Effect of Nebivolol on MIBG Parameters and Exercise in Heart Failure with Normal Ejection Fraction / Efeito do Nebivolol sobre Parâmetros do MIBG e Exercício na Insuficiência Cardíaca com Fração de Ejeção Normal

Abstract Background: More than 50% of the patients with heart failure have normal ejection fraction (HFNEF). Iodine-123 metaiodobenzylguanidine (123I-MIBG) scintigraphy and cardiopulmonary exercise test (CPET) are prognostic markers in HFNEF. Nebivolol is a beta-blocker with vasodilating properties. Objectives: To evaluate the impact of nebivolol therapy on CPET and123I-MIBG scintigraphic parameters in patients with HFNEF. Methods: Twenty-five patients underwent 123I-MIBG scintigraphy to determine the washout rate and early and late heart-to-mediastinum ratios. During the CPET, we analyzed the systolic blood pressure (SBP) response, heart rate (HR) during effort and recovery (HRR), and oxygen uptake (VO2). After the initial evaluation, we divided our cohort into control and intervention groups. We then started nebivolol and repeated the tests after 3 months. Results: After treatment, the intervention group showed improvement in rest SBP (149 mmHg [143.5-171 mmHg] versus 135 mmHg [125-151 mmHg, p = 0.016]), rest HR (78 bpm [65.5-84 bpm] versus 64.5 bpm [57.5-75.5 bpm, p = 0.028]), peak SBP (235 mmHg [216.5-249 mmHg] versus 198 mmHg [191-220.5 mmHg], p = 0.001), peak HR (124.5 bpm [115-142 bpm] versus 115 bpm [103.7-124 bpm], p= 0.043), HRR on the 1st minute (6.5 bpm [4.75-12.75 bpm] versus 14.5 bpm [6.7-22 bpm], p = 0.025) and HRR on the 2nd minute (15.5 bpm [13-21.75 bpm] versus 23.5 bpm [16-31.7 bpm], p = 0.005), but no change in peak VO2 and 123I-MIBG scintigraphic parameters. Conclusion: Despite a better control in SBP, HR during rest and exercise, and improvement in HRR, nebivolol failed to show a positive effect on peak VO2 and 123I-MIBG scintigraphic parameters. The lack of effect on adrenergic activity may be the cause of the lack of effect on functional capacity.

Resumo Fundamento: Mais de 50% dos pacientes com insuficiência cardíaca têm fração de ejeção preservada (ICFEN). A cintilografia marcada com iodo 123 com metaiodobenzilguanidina (123I-MIBG) e o teste cardiopulmonar do exercício (TCPE) são marcadores de prognóstico da ICFEN. O nebivolol é um betabloqueador com propriedade vasodilatadora. Objetivos: Avaliar o impacto da terapia com nebivolol sobre as variáveis da cintilografia com 123I-MIBG e do TCPE em pacientes com ICFEN. Métodos: Vinte e cinco pacientes realizaram cintilografia com 123I-MIBG para avaliar a taxa de washout e a relação coração/mediastino precoce e tardia. Durante o TCPE, foi analisado o comportamento da pressão arterial sistólica (PAS), frequência cardíaca (FC) durante o esforço e a recuperação (FCR) e o consumo de oxigênio (VO2). Após avaliação inicial, separamos nossa amostra em grupos controle versus intervenção, iniciamos o nebivolol e repetimos os exames após 3 meses. Resultados: Após o tratamento, o grupo intervenção apresentou melhora na PAS (149 mmHg [143,5-171 mmHg] versus 135 mmHg [125-151 mmHg, p = 0,016]), FC em repouso (78 bpm [65,5-84 bpm] versus 64,5 bpm [57,5-75,5 bpm, p = 0,028]), PAS no pico do esforço (235 mmHg [216,5-249 mmHg] versus 198 mmHg [191-220,5 mmHg], p = 0,001), FC no pico do esforço (124,5 bpm [115-142 bpm] versus 115 bpm [103,7-124 bpm], p = 0,043) e FCR no 1º minuto (6,5 bpm [4,75-12,75 bpm] versus 14,5 bpm [6,7-22 bpm], p = 0,025) e no 2º minuto (15,5 bpm [13-21,75 bpm] versus 23,5 bpm [16-31,7 bpm], p = 0,005), porém não apresentou mudança no VO2 de pico e nos parâmetros da cintilografia com 123I-MIBG. Conclusão: Apesar de um melhor controle da PAS e na FC em repouso e durante o esforço e uma melhora na FCR, o nebivolol não ocasionou efeito positivo sobre o VO2 de pico e nos parâmetros da cintilografia com 123I-MIBG. A ausência de efeito sobre a atividade adrenérgica pode ser a causa da falta de efeito sobre a capacidade funcional.

Effect of Nebivolol on MIBG Parameters and Exercise in Heart Failure with Normal Ejection Fraction.

BACKGROUND: More than 50% of the patients with heart failure have normal ejection fraction (HFNEF). Iodine-123 metaiodobenzylguanidine (123I-MIBG) scintigraphy and cardiopulmonary exercise test (CPET) are prognostic markers in HFNEF. Nebivolol is a beta-blocker with vasodilating properties. OBJECTIVES: To evaluate the impact of nebivolol therapy on CPET and123I-MIBG scintigraphic parameters in patients with HFNEF. METHODS: Twenty-five patients underwent 123I-MIBG scintigraphy to determine the washout rate and early and late heart-to-mediastinum ratios. During the CPET, we analyzed the systolic blood pressure (SBP) response, heart rate (HR) during effort and recovery (HRR), and oxygen uptake (VO2). After the initial evaluation, we divided our cohort into control and intervention groups. We then started nebivolol and repeated the tests after 3 months. RESULTS: After treatment, the intervention group showed improvement in rest SBP (149 mmHg [143.5-171 mmHg] versus 135 mmHg [125-151 mmHg, p = 0.016]), rest HR (78 bpm [65.5-84 bpm] versus 64.5 bpm [57.5-75.5 bpm, p = 0.028]), peak SBP (235 mmHg [216.5-249 mmHg] versus 198 mmHg [191-220.5 mmHg], p = 0.001), peak HR (124.5 bpm [115-142 bpm] versus 115 bpm [103.7-124 bpm], p= 0.043), HRR on the 1st minute (6.5 bpm [4.75-12.75 bpm] versus 14.5 bpm [6.7-22 bpm], p = 0.025) and HRR on the 2nd minute (15.5 bpm [13-21.75 bpm] versus 23.5 bpm [16-31.7 bpm], p = 0.005), but no change in peak VO2 and 123I-MIBG scintigraphic parameters. CONCLUSION: Despite a better control in SBP, HR during rest and exercise, and improvement in HRR, nebivolol failed to show a positive effect on peak VO2 and 123I-MIBG scintigraphic parameters. The lack of effect on adrenergic activity may be the cause of the lack of effect on functional capacity.

Anti-atherogenic effects of a new thienylacylhydrazone derivative, LASSBio-788, in rats fed a hypercholesterolemic diet.

The compound LASSBio-788 (N-Allyl (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine) is a thienylacylhydrazone derivative shown to have antiplatelet, vasodilatory, and anti-inflammatory properties in vitro. We hypothesize that LASSBio-788 may exert beneficial effects on atherosclerosis. Male wistar rats were divided into 4 groups: Control group received standard rat chow, hypercholesterolemic group (HC) and HC+788 (compound LASSBio-788 group) received hypercholesterolemic diet for 45 days. HC+788 group received compound LASSBio-788 (100 µmol/kg) once daily in the last 15 days. LASSBio-788 reduced the levels of total cholesterol (109.1 ± 4.3 vs. 361.0 ± 12.8 mg/dl), triglycerides (66.1 ± 1.1 vs. 186.9 ± 17.7 mg/dl), LDLc (63.2 ± 6.1 vs. 330.9 ± 9.7 mg/dl), VLDLc (9.8 ± 1.1 vs. 45.0 ± 4.6 mg/dl) and malondialdehyde (4.8 ± 0.3 vs. 9.4 ± 0.5 nmol/ml) compared to the HC group. LASSBio-788 presented antiplatelet properties and decreased inflammatory markers levels. LASSBio-788 promoted a decrease in contractile response to phenylephrine and an improvement in endothelium-dependent vasorelaxant response by increasing two-fold the expression of nitric oxide synthase (eNOS). Our results suggest that the compound LASSBio-788 represents a new multi-targeted drug candidate for the treatment of atherosclerosis.

