Myosin light chain phosphorylation at resting level and the composition ofmyosin isoforms in the bladder body and urethra

Bladder filling depends upon the coordinated control of a storage chamber, the bladder body, and its outlet, the bladder base and urethra. Bladder emp tying results from development of force in the bladder body and relaxation of the outlet. Muscle strips from bladder body reveal phasic characteristic s, whereas the strips from urethral wall are tonic. To determine whether th e compositions of myosin heavy chain (MHC) isoforms and the level of myosin light chain (MLC) phosphorylation contribute to the regional variation in the contractile states of the bladder smooth muscle, we analyzed the levels of MLC phosphorylation and the expression of myosin isoforms in smooth mus cle tissues from different regions of the urinary bladder. Strips of bladde r from the dome, mid body, base of the bladder and urethra were removed and analyzed for the levels of MLC phosphorylation at the resting tone. The ex pression of MHC isoforms that differ in the C-terminus (SM1 and SM2) and in the N-terminal region (SM-A and SM-B), formed by alternative splicing of t he pre-mRNA at either the 3' end or the 5' end, respectively, was analyzed. The expression of these isoforms was characterized at the mRNA and protein levels using reverse transcriptase-polymerase chain reaction (RT-PCR), SDS -PAGE, and Western blotting. The levels of MLC phosphorylation were 35.5 +/ - 4.6, 24.7 +/- 2.2, 13.6 +/- 2.1, and 12.8 +/- 2.7 for dome, mid bladder b ody, base and urethra respectively. Almost 100% of the MHC mRNA in the dome , mid bladder body, and base contains a 7-amino acid insert near the ATP-bi nding region, whereas the MHC in the urethral smooth muscle is only 81% ins erted. Prior studies have shown that inserted myosin has a two-fold higher actin-activated ATPase activity compared to the myosin isoform that lacks t he insert, and the maximum velocity of shortening of smooth muscle containi ng this insert is high compared to muscle that do not contain the insert. T he expression of SM1 and SM2 were not significantly different. Our data sug gests the presence of a high degree of inserted myosin and LC20 phosphoryla tion in the bladder dome and mid-body helps to facilitate rapid force devel opment and emptying. Non-inserted myosin and the low level of MLC phosphory lation in the urethra may contribute to slowly or non-cycling myosin cross bridges and the maintenance of a tonic or contracted state during bladder f illing.