Analysis of expression of cGMP-dependent protein kinase in rabbit heart cells.
We previously showed that stimulation of cGMP-dependent protein kinase (PKG) stimulates L-type calcium current in newborn but not in adult rabbit ventricular myocytes. We have now isolated rabbit PKG type Ialpha cDNA (+1 to 2095), determined the sequence, and analyzed specific expression of PKG in adult and newborn rabbit heart by Western and Northern analyses to elucidate the developmental decline in the significance of PKG in cardiac function. We obtained full-length cDNA of PKG Ialpha from newborn rabbit heart mRNA with reverse transcription-polymerase chain reaction. The coding region of rabbit PKG Ialpha showed 94% homology to sequences of human and bovine PKG Ialpha. The deduced amino acid sequence of 671 amino acids showed seven substitutions between rabbit and either human or bovine PKG Ialpha. The major substitutions were found in the cGMP-binding domain. The cloned PKG 1alpha cDNA was expressed in COS cells. Expressed PKG showed cGMP stimulated PKG activity and immunoreactivity. Northern blot analysis of cardiac tissue demonstrated PKG Ialpha mRNA of 6.8 kb, with much higher levels in newborn than in adult cells. Western analysis in homogenates from ventricular tissues and isolated ventricular myocytes of rabbit heart showed much higher expression of PKG type I protein in newborn compared with adult cells. These findings suggest that PKG is developmentally regulated in rabbit heart and is expressed at a much higher level in newborn than in adult cells. The greater expression of PKG in newborn cells could be responsible for differences in the significance of cGMP in adult and newborn rabbit cells.
Kumar
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Todd Franklin Cardiac Research Laboratory, Children's Heart Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA. Rajiv@cellbio.emory.edu
Joyner
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Komalavilas
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Lincoln
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D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis; D005260:Female; D006321:Heart / Q000254:growth & development; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000096:biosynthesis; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits
D015220:Calcium Channels; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D017869:Cyclic GMP-Dependent Protein Kinases