Interleucina 1β correlaciona-se com MIBG e parâmetros do exercício, na insuficiência cardíaca? / Do interleukin-1β levels correlate with MIBG and exercise parameters in heart failure?

FUNDAMENTO: Na insuficiência cardíaca, níveis de interleucina 1β (IL 1β) se associam a prognóstico. A atividade adrenérgica cardíaca avaliada através da cintilografia com metiodobenzilguanidina (I123 MIBG) e parâmetros do exercício são importantes preditores de prognóstico. A relação entre essas variáveis não está bem definida. OBJETIVO: Avaliar associação entre níveis de IL 1β com parâmetros do exercício e do I123 MIBG. MÉTODOS: Estudo observacional transversal, com avaliação de 25 pacientes consecutivos com insuficiência cardíaca e fração de ejeção menor que 45%, através de: dosagem de IL 1β; parâmetros do I123 MIBG [relação coração/mediastino precoce e tardia, taxa de washout (WO)]; e teste ergométrico em esteira pelo protocolo de Rampa. RESULTADOS: Separados em dois grupos pelos níveis de IL 1β (normal vs. elevado), o grupo com níveis aumentados apresentava menor reserva de duplo produto (RDP), menor capacidade funcional (CF) e recuperação mais lenta da frequência cardíaca no 1º (RFC 1º) e 2º minuto (RFC 2º), e maior WO. Na análise univariada, todas as variáveis se correlacionaram com a IL 1β; RDP: r = 0,203, p = 0,024; CF: r = 0,181, p = 0,034; RFC 1º: r = 0,182, p = 0,034; RFC 2º: r = 0,204, p = 0,023; WO: r = 0,263, p = 0,009. Na multivariada, apenas a WO permaneceu com correlação significativa (r2 = 0,263, p = 0,009). CONCLUSÃO: A hipertonia adrenérgica foi o principal determinante dos níveis de IL 1β, demonstrando que a atividade simpática excessiva influencia a atividade inflamatória sistêmica. As variáveis do teste ergométrico não foram capazes de identificar pacientes com níveis elevados de IL 1β.

BACKGROUND: Interleukin 1β (IL 1β) levels are associated with prognosis in heart failure. The cardiac adrenergic activity as assessed by metaiodobenzylguanidine (I123 MIBG) scintigraphy along with exercise parameters are important predictors of prognosis. The relationship between these variables is not fully established. OBJECTIVE: To evaluate the association of IL 1β levels with exercise and I123 MIBG parameters. METHODS: Cross-sectional observational study evaluating 25 consecutive patients with heart failure and ejection fraction lower than 45% by means of: determination of IL 1β levels; I123 MIBG parameters [early and late heart/mediastinum ratio, washout rate (WO)]; and treadmill exercise test using the ramp protocol. RESULTS: The patients were divided into two groups according to their IL 1β levels (normal vs. increased). The group with increased levels showed lower double-product reserve (DPR); lower functional capacity (FC); slower heart rate recovery at the first (HRR 1º) and second minute (HRR 2º); and higher WO. In the univariate analysis, all variables correlated with IL 1β; DPR: r = 0.203, p = 0.024; FC: r = 0.181, p = 0.034; HRR 1º: r = 0.182, p = 0.034; HRR 2º: r = 0.204, p = 0.023; WO: r = 0.263, p = 0.009. In the multivariate analysis, only WO maintained a significant correlation (r² = 0.263, p = 0.009). CONCLUSION: Adrenergic overactivity was the main determinant of IL 1β levels, thus demonstrating that an excessive sympathetic activity influences the systemic inflammatory response. Exercise test variables were not able to identify patients with high IL 1β levels.

