Metabolic characteristics of alpha-toxin-permeabilized smooth muscle.
Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 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 hydrolyzing 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 control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 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 the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.
Trinkle-Mulcahy
L
L
Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
Siegman
M J
MJ
Butler
T M
TM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002199:Capillary Permeability; D005260:Female; D006706:Homeostasis; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011169:Portal Vein / Q000378:metabolism; D011817:Rabbits; D012146:Rest; D019810:Sodium Azide; D010738:Type C Phospholipases / Q000494:pharmacology*; D014661:Vasoconstriction
D001386:Azides; D009711:Nucleotides; D019810:Sodium Azide; D010738:Type C Phospholipases; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid