Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca2+ from sarcoplasmic reticulum. However, the additional Ca2+-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca2+-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.

1 The signal transduction pathways involved in carbachol (CCh)-induced calcium sensitization in b-escin permeabilized rat and guinea-pig bladder smooth muscles were investigated and the results were compared with guinea-pig taenia caecum. 2 Calcium contractions elicited cumulatively (pCa 7.5-5) in the presence of calmodulin were significantly increased in all three tissues when CCh (50 mM) was added to the medium. 3 Under constant [Ca 2 þ ] i conditions (pCa 6), calmodulin (1 mM) and then GTP (100 mM) initiated significant contractions. CCh (50 mM) added to the bath caused a further contraction in all three tissues -calcium sensitization. This sensitization was significantly inhibited by atropine (50 mM). 4 The incubation of the tissues with the IP 3 -receptor blocker 2-APB (30 mM) reduced the subsequent development of calcium sensitization by CCh in rat bladder but did not affect it in guinea-pig bladder and taenia ceacum. 5 The Rho kinase (ROK) inhibitor Y-27632 (5 mM) added in the presence of CCh reversed the calcium sensitization in rat bladder, whereas a transient contraction followed by a relaxation to a level not significantly different from the CCh contraction was seen in both guinea-pig bladder and taenia caecum. Y-27632 (1 mM) continuously present significantly inhibited the CCh-induced Ca 2 þ sensitization in rat bladder but not in guinea-pig bladder or taenia caecum. 6 In the presence of cyclopiazonic acid (CPA) (1 mM) and calmodulin (1 mM), Y-27632 (5 mM) did not change the calcium response curve (3 Â 10 À7 -10 À5 M) in rat bladder but increased the contractile responses significantly in both guinea-pig bladder and taenia caecum. 7 The protein kinase C (PKC) inhibitor GF 109203X (5 mM) added in the presence of CCh inhibited the calcium sensitization induced by this muscarinic agonist in all three tissues in different ratios. 8 In conclusion, muscarinic receptor activation induces calcium sensitization in rat and guinea-pig detrusor smooth muscles but there are differences in their pathways.

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