Cholinergic-induced Ca²⁺ signaling in interstitial cells of Cajal from the guinea pig bladder

Abstract

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor 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 bladder detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca2+ indicator fluo 4AM. ICC fired Ca2+ transients in response to stimulation by carbachol (1/10 μM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 μM), an M3 receptor antagonist, but not by the M2 receptor antagonist methoctramine (1 μM). The source of Ca2+ underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 μM) or Ni2+ (30–100 μM) to block Ca2+ channels or the removal of external Ca2+ reduced the amplitude of the carbachol transients. Application of ryanodine (30 μM) or tetracaine (100 μM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 μM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 μM) caused a significant reduction and Xestospongin C (1 μM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca2+ indicator showed distinctively different patterns of spontaneous Ca2+ events in smooth muscle cells 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 Ca2+ transients.

Detrusor myocyte activity and afferent signaling

“…Some of these cells were closely associated with intramural nerves. 54,55 ICCs are c-kit positive in the guinea-pig bladder 51 and are also highly immunoreactive for cGMP. 50,52 Kubota et al 56 assessed the possible role of these c-kit positive cells in the bladder, and investigated the effects of the c-kit tyrosine kinase inhibitor, imatinib mesylate (Glivec 1 ), on spontaneous excitation and ion channel activity in detrusor smooth muscle using intracellular microelectrodes, isometric muscle tension recordings, and patch-clamp techniques.…”

“…They have been found not only in the suburothelium (lamina propria), where they may serve sensory functions, 46 -49 but also as a network within the detrusor muscle. 50 -55 Hashitani et al 9 and Johnston et al 55 found that within the guinea pig detrusor, based on their location, ICC could be divided into three subpopulations: those adjacent to the boundary of muscle bundles (boundary ICC or ICC-IB), those scattered amongst the smooth muscle cells within the muscle bundles (intramuscular ICC, ICC-IM), and those distributed in connective tissues between muscle bundles (interbundle ICC). Some of these cells were closely associated with intramural nerves.…”

“…7 Support for such an action is accumulating. 51 Immunohistochemical labeling and confocal microscopy showed that the ICC-IM and ICC-IB in the detrusor lie in close proximity to intramural nerves, 51,55 and it has been reported that both enzymatically dispersed detrusor ICC and ICC within tissue preparations respond to cholinergic stimulation by firing Ca 2þ transients. This suggests that nerve-released ACh can activate detrusor myocytes directly, but also indirectly via activation of ICC.…”

“…As in detrusor myocytes, the muscarinic receptor subtype involved in activation seems to be the M3 receptor. 51,55 It may be speculated that detrusor ICC are involved not only in the generation of contractile activity of detrusor muscle during filling (spontaneously generated Ca 2þ signals), but also in parasympathetically mediated voiding contractions (AChinduced signals), contributing to the coordination of the activity of contractile units produced by the nerve input. Interestingly, the suburothelial ICCs also may be able to affect the activity of the detrusor myocytes.…”

Control of Urinary Drainage and Voiding

“…In the last decade, these have been identified as bladder ICCs (BICCs) and the emerging consensus is that they are likely to play an important role in modulating bladder motility (74)(75)(76)(77)(78)(79)(80)(81). ICCs were first identified in the gastrointestinal tract where they function as pacemakers, neuron transducers, and mechanosensors to modulate smooth muscle motility (82)(83)(84)(85), and more than a dozen gut disorders are linked to ICC dysfunction, including gastroparesis and intestinal pseudo-obstruction (86,87).…”