RESUMEN
BACKGROUND: The aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens function, leading to compromised reproductive capacity. Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life. METHODOLOGY/PRINCIPAL FINDINGS: Young adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca(2+)-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca(2+) affinity and regulation of Ca(2+) handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca(2+) release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca(2+) handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca(2+)-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation. CONCLUSIONS/SIGNIFICANCE: The functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the vas deferens were an accentuated reduction in fertility and fecundity.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Desnutrición/patología , Estrés Oxidativo , Reproducción , Conducto Deferente/metabolismo , Conducto Deferente/patología , Envejecimiento/patología , Animales , Transporte Biológico , Peso Corporal , ATPasas Transportadoras de Calcio/metabolismo , Recuento de Células , Supervivencia Celular , Enfermedad Crónica , Epidídimo/patología , Haploidia , Cinética , Masculino , Desnutrición/enzimología , Músculos/patología , Tamaño de los Órganos , Oxidación-Reducción , Fosforilación , Ratas , Ratas Wistar , Espermatozoides/patología , Testículo/patología , Conducto Deferente/enzimologíaRESUMEN
The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Calcio/metabolismo , Retículo Sarcoplasmático/enzimología , Conducto Deferente/inervación , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Membrana Celular/enzimología , Membrana Celular/metabolismo , Desnervación , Homeostasis , Masculino , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Conducto Deferente/enzimología , Conducto Deferente/metabolismoRESUMEN
In the rat vas deferens, an organ richly innervated by peripheral sympathetic neurons, we have demonstrated recently the expression of alpha(1) and alpha(2), but not alpha(3) isoforms of the alpha subunit of Na(+)/K(+)-ATPase (EC 3.6.1.37), a membrane-bound enzyme of vital function for living cells (Noël et al., Biochem Pharmacol 55: 1531-1535, 1998). In the present work, we characterized, qualitatively and quantitatively, Na(+)/K(+)-ATPase alpha isoforms in denervated rat vasa deferentia. [(3)H]Ouabain binding at concentrations defined for high-affinity isoforms (alpha(2) and/or alpha(3)) detected only one class of specific binding sites in control (C) and denervated (D) vas deferens. Although the dissociation constant was similar for both groups [K(d) = 138 +/- 14 nM (C) and 125 +/- 8 nM (D)], a marked decrease in density was observed after denervation [716 +/- 81 fmol.mg protein(-1) (C) and 445 +/- 34 fmol.mg protein(-1) (D), P < 0.05]. In addition, western blotting revealed that denervated vasa deferentia produce the alpha(1) and alpha(2) isoforms but not alpha(3), just as we reported for the controls previously (Noël et al., Biochem Pharmacol 55: 1531-1535, 1998). Densitometric analysis showed a decrease of the alpha(2) isoform by about 40% in denervated organs, in very good agreement with what was shown with the [(3)H]ouabain binding technique, but no significant change in alpha(1) isoform density. Truncated alpha(1) (alpha(1)T), an isoform suggested to exist in the guinea pig vas deferens, was not detected. Altogether, our results demonstrated that Na(+)/K(+)-ATPase alpha(2) is down-regulated after sympathetic denervation of the rat vas deferens.
Asunto(s)
Isoenzimas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Conducto Deferente/enzimología , Animales , Anticuerpos , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Isoenzimas/inmunología , Masculino , Ouabaína/farmacología , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/inmunología , Tritio , Conducto Deferente/inervación , Conducto Deferente/metabolismo , Conducto Deferente/cirugíaRESUMEN
Binding assays were performed with [3H]ouabain to investigate the presence of, and to characterize, a Na+/K(+)-ATPase isoform with high affinity for cardiac glycosides in the rat vas deferens. Nonlinear regression analysis of equilibrium experiments carried out with crude preparations in a Mg-Pi medium indicated the presence of high-affinity sites characterized with good precision (individual coefficients of variation = 11-35%) by their density (Bmax = 0.42 to 0.72 pmol/mg protein) and dissociation constant (Kd = 0.069 to 0.136 microM) values. The values of the dissociation rate constant (kappa-1) and the association rate constant (kappa+1) for these sites were 0.151 to 0.267 min-1 and 2.87 to 3.60 microM-1.min-1, respectively. A higher number of low-affinity sites (Kd around 15 microM), supposed to correspond to the alpha 1 isoform, was also identified, but their Kd and Bmax values were not quantified precisely in this crude preparation. Western blot assays indicated hybridization with specific anti-alpha 1 and anti-alpha 2 isoform antibodies but not with anti-alpha 3 isoform antibody. Taken together, the present results indicate the existence of a low proportion of the alpha 2 isoform of Na+/K(+)-ATPase in the rat vas deferens that can be quantified precisely by [3H]ouabain binding even in a crude membrane preparation that is suitable for studies under conditions of plasticity.
Asunto(s)
Isoenzimas/metabolismo , Ouabaína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Conducto Deferente/enzimología , Animales , Sitios de Unión , Encéfalo/enzimología , Fraccionamiento Celular , Isoenzimas/inmunología , Isoenzimas/aislamiento & purificación , Riñón/enzimología , Cinética , Masculino , Miocardio/enzimología , Ratas , Ratas Wistar , Análisis de Regresión , Reproducibilidad de los Resultados , ATPasa Intercambiadora de Sodio-Potasio/inmunología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificaciónRESUMEN
The endothelin receptors controlling sympathetic neurotransmission and the presence of endothelin-converting enzyme were investigated in the mouse vas deferens. Endothelin-1 or endothelin-3 (0.01-100 nM) enhanced contractions evoked by field stimulation, yielding EC50 (geometric mean and 95% confidence limits) of 0.7 nM (0.4-1.6) and 13.7 nM (10.2-14.1) and Emax (mean +/- S.E.M. increase in twitch tension, in mg/10 mg wet tissue) of 473 +/- 35 and 520 +/- 51, respectively. The selective endothelin ETB receptor agonists IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1) and sarafotoxin S6c were inactive up to 100 nM. Responses to endothelin-3 were progressively inhibited by the selective endothelin ETA receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]) (10, 30 and 100 nM). At 100 nM, BQ-123 almost abolished the response to endothelin-3 (100 nM). In contrast, at 100, 300 nM and 1 microM, BQ-123 shifted the curve to endothelin-1 to the right only 2-, 5- and 6-fold, respectively. The selective endothelin ETB receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-++ +methoxycarbonyltryptophanyl-D-norleucine) (100 nM) did not modify responses to endothelin-1 or endothelin-3 (0.01-100 nM). Big-endothelin-1 (0.3-30 nM) was 10-fold less potent than endothelin-1 in increasing neurogenic responses (EC50 6.8 nM, 4.7-9.6; Emax 457 +/- 37 mg/10 mg wet tissue). Preincubation with phosphoramidon (100 microM) reduced responses to big-endothelin-1, but not endothelin-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Receptores de Endotelina/fisiología , Conducto Deferente/fisiología , Animales , Estimulación Eléctrica , Enzimas Convertidoras de Endotelina , Endotelinas/farmacología , Masculino , Metaloendopeptidasas , Ratones , Contracción Muscular , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , Receptores de Endotelina/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Conducto Deferente/enzimologíaRESUMEN
Routine histological fixatives barely preserve tyrosine-hydroxylase immunoreactivity in paraffin sections. fixation in 5% acrolein in phosphate buffer, pH 7.4, resulted in good preservation of the enzyme in the tissues investigated.