Do interleukin-1ß levels correlate with MIBG and exercise parameters in heart failure?

BACKGROUND: Interleukin 1ß (IL 1ß) levels are associated with prognosis in heart failure. The cardiac adrenergic activity as assessed by metaiodobenzylguanidine (I123 MIBG) scintigraphy along with exercise parameters are important predictors of prognosis. The relationship between these variables is not fully established. OBJECTIVE: To evaluate the association of IL 1ß levels with exercise and I123 MIBG parameters. METHODS: Cross-sectional observational study evaluating 25 consecutive patients with heart failure and ejection fraction lower than 45% by means of: determination of IL 1ß levels; I123 MIBG parameters [early and late heart/mediastinum ratio, washout rate (WO)]; and treadmill exercise test using the ramp protocol. RESULTS: The patients were divided into two groups according to their IL 1ß levels (normal vs. increased). The group with increased levels showed lower double-product reserve (DPR); lower functional capacity (FC); slower heart rate recovery at the first (HRR 1º) and second minute (HRR 2º); and higher WO. In the univariate analysis, all variables correlated with IL 1ß; DPR: r = 0.203, p = 0.024; FC: r = 0.181, p = 0.034; HRR 1º: r = 0.182, p = 0.034; HRR 2º: r = 0.204, p = 0.023; WO: r = 0.263, p = 0.009. In the multivariate analysis, only WO maintained a significant correlation (r² = 0.263, p = 0.009). CONCLUSION: Adrenergic overactivity was the main determinant of IL 1ß levels, thus demonstrating that an excessive sympathetic activity influences the systemic inflammatory response. Exercise test variables were not able to identify patients with high IL 1ß levels.

Ativação adrenérgica alterada se associa à recuperação anormal da Frequência Cardíaca? / Is abnormal adrenergic activation associated with abnormal Heart Rate Recovery?

FUNDAMENTO: Frequência Cardíaca de Recuperação (RFC) reflete a capacidade do sistema cardiovascular de reverter a supressão vagal ocasionada pelo exercício. A cintilografia com metaiodobenzilguanidina (I¹²³ MIBG) avalia a inervação e ativação adrenérgica cardíaca. A associação entre esses dois métodos não está bem esclarecida. OBJETIVO: Avaliar associação entre RFC e Taxa de "Washout" (WO) de I¹²³ MIBG em pacientes com Insuficiência Cardíaca (IC). MÉTODOS: Vinte e cinco pacientes com fração de ejeção < 45% realizaram teste ergométrico, sendo analisada a variação da RFC do 1º ao 8º minuto pós-esforço. Submetidos a I¹²³ MIBG foram separados em grupos pela WO: G1) < 27% e G2) > 27%. Para análise estatística, foi utilizado teste U de Mann Whitney e o coeficiente de correlação de Spearman. RESULTADOS: G2 demonstrou uma variação mais lenta da RFC: 1º minuto: G1: 21,5 (16,12 - 26,87) vs G2: 11,00 (8,5 - 13,5) bpm, p = 0,001; 2º minuto: G1: 34 (29 - 39) vs G2: 20 (14 - 26) bpm, p = 0,001; 3º minuto: G1: 46 (37,8 - 54,1) vs G2: 30 (22 - 38) bpm, p = 0,005; 5º minuto: G1: 51,5 (42 - 61) vs G2: 39 (31,5 - 46,5) bpm, p = 0,013; e no 8º minuto: G1: 54,5 (46,5 - 62,5) vs G2: 43 (34 - 52) bpm, p = 0,037. A RFC no 1º (r = -0,555; p = 0,004), e 2º minuto (r = -0,550; p = 0,004) apresentaram correlação negativa com WO. CONCLUSÃO: Pacientes com IC e WO elevado apresentaram uma RFC anormal em comparação com pacientes com WO normal. Tais achados sugerem que a ativação adrenérgica pode influenciar na RFC.

