IP(3) receptor function and localization in myotubes: an unexplored Ca(2+) signaling pathway in skeletal muscle.
We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca(2+) signaling pathway in skeletal muscle. RT-PCR methods confirm expression of all three known isotypes of the inositol trisphosphate receptor in cultured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, reveals that the receptors are localized to the I band of the sarcoplasmic reticulum, and this staining is continuous with staining of the nuclear envelope region. These results suggest that the receptors are positioned to mediate a slowly propagating Ca(2+) wave that follows the fast Ca(2+) transient upon K(+) depolarization. This slow wave, imaged using fluo-3, resulted in an increase in nucleoplasmic Ca(2+) lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122. To test the hypothesis that these slow Ca(2+) signals are involved in signal cascades leading to regulation of gene expression, we assayed for early effects of K(+) depolarization on mitogen-activated protein kinases, specifically extracellular-signal related kinases 1 and 2 and the transcription factor cAMP response element-binding protein (CREB). Within 30-60 seconds following depolarization, phosphorylation of both the kinases and CREB was evident and could be inhibited by 2-aminoethoxydiphenyl borate. These results suggest a signaling system mediated by Ca(2+) and inositol trisphosphate that could regulate gene expression in muscle cells.
Powell
J A
JA
Department of Biological Sciences, Smith College, Northampton, MA 01063, USA. jpowell@smith.edu
Carrasco
M A
MA
Adams
D S
DS
Drouet
B
B
Rios
J
J
Müller
M
M
Estrada
M
M
Jaimovich
E
E
D000185:Actinin / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D020033:Protein Isoforms; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D016895:Culture Media, Serum-Free; D017362:Cyclic AMP Response Element-Binding Protein; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D014966:Xanthenes; D000185:Actinin; C059715:Fluo-3; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009218:Myosins; D000252:Calcium-Transporting ATPases; D002118:Calcium