assays, followed by cloning and sequencing, demonstrated the presence of alpha(1A)- and alpha(1B)-adrenoceptor subtypes. NE-treated cells (24 or 72 h) were used in competition assays, and showed no significant change in the competition curves of alpha(1)-adrenoceptors as compared with control curves. Other adrenoceptor subtypes were not identified in these cells, and NE pretreatment did not induce their expression. In conclusion, the activation of SK-Mel 23 human melanoma alpha(1)-adrenoceptors elicit biological effects, such as proliferation decrease and tyrosinase activity increase. Desensitization or expression of other adrenoceptor subtypes after chronic NE treatment were not observed.

Adrenergic alpha-1 Receptor Agonists↗

PubMed2004

Differential expression and antibacterial activity of epididymis protein 2 isoforms in the male reproductive tract of human and rhesus monkey (Macaca mulatta).

The epididymis protein 2 (EP2) gene, the fusion of two ancestral beta-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.

Aged↗

PubMed2004

Effect of estrogen on muscarinic acetylcholine receptor expression in rat myometrium.

We report the effect of acute estrogen treatment in the expression of muscarinic acetylcholine receptors (mAChRs) in myometrium. Strips were obtained from rats in estrus (control) and treated with estrogen, 24h before the experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed and m2, m3 and m5 mAChR mRNA subtypes were detected in myometrium from both groups. [(3)H]Quinuclidinyl benzilate [(3)HQNB] binding studies indicated that estrogen treatment did not change the affinity and density of mAChRs in myometrial membranes. Displacement curves of [(3)HQNB] with different mAChRs antagonists indicated a one-site fit for all antagonists tested. Comparison of pK(i) values indicated a significant correlation to M(2)-mAChR subtype. Functional studies, however, showed that estrogen treatment increased myometrium sensitivity to carbachol and the calculated apparent affinity values were significantly correlated to M(3)-mAChR. Furthermore, the pharmacological profile of the two populations of mAChR was not affected by estrogen. In conclusion, these results provide evidence for the presence of M(2)- and M(3)-mAChR, at the mRNA and protein level, in the rat myometrium and indicate that estrogen induces an increase in myometrial responsiveness to mAChR agonists.

Animals↗

PubMed2002

Alpha1-adrenoceptor subtypes in rat epididymis and the effects of sexual maturation.

We have characterized the expression of alpha1-adrenoceptor in epididymis from rats in different stages of sexual maturation: 40 (immature), 60 (young adult), and 120 (adult) days of age. Plasma testosterone levels were low in the immature animals but increased significantly in the 60- and 120-day-old animals. These changes were followed by a progressive increase in rat body weight and in caput and cauda epididymis relative weight. Reverse transcription polymerase chain reaction assay indicated that alpha1a-, alpha1b-, and alpha1d-adrenoceptor transcripts were present in both caput and cauda epididymis from adult rats. Ribonuclease protection assays further indicated that the expression of these alpha1-adrenoceptor transcripts differed with age and epididymal region analyzed. Prazosin (nonselective alpha1 antagonist), 5-methyl urapidil (alpha1A-selective), and BMY 7378 (alpha1D-selective) displaced [3H]prazosin binding curves in caput and cauda epididymis from 40- and 120-day-old rats. The potency order for these antagonists, as calculated from the negative logarithm of the inhibition constant (pK(i)) values for the high-affinity sites, indicated a predominant population of alpha1A-adrenoceptor subtype in caput and cauda epididymis from adult animals. Differences in pK(i) values in caput and cauda epididymis from immature and adult animals also suggested that the relative amount of alpha1-adrenoceptors, at the protein level, is modulated by sexual maturation. Taken together, the changes in alpha1-adrenoceptor expression during sexual maturation may suggest specific roles for these receptors in epididymal function.

Adrenergic alpha-Antagonists↗

PubMedDate not supplied

Segment-specific decrease of both catecholamine concentration and acetylcholinesterase activity are accompanied by nerve refinement in the rat cauda epididymis during sexual maturation.

In the present work, histochemical and biochemical studies were conducted to analyze changes in the pattern of autonomic innervation during sexual maturation, using the rat epididymis as a model. Glyoxylic acid histochemistry and immunohistochemical studies against dopamine beta-hydroxylase (DbetaH) and acetylcholinesterase (AChE) indicated a reduction in the amount of catecholaminergic and AChE-positive neurons, fibers, and puncta detected in the cauda epididymis of adult rats (120 days old), when compared to immature (40 days) and young adult (60 days) animals. No obvious age-related variations were detected in the few catecholaminergic and AChE-positive fibers and puncta present in the caput region. AChE-positive fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen of the cauda region of adult rats. Furthermore, a positive staining for AChE in epithelial cells was also detected in the caput and cauda epididymis in all ages studied. Biochemical analysis confirmed a significant decrease in noradrenaline concentration as well as AChE activity in the cauda epididymis with sexual maturation. Immunohistochemical studies against microtubule-associated protein 1B (MAP 1B), a neuronal cytoskeletal marker, further substantiated the quantitative changes observed in catecholaminergic and AChE-positive neuronal elements in the cauda epididymis. Thus, our results documented segment-specific variations in noradrenaline concentration and AChE activity during epididymal sexual maturation and suggest that such variations result, at least in part, from the refinement of the autonomic innervation pattern with age.

Acetylcholinesterase↗