Synaptotagmin 1 is required for vesicular Ca²âº/H⁺-antiport activity.

A low-affinity Ca²âº/H⁺-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 Ca²âº-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 Ca²âº/H⁺-antiport. We measured Ca²âº/H⁺-antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca²âº-induced dissipation of the vesicular H⁺-gradient; (ii) bafilomycin-sensitive calcium accumulation and (iii) pH-jump-induced calcium accumulation. The results were congruent and highly significant: Ca²âº/H⁺-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 Ca²âº/H⁺-antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca²âº sequestration through a Ca²âº/H⁺ antiport.

Functional transient receptor potential vanilloid 1 is expressed in human urothelial cells.

PURPOSE: We investigated the expression and functional status of TRPV1 receptor in human urothelial cells. MATERIAL AND METHODS: Human urothelium was cultured and TRPV1 receptor expression was studied by immunocytochemistry and reverse transcriptase-polymerase chain reaction. The influence of inflammatory mediators on TRPV1 mRNA levels was also studied. Functional assays (cobalt uptake measurements and whole cell voltage clamp records) were used to study the response of urothelial cells to capsaicin, temperature, low pH and inflammatory mediators. Capsaicin induced adenosine triphosphate release from urothelial cells was assessed by bioluminescence. RESULTS: TRPV1 protein and mRNA were detected in human urothelial cells and mRNA more than tripled in the presence of inflammatory mediators. Nerve growth factor treatment alone did not affect TRPV1 mRNA expression. Capsaicin (100 nM and 1 microM) and heat (41C and 45C) evoked cobalt uptake and inflammatory mediators lowered the temperature threshold for TRPV1 activation to 37C. Capsaicin (1 microM) induced TRPV1 desensitization to further applications of the agonist. In whole cell patch clamp experiments 1 microM capsaicin and a heat ramp from 37C to 50C caused inward currents. The same concentration of capsaicin induced the release of about 7 fmol adenosine triphosphate per mg. CONCLUSIONS: TRPV1 receptors expressed by human urothelial cells respond to capsaicin and thermal stimuli. Capsaicin evoked release of adenosine triphosphate suggests that human urothelial TRPV1 is involved in the afferent branch of the micturition reflex. Inflammatory mediators decrease the TRPV1 thermal threshold of activation to body temperature and increase its expression. This finding may be relevant for symptoms associated with cystitis.