beta-escin and protein-kinase-inhibitor-peptide

We established a beta-escin-permeabilized gland model with the use of rabbit isolated gastric glands. The glands retained an ability to secrete acid, monitored by [14C]aminopyrine accumulation, in response to cAMP, forskolin, and histamine. These responses were all inhibited by cAMP-dependent protein kinase inhibitory peptide. Myosin light-chain kinase inhibitory peptide also suppressed aminopyrine accumulation, whereas the inhibitory peptide of protein kinase C or that of calmodulin kinase II was without effect. Guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) abolished cAMP-stimulated acid secretion concomitantly, interfering with the redistribution of H+-K+-ATPase from tubulovesicles to the apical membrane. To identify the targets of GTPgammaS, effects of peptide fragments of certain GTP-binding proteins were examined. Although none of the peptides related to Rab proteins showed any effect, the inhibitory peptide of Arf protein inhibited cAMP-stimulated secretion. These results demonstrate that our new model, the beta-escin-permeabilized gland, allows the introduction of relatively large molecules, e.g., peptides, into the cell, and will be quite useful for analyzing signal transduction of parietal cell function.

Topics: Aminopyrine; Animals; Biological Transport; Enzyme Inhibitors; Escin; Gastric Acid; Gastric Mucosa; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); H(+)-K(+)-Exchanging ATPase; In Vitro Techniques; Peptide Fragments; Peptides; Permeability; Protein Kinases; Rabbits

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)

Topics: Adenosine Diphosphate Ribose; ADP Ribose Transferases; Aluminum Compounds; Animals; Botulinum Toxins; Calcium; Escin; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Norepinephrine; Peptides; Phorbol 12,13-Dibutyrate; Protein Kinase C; rho GTP-Binding Proteins; Vas Deferens