RESUMO
PROBLEM: Inflammation and genital infections promote the increase in leukocytes, pro-inflammatory cytokines, and oxygen reactive species, impairing sperm functions such as motility, capacitation, and acrosome reaction. All these functions are primarily regulated by cytoplasmic concentration of Ca(2+) ([Ca(2+) ]cyto ). This study evaluated the effect of tumor necrosis factor (TNF)-α on the [Ca(2+) ]cyto and its regulation in human sperm. METHOD OF STUDY: Sperm loaded with fura-2 were incubated with or without TNF-α (0-500 pg/mL) from 0 to 120 min. After incubation, the basal [Ca(2+) ]cyto and membrane permeability to Ca(2+) were evaluated by spectrofluorometry, before and after Ca(2+) addition to the extracellular medium. RESULTS: Without TNF-α, the addition of Ca(2+) promotes an transitory increase in [Ca(2+) ]cyto in the spermatozoa, that returns in a few minutes to a basal level, indicating calcium regulation activation. TNF-α decreases the Ca(2+) permeation and increases the basal level of [Ca(2+) ]cyto after a Ca(2+) pulse (P < 0.04); affecting calcium regulation in a way that is time and concentration dependent. TNF-α effect was partially prevented by the addition of an antioxidant (butylated hydroxytoluene) (P < 0.03). CONCLUSION: Tumor necrosis factor-α decreases membrane permeability to Ca(2+) and affects Ca(2+) regulation in sperm cells in vitro, probably via lipid peroxidation, which may explain the decrease in sperm fertilizing capacity during inflammatory and infectious processes.
Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Reação Acrossômica , Adulto , Membrana Celular/metabolismo , Células Cultivadas , Fertilização , Fura-2/farmacologia , Homeostase , Humanos , Masculino , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/metabolismo , Adulto JovemRESUMO
We have recently synthesized a new series of hybrid compounds having the moieties of tacrine, a potent inhibitor of brain and peripheral acetylcholinesterase (AChE), and nimodipine, a blocker of L-type voltage-dependent calcium channels (VDCCs). These compounds were designed to target AChE and L calcium channels in the brain, as potential therapeutic agents in Alzheimer's disease. We performed the present study to determine the main peripheral side effects of two of these compounds, ITH12117 and ITH12118. We have here shown that in rat vas deferens these compounds inhibited AChE with a potency about 1000-fold lower than that of physostigmine or tacrine. Furthermore, the hybrid compounds enhanced contractions evoked by acetylcholine, with a potency about 100-fold lower than that of physostigmine or tacrine. Additionally, contractions induced by Ca2+ on depolarized vas deferens were blocked by nimodipine with greater efficacy, compared with ITH12117 and ITH12118. Compound ITH12118 (1 µM) caused a pronounced inhibition of the tonic (but not phasic) contraction elicited by electrical field stimulation. Furthermore, the same dose of nimodipine and ITH12118 blocked by 75% cytosolic Ca2+ elevations produced by acetylcholine, noradrenaline, or ATP. As a matter of comparison, we showed that rat brain cortex AChE was inhibited by ITH12118 with a potency 10 to 20-fold higher than that for vas deferens. This study shows that ITH12118 could be a paradigmatic multitarget compound having selective brain effects with smaller peripheral side effects. This may help to orient the search of new neuroprotective compounds with potential therapeutic application in Alzheimer's disease.
Assuntos
Acetilcolinesterase/metabolismo , Cálcio/metabolismo , Inibidores da Colinesterase/farmacologia , Contração Muscular/efeitos dos fármacos , Tacrina/farmacologia , Ducto Deferente/metabolismo , Ducto Deferente/fisiologia , Acetilcolina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Butirilcolinesterase/metabolismo , Cálcio/farmacologia , Córtex Cerebral/enzimologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Estimulação Elétrica , Fura-2/farmacologia , Humanos , Técnicas In Vitro , Masculino , Nimodipina/farmacologia , Norepinefrina/farmacologia , Fisostigmina/farmacologia , Ratos , Ratos Wistar , Ducto Deferente/citologia , Ducto Deferente/efeitos dos fármacosRESUMO
We previously reported developmental differences in the response of cytosolic free calcium ([Cai]) to extracellular potassium ([Kex]) depolarization (cardioplegia) in juvenile (4 weeks post partum) and mature (12 to 18 months post partum) cardiac myocytes (rabbit). Our present study explored the physiologic basis for these observations. Single calcium-tolerant cardiac myocytes were isolated by sequential exposure to proteolytic agents, loaded with a fluorescent probe for calcium (fura-2) and [Cai] measured by standard fluorescence techniques. The response of [Cai] to [Kex] depolarization (30 mmol/L) was determined in the presence of varying levels of extracellular calcium [Caex], verapamil, ouabain, and amiloride. At nominal levels of [Caex] (0 mmol/L), no significant increases in [Cai] from the juvenile (2.1% +/- 3.3%) or mature (8.5% +/- 3.1%) myocytes were seen. At increasing [Caex], there was a progressive increase in the response of [Cai] to [Kex] depolarization in mature animals' cells (191.8% +/- 40.7%) but not in the juveniles' cells (28.0% +/- 11.5%). Exposure to verapamil resulted in an approximate 80% reduction in relative increase in [Cai] in the mature compared with 60% in the juvenile myocytes. Finally, ouabain exposure resulted in a significant increase in the relative change of [Cai] in juvenile cells (30% to 126.4% +/- 39.4%) but not in the adults' cells. This increase in the juvenile myocytes was blocked by amiloride. We conclude that increases in cardiac myocyte [Cai] after [Kex] depolarization occur predominantly through the calcium channel in the mature animal and through sodium-calcium exchange in the juvenile.