Cholinergic-induced [Ca.sup.2+] signaling in interstitial cells of Cajal from the guinea pig bladder

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detmsor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladde,' detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the [Ca.sup.2+] indicator fluo 4AM. ICC fired [Ca.sup.2+] transients in response to stimulation by carbachol (1/10 [micro]M). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 [micro]M), an [M.sub.3] receptor antagonist, but not by the [M.sub.2] receptor antagonist methoctramine (1 [micro]M). The source of [Ca.sub.2+] underlying the carbachol transients in isolated 1CC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 [micro]M) or [Ni.sup.2+] (30-100 [micro]M) to block [Ca.sup.2+] channels or the removal of external [Ca.sup.2+] reduced the amplitude of the carbachol transients. Application of ryanodine (30 [micro]M) or tetracaine (100 [micro]M) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 [micro]M), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 [micro]M) caused a significant reduction and Xestospongin C (1 [micro]M) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a [Ca.sup.2+] indicator showed distinctively different patterns of spontaneous [Ca.sup.2+] events in smooth muscle ceils and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing [Ca.sup.2+] transients. confocal microscopy; c-Kit