Receptor-coupled shortening of alpha-toxin-permeabilized single smooth muscle cells from the guinea-pig stomach.
1. Isolated single smooth muscle cells from the fundus of the guinea-pig stomach were permeabilized by use of Staphylococcus aureus alpha-toxin. Receptor-coupled shortening of individual cells was monitored under phase contrast microscopy. 2. Most of the isolated cells responded to 0.6 microM Ca2+, but not to 0.3 microM Ca2+, with a resulting maximal shortening to approximately 65% of the resting cell length. The contractile activity of these permeabilized cells lasted for several hours and repeated shortening was readily achieved after washing out. 3. Addition of acetylcholine (ACh) at a maximal concentration (10 microM) resulted in a marked decrease in the concentration of Ca2+ required to trigger a threshold response from 0.6 microM to 0.2 microM, and 1 mM guanosine 5'-diphosphate (GDP) blocked this decrease. Moreover, treatment with 100 microM guanosine 5'-triphosphate (GTP) mimicked the action of ACh. 4. Addition of 100 microM inositol 1,4,5-trisphosphate (InsP3) with 0.2 microM Ca2+ did not cause cell shortening, whereas 10 microM ACh with 0.2 microM Ca2+ did, suggesting that InsP3-induced Ca2+ release is not involved in ACh-operated cell shortening. 5. The present study demonstrates an alpha-toxin-permeabilized single smooth muscle cell preparation which retains its receptor function and also provides an insight into mechanisms leading to augmentation of Ca2+ sensitivity by stimulation of muscarinic receptors or GTP-binding proteins.
Ono
T
T
Department of Molecular Pharmacology, Meiji College of Pharmacy, Tokyo, Japan.
Mita
M
M
Suga
O
O
Hashimoto
T
T
Oishi
K
K
Uchida
M K
MK
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000502:physiology*; D013211:Staphylococcus aureus; D013270:Stomach / Q000166:cytology; D010738:Type C Phospholipases / Q000494:pharmacology*
D011976:Receptors, Muscarinic; D010738:Type C Phospholipases; D000109:Acetylcholine; D002118:Calcium