METABOLIC CHARACTERISTICS OF ALPHA-TOXIN-PERMEABILIZED SMOOTH-MUSCLE

Rabbit portal veins were permeabilized using Staphylococcus aureus alp ha-toxin, and adenosinetriphosphatase (ATPase) was measured as the for mation of [H-3]ADP, [H-3]AMP, and [H-3]adenosine from [H-3]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is similar to 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleo tide accumulating as ADP, AMP, and adenosine. The ATPase activity is a lso seen when the intact muscle is incubated in a Krebs solution conta ining 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydr olyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the con trol of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocr eatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the int act muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatost ilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabiliz ation and inclusion Of sodium azide in subsequent solutions reduces th e ecto-ATPase by similar to 70%. Addition of PCr and creatine kinase t hen results in the maintenance of high [ATP] and low [ADP] in the musc le, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscl e in pCa 4.5. In general, the force output in high Ca2+ increased as t he metabolic profile of the muscle improved. When ATPase was measured as the appearance of [P-32]P-i from [P-32]PCr and [gamma-P-32]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment sho wed only similar to 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that t he reduction of the basal ATPase by the DIDS-azide treatment permits b oth rigorous control of nucleotide contents and accurate measurement o f ATPase activity in alpha-toxin-permeabilized smooth muscle.