Involvement of heterotrimeric GTP-binding protein and rho protein, but not protein kinase C, in agonist-induced Ca2+ sensitization of skinned muscle of guinea pig vas deferens.
We studied the involvement of protein kinase C (PKC) and a small GTP-binding protein (G-protein), rho, in receptor-mediated Ca2+ sensitization of the contractile apparatus of smooth muscle of guinea pig vas deferens. In beta-escin-permeabilized smooth muscle strips, norepinephrine (NE) in the presence of GTP caused further contraction of the preparations at a constant Ca2+ level (Ca2+ sensitization). Prazosin and GDP beta S, a nonhydrolyzable GDP analogue, inhibited NE-induced Ca2+ sensitization, indicating an alpha-1 adrenoceptor/G-protein mediated response. GTP alone (> 10 microM) and GTP gamma S, a non-hydrolyzable GTP analogue, also induced Ca2+ sensitization. Pretreatment of preparations with C3 exoenzyme of Clostridium botulinum, which is known to ADP-ribosylate rho family proteins, with NAD resulted in complete inhibition of NE- and GTP (GTP gamma S)-induced Ca2+ sensitization. AIF4-, which activates heterotrimeric G-, but not small G-protein also induced Ca2+ sensitization. Interestingly, AIF4(-)-induced Ca2+ sensitization was inhibited by not only GDP beta S but also C3-treatment, suggesting that activation of heterotrimeric GTP-binding protein precedes activation of rho protein. On the other hand, phorbol 12,13-dibutyrate, like NE, also induced Ca2+ sensitization. The sensitization was inhibited by PKC(19-31), a PKC inhibitor peptide. However, PKC(19-31) did not have any effect on NE- or AIF4(-)-induced Ca2+ sensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
Fujita
A
A
Department of Veterinary Pharmacology, College of Agriculture, University of Osaka Prefecture Sakai, Japan.
Takeuchi
T
T
Nakajima
H
H
Nishio
H
H
Hata
F
F
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001905:Botulinum Toxins; D002118:Calcium / Q000378:metabolism*; D004928:Escin / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014649:Vas Deferens / Q000187:drug effects / Q000378:metabolism*; D020741:rho GTP-Binding Proteins
D017607:Aluminum Compounds; D010455:Peptides; C061479:protein kinase inhibitor peptide; D000246:Adenosine Diphosphate Ribose; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004928:Escin; D006160:Guanosine Triphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011493:Protein Kinase C; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D005459:Fluorides; D002118:Calcium; D009638:Norepinephrine; C032311:aluminum fluoride