BACKGROUND: Heart Rate Recovery (HRR) reflects the capacity of the cardiovascular system to reverse the vagal withdrawal caused by exercise. Scintigraphy with metaiodobenzylguanidine (I¹23 MIBG) evaluates innervation and cardiac adrenergic activation. The association of these two methods is not well established. OBJECTIVE: To evaluate the association between HRR and washout rate (WO) of I¹²³ MIBG in patients with heart failure (HF). METHODS: wenty-five patients with ejection fraction < 45% underwent exercise testing, and analysis of the variation of HRR from the 1st to the 8th minute after exertion. Submitted to I¹²³ MIBG, they were separated into groups by WO: G1) <27% and G2) > 27%. For the statistical analysis Mann-Whitney's U test and Spearman's correlation coefficient were used. RESULTS: G2 showed a slower variation of HRR: 1st minute: G1: 21.5 (16.12 to 26.87) vs. G2: 11.00 (8.5 to 13.5) bpm, p = 0.001; 2nd minute: G1: 34 (29-39) vs. G2: 20 (14 - 26) bpm, p = 0.001; 3rd minute: G1: 46 (37.8 - 54.1) vs. G2: 30 (22 - 38) bpm, p = 0.005; 5th minute: G1: 51.5 (42 - 61) vs. G2: 39 (31.5 to 46.5) bpm, p = 0.013, and in the 8th minute: G1: 54.5 (46.5 - 62.5) vs. G2: 43 (34 - 52) bpm, p = 0.037. HRR in the 1st (r = -0.555, p = 0.004), and in the 2nd minute (r = -0.550, p = 0.004) were negatively correlated with WO. CONCLUSION: Patients with high HF and WO showed an abnormal HRR compared with patients with normal WO. These findings suggest that adrenergic activation may influence the HRR.

Is abnormal adrenergic activation associated with abnormal Heart Rate Recovery?

BACKGROUND: Heart Rate Recovery (HRR) reflects the capacity of the cardiovascular system to reverse the vagal withdrawal caused by exercise. Scintigraphy with metaiodobenzylguanidine (I(123) MIBG) evaluates innervation and cardiac adrenergic activation. The association of these two methods is not well established. OBJECTIVE: To evaluate the association between HRR and washout rate (WO) of I(123) MIBG in patients with heart failure (HF). METHODS: Twenty-five patients with ejection fraction < 45% underwent exercise testing, and analysis of the variation of HRR from the 1st to the 8th minute after exertion. Submitted to I(123) MIBG, they were separated into groups by WO: G1) <27% and G2) ≥ 27%. For the statistical analysis Mann-Whitney's U test and Spearman's correlation coefficient were used. RESULTS: G2 showed a slower variation of HRR: 1st minute: G1: 21.5 (16.12 to 26.87) vs. G2: 11.00 (8.5 to 13.5) bpm, p = 0.001; 2nd minute: G1: 34 (29-39) vs. G2: 20 (14 - 26) bpm, p = 0.001; 3rd minute: G1: 46 (37.8 - 54.1) vs. G2: 30 (22 - 38) bpm, p = 0.005; 5th minute: G1: 51.5 (42 - 61) vs. G2: 39 (31.5 to 46.5) bpm, p = 0.013, and in the 8th minute: G1: 54.5 (46.5 - 62.5) vs. G2: 43 (34 - 52) bpm, p = 0.037. HRR in the 1st (r = -0.555, p = 0.004), and in the 2nd minute (r = -0.550, p = 0.004) were negatively correlated with WO. CONCLUSION: Patients with high HF and WO showed an abnormal HRR compared with patients with normal WO. These findings suggest that adrenergic activation may influence the HRR.

Muscarinic acetylcholine receptor subtypes in the male reproductive tract: expression and function in rat efferent ductules and epididymis.

