RESUMO
A low-affinity Ca²âº/Hâº-antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation-release coupling in rapid synapses. Synaptotagmin-1, a vesicular protein interacting with membranes upon low-affinity Ca²âº-binding, plays a major role in excitation-release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin-1 is necessary for expression of the vesicular Ca²âº/Hâº-antiport. We measured Ca²âº/Hâº-antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca²âº-induced dissipation of the vesicular Hâº-gradient; (ii) bafilomycin-sensitive calcium accumulation and (iii) pH-jump-induced calcium accumulation. The results were congruent and highly significant: Ca²âº/Hâº-antiport activity is detectable only in acidic organelles expressing functional synaptotagmin-1. In contrast, synaptotagmin-1-deficient cells--and cells where transgenically encoded synaptotagmin-1 was acutely photo-inactivated--were devoid of any Ca²âº/Hâº-antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca²âº sequestration through a Ca²âº/H⺠antiport.
Assuntos
Antiporters/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sinaptotagmina I/fisiologia , Antiporters/antagonistas & inibidores , Química Encefálica/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Células Clonais , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Corantes Fluorescentes , Humanos , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Ionomicina/farmacologia , Macrolídeos/farmacologia , Células PC12 , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética , Transfecção , Proteína 1 Associada à Membrana da Vesícula/antagonistas & inibidores , Proteína 1 Associada à Membrana da Vesícula/imunologiaRESUMO
Mediators of neuromuscular transmission in rat bladder strips were dissected pharmacologically to examine their susceptibilities to inhibition by botulinum neurotoxins (BoNTs) and elucidate a basis for the clinical effectiveness of BoNT/A in alleviating smooth muscle spasms associated with overactive bladder. BoNT/A, BoNT/C1, or BoNT/E reduced peak and average force of muscle contractions induced by electric field stimulation (EFS) in dose-dependent manners by acting only on neurogenic, tetrodotoxin-sensitive responses. BoNTs that cleaved vesicle-associated membrane protein proved to be much less effective. Acetylcholine (ACh) and ATP were found to provide virtually all excitatory input, because EFS-evoked contractions were abolished by the muscarinic receptor antagonist, atropine, combined with either a desensitizing agonist of P2X(1) and P2X(3) or a nonselective ATP receptor antagonist. Both transmitters were released in the innervated muscle layer and, thus, persisted after removal of urothelium. Atropine or a desensitizer of the P2X(1) or P2X(3) receptors did not alter the rate at which muscle contractions were weakened by BoNT/A. Moreover, although cholinergic and purinergic signaling could be partially delineated by using high-frequency EFS (which intensified a transient, largely atropine-resistant spike in muscle contractions that was reduced after P2X receptor desensitization), they proved equally susceptible to BoNT/A. Thus, equi-potent blockade of ATP co-released with ACh from muscle efferents probably contributes to the effectiveness of BoNT/A in treating bladder overactivity, including nonresponders to anticholinergic drugs. Because purinergic receptors are known mediators of sensory afferent excitation, inhibition of efferent ATP release by BoNT/A could also help to ameliorate acute pain and urgency sensation reported by some recipients.