Hypothesis / aims of study Carbachol (CCh)-induced detrusor contractions are mainly mediated via M3 receptor subtype^[1] and depend not only on Ca²⁺-release from the intracellular Ca²⁺ stores but also on Ca²⁺-influx via L-type Ca²⁺-channels. The proposed cellular mechanism for M₃-mediated contractions involves inositol trisphosphate (IP3)-induced Ca²⁺ release from intracellular

stores, Ca²⁺-influx via nifedipine-sensitive L-type Ca²⁺-channels and increased sensitivity of the contractile machinery to Ca²⁺ via inhibition of myosin light chain phosphatase through activation of rho kinase.^[2] The contribution of Ca²⁺influx was recently evaluated in Ca²⁺-channel (Cav1.2) knockout mice which had significantly smaller contractions induced by CCh compared to wildtype.^[3] The purpose of this study was to examine the different contribution of Ca²⁺-influx and possible other mechanisms underlying muscarinic receptor-mediated contractions in human, pig and mouse detrusor.

Study design, materials and methods
Contractility was measured in urothelium-denuded muscle strips from patients undergoing radical cystectomy for
treatment of urinary bladder cancer; from pigs and from C57Bl6 mice.
Contractions were induced by increasing concentrations of the muscarinic receptor agonist CCh. After equilibration,
cumulative concentration response curves (CRC) for CCh were generated followed by a washout period of 1 hour.
Then test drugs were added and a second CRC for CCh in the presence of the test compound was started after one
further hour. Time-matched control (TMC) experiments were run separately without any drug added.

Results The L-type Ca²⁺-channel blocker nifedipine impaired detrusor contraction in man by no more than 25 % but by 80 to 90 % in pig and mouse resulting in the following order of efficacy in the three studied species: man < pig = mouse. The effects on maximum responses were comparable in pig and mouse detrusor. On the other hand, contractions elicited by high KCl were blocked to 75-80 % by nifedipine in all three species, confirming their Ca²⁺-influx-dependent nature. 2-aminoethoxydiphenyl borate (2-APB) impaired CCh-induced contractions to various degrees: 300 µM 2-APB blocked 40% of maximum response in man, 65% in pig, but about 80% in mouse. In addition, 300 µM 2-APB tended to shift the CRC for CCh to higher concentrations in human and pig detrusor, although it did not reach the level of statistical significance. 300 µM 2-APB also completely reduced nifedipine-insensitive contractions in human detrusor. Block of the Ca²⁺-induced Ca²⁺-release with ryanodine had no significant effect on CCh contractions in all three species. In pig about 50% of CCh-induced contraction amplitude was suppressed by thapsigargin (0.3-10 µM). Thapsigargin impaired CCh-induced force of contraction to a lesser extent in human and pig than in mouse detrusor, in which addition of CCh in the presence of 1 or 10 µM thapsigargin did not further increase contraction. 10 µM thapsigargin did not further reduce nifedipine-insensitive contractions in human detrusor.

Interpretation of results In the present study, block of Ca²⁺-influx indeed suppressed CCh-induced contractions, and did so to a lesser degree in human than in pig and mouse detrusor with complete block of contraction by nifedipine in the latter species. On the other hand, contractions elicited by high KCl were blocked similarly by nifedipine in all three species, confirming their Ca²⁺-influx-dependent nature. This suggests that muscarinic receptor-stimulated contractions of human detrusor are less dependent on Ca²⁺-influx via L-type Ca²⁺-channels than in pig and mouse. Beside inhibition of IP3-induced Ca²⁺-release 2-APB is also known to influence [Ca²⁺]i by (i) direct inhibition of store-operated Ca²⁺-influx or (ii) activation of a novel Ca²⁺-permeable cation channel. Since influence of store-operated channels and phospholipase C on muscarinic receptor-mediated human detrusor contraction was excluded,^[2] the other two mechanisms could be involved. Due to the fact that nifedipine has only little effect in human detrusor and 2APB reduces nifedipine-insensitive contractions, we conclude, that beside Ca²⁺-influx via L-type Ca²⁺-channels additional processes are involved for elevating the intracellular Ca²⁺-concentration during muscarinic receptor mediated detrusor contraction in this species. Lack of sensitivity of contraction to ryanodine suggests, that Ca²⁺-induced Ca²⁺-release probably does not contribute to CCh-mediated detrusor contractions in the three species. Our measurements with thapsigargin reveal that at the used concentrations it may directly inhibits Ca²⁺-influx via L-type calcium currents. Other reports have predicted that thapsigargin may also stimulates release of arachidonic acid.

Concluding message In conclusion, muscarinic receptor-mediated contractions in human, pig and mouse detrusor depend on Ca²⁺-influx via L-type Ca²⁺-channels but to a different extent. Beside the possible involvement of IP3-induced Ca²⁺-release it cannot be excluded that additional processes may play a role for elevating the intracellular Ca²⁺-concentration during muscarinic receptor mediated detrusor contraction. Our study has shown considerable species differences between man, pig and mouse and it may has relevance for drawing conclusions about human urinary bladder contraction when using different animal models.

References

[1] J. Auton. Pharmacol. 2001, 21, 243-248.

[2] J. Pharmacol. Exp. Therap. 2004, 309, 1148-1153.

[3] FASEB J. 2004, 18, 1159-1161.

FUNDING: NONE DISCLOSURES: NONE HUMAN SUBJECTS: This study was approved by the Ethics committee of the University Clinics, Dresden University of Technology and followed the Declaration of Helsinki Informed consent was obtained from the patients.

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