In mammals, at least five different muscarinic acetylcholine receptor subtypes (mAChRs; M(1)-M(5)) are known to be widely expressed and distributed in different tissues from different species. They mediate distinct physiological functions according to their location and receptor subtype. Multiple events are associated with the regulation of intracellular signaling by mAChRs, and a coordinated balance of the molecular mechanisms governing receptor signaling, desensitization, resensitization, and mitogenic signaling is known to occur in various cell types. Most of the actions of acetylcholine (ACh) in the male reproductive tract are induced by its effects on mAChRs, but the role of specific mAChR subtypes on male reproductive function and fertility are still not well understood. The rat efferent ductules and epididymis are androgen-dependent tissues of the male reproductive tract, with important roles in the process to form a viable and fertile sperm. In the present study, aspects of the expression, localization, and potential function of mAChR subtypes in rat efferent ductules and epididymis are reviewed. Furthermore, evidences for the implication of mAChRs in the regulation of protein synthesis and secretion in these tissues are presented. Taken together, the studies contribute to our understanding about physiological aspects of mAChR and mechanisms by which the cholinergic system affects male reproduction.

Effects of the antiestrogen fulvestrant (ICI 182,780) on gene expression of the rat efferent ductules.

The efferent ductules express the highest amount of estrogen receptors ESR1 (ERalpha) and ESR2 (ERbeta) within the male reproductive tract. Treatment of rats with the antiestrogen fulvestrant (ICI 182,780) causes inhibition of fluid reabsorption in the efferent ductules, leading to seminiferous tubule atrophy and infertility. To provide a more comprehensive knowledge about the molecular targets for estrogen in the rat efferent ductules, we investigated the effects of ICI 182,780 treatment on gene expression using a microarray approach. Treatment with ICI 182,780 increased or reduced at least 2-fold the expression of 263 and 98 genes, respectively. Not surprisingly, several genes that encode ion channels and macromolecule transporters were affected. Interestingly, treatment with ICI 182,780 markedly altered the expression of genes related to extracellular matrix organization. Matrix metalloproteinase 7 (Mmp7), osteopontin (Spp1), and neuronal pentraxin 1 (Nptx1) were among the most altered genes in this category. Upregulation of Mmp7 and Spp1 and downregulation of Nptx1 were validated by Northern blot. Increase in Mmp7 expression was further confirmed by immunohistochemistry and probably accounted for the decrease in collagen content observed in the efferent ductules of ICI 182,780-treated animals. Downregulation of Nptx1 probably contributed to the extracellular matrix changes and decreased amyloid deposition in the efferent ductules of ICI 182,780-treated animals. Identification of new molecular targets for estrogen action may help elucidate the regulatory role of this hormone in the male reproductive tract.

Muscarinic acetylcholine receptor subtypes in the rat seminal vesicle.

The aim of the present study was to identify the muscarinic acetylcholine receptor (mAChR) mRNA subtypes in the rat seminal vesicle. Furthermore, the mAChR subtypes involved in the contraction of the seminal vesicle were also explored. Reverse transcriptase-polymerase chain reaction (PCR) was performed and five PCR products corresponding to M1-M5 mAChR mRNA subtypes were detected in this tissue. Functional pharmacological studies indicated that the rank order of mAChR antagonists in blocking the contractile effects of carbachol was p-fluoro-hexahydro-sila-difenidol (pF-HHSiD) >> tropicamide > methoctramine = pirenzepine. This antagonist profile indicates that M3 mAChR subtype is predominantly involved in the seminal vesicle contraction. Furthermore, immunohistochemical studies confirmed the presence of the M3 mAChR subtype in the smooth muscle layers. M2 mAChR subtype was also immunolocalized in smooth muscle cells and may be involved in the contraction of this tissue. The presence of M2 and M3 mAChR subtypes in the epithelial cells suggests that these receptors could be involved in the protein secretion. Taken together, the cholinergic neurotransmitter may be a factor controlling contractility and protein secretion in this tissue.

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.

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.

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.