title: Developmental changes in Ca2+ homeostasis and contractility in gallbladder smooth muscle.
abstract: Relatively little is known about the contribution of Ca(2+)-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied Ca(2+) homeostasis and Ca(2+) sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular Ca(2+) concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased Ca(2+) mobilization, suggesting immaturity of Ca(2+) sensitization mechanisms. The enhanced Ca(2+) release in the newborn gallbladder was the result of the increase in the size of the releasable Ca(2+) pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane Ca(2+) pump nor the Na(+)/Ca(2+) exchanger collaborate in the extrusion of Ca(2+). In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump. The reduced Ca(2+) sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation.
authors.affiliation: Dept. of Physiology, Nursing School, Avda Universidad, Cáceres 10071, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Camello-Almaraz
authors.forename: Beatriz
authors.initials: B
authors.lastname: Macias
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Gomez-Pinilla
authors.forename: Soledad
authors.initials: S
authors.lastname: Alcon
authors.forename: Francisco E
authors.initials: FE
authors.lastname: Martin-Cano
authors.forename: Akemishi
authors.initials: A
authors.lastname: Baba
authors.forename: Toshio
authors.initials: T
authors.lastname: Matsuda
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Camello
authors.forename: María J
authors.initials: MJ
authors.lastname: Pozo
mesh_terms: D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002766:Cholecystokinin / Q000378:metabolism; D005704:Gallbladder / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D006168:Guinea Pigs; D006706:Homeostasis; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D024510:Muscle Development; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D002766:Cholecystokinin; D043263:Myosin-Light-Chain Phosphatase; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019831:Sodium-Calcium Exchanger; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000099211:Phospholamban