Signal Transduction Underlying Carbachol-Induced Contraction of Rat Urinary Bladder. I. Phospholipases and Ca²⁺Sources

Abstract

We have reexamined the muscarinic receptor subtype mediating carbachol-induced contraction of rat urinary bladder and investigated the role of phospholipase (PL)C, D, and A 2 and of intra-and extracellular Ca 2ϩ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M 2 receptorselective (R)-4-{2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl}-piperidine-1-carboxylic acid amide , and the highly M 3 receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M 3 receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1- Carbachol had only little effect on PLD activity in bladder slices, but the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol (0.3% each), caused detectable inhibition of carbachol-induced bladder contraction. The cytosolic PLA 2 inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbacholinduced contraction at a concentration of 300 M, but the cyclooxygenase inhibitor indomethacin (1-10 M) remained without effect. The Ca 2ϩ entry blocker nifedipine (10 -100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast, 1-[␤-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 M), an inhibitor of storeoperated Ca 2ϩ channels, caused little inhibition. We conclude that carbachol-induced contraction of rat bladder largely depends on Ca 2ϩ entry through nifedipine-sensitive channels and, perhaps, PLD, PLA 2 , and store-operated Ca 2ϩ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.Muscarinic acetylcholine receptors are the physiologically most important mechanisms to elicit contraction of the urinary bladder (Andersson, 1993). In the bladder of various mammalian species, including humans, M 2 and M 3 muscarinic receptors coexist, but the expression of M 2 receptors is much greater than that of the M 3 receptors (Wang et al., 1995;Goepel et al., 1998;Yamanishi et al., 2000;Kories et al., 2003). Nevertheless, the contractile response to the exogenous agonist carbachol and to endogenous agonists released by field stimulation have been attributed predominantly, if not exclusively, to M 3 receptors in rats (Longhurst et al., 1995;Hegde et al., 1997;Tong et al., 1997;Braverman et al., 1998;Choppin et al., 1998;Longhurst and Levendusky, 2000;Kories et al., 2003), mice (Choppin and Eglen, 2001b), pigs (Yamanishi et al., 2000), dogs (Choppin and Eglen, 2001a), and humans (Chess-Williams et al., 2001;Fetscher et al., 2002). Moreover, at least male M 3 (but not M 2 ) receptorknockout mice exhibit bladder distension and develop urinary retention (Matsui et al., 2000). On the other hand, it should be considered that hitherto available antagonists have only modest subtype selectivity and/or do not act in a purely competitive manner; hence, they were not well suited for detecting a pot...

Signal Transduction Underlying Carbachol-Induced Contraction of Rat Urinary Bladder. II. Protein Kinases

“…Although rat bladder expresses more M 2 than M 3 receptors, contractile responses to the exogenous muscarinic agonist carbachol and to endogenous agonists released by field stimulation occur predominantly, if not exclusively, via M 3 receptors in rats (Longhurst et al, 1995;Hegde et al, 1997;Tong et al, 1997;Braverman et al, 1998;Choppin et al, 1998;Longhurst and Levendusky, 2000;Kories et al, 2003). In the accompanying paper, we have confirmed the involvement of M 3 but not M 2 receptors in carbachol-induced contraction and demonstrated that this involves nifedipine-sensitive Ca 2ϩ channels and, to a lesser degree, phospholipases D and A 2 and store-operated Ca 2ϩ channels but not cyclooxygenase or, surprisingly, phospholipase C (Schneider et al, 2004). Overall, this pattern of proximal signaling mediating urinary bladder smooth muscle contraction shares many properties with that of other types of smooth muscle, such as vascular smooth muscle.…”

“…In the present study, three chemically distinct PKC inhibitors failed to significantly affect carbachol-induced contraction of rat urinary bladder despite being tested in high concentrations. Since PKC activation, particularly of the classical PKC isoforms frequently occurs secondary to phospholipase C activation, and since experiments shown in the accompanying paper did not detect a role for phospholipase C in urinary bladder contraction (Schneider et al, 2004), these data demonstrate that the phospholipase C/PKC cascade may be activated by muscarinic receptors in rat bladder (Livak and Schmittgen, 2001;Schneider et al, 2004) but is not crucial for induction of contraction.…”

“…The accompanying paper described the relative roles of proximal signaling pathways in mediating this response (Schneider et al, 2004). In other types of smooth muscle, these proximal pathways are linked to smooth muscle contraction by a network of protein kinases, but the specific wiring depends on the receptor and type of smooth muscle under investigation.…”

A possible role of the cholinergic and purinergic receptor interaction in the regulation of the rat urinary bladder function

“…Acetylcholine acts via M 3 receptor activation, generation of intracellular inositol-1,4,5-trisphosphate (IP 3 ) and Ca 2+ release from intracellular stores. There is also evidence that muscarinic activation leads to Ca 2+ influx (Schneider et al, 2004). Conversely, ATP binds to ionotropic P2X receptors, depolarizing the cell membrane and instigating Ca 2+ influx through voltage-activated (mainly L-type) Ca 2+ channels (Andersson and Arner, 2004 ] i ) remains a common pathway to regulate detrusor contractility, with additional modulation via alterations to the Ca 2+ sensitivity of contractile proteins (Andersson and Arner, 2004;Arner et al 2007).…”

Bradykinin Contracts Rat Urinary Bladder Largely Independently of Phospholipase C

“…This has also been assumed to be the molecular mechanism underlying bladder contraction (Ouslander, 2004). Indeed, stimulation of M 3 muscarinic receptors in the bladder activates PLC (Kories et al, 2003;Schneider et al, 2004b). Surprisingly, however, several studies in mice (Wegener et al, 2004), rats (Schneider et al, 2004b;Frazier et al, 2007), and humans (Schneider et al, 2004a) demonstrate that such activation contributes only little to bladder contraction.…”