Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.
Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.
Shibata
H
H
Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-0615.
Robinson
F W
FW
Benzing
C F
CF
Kono
T
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D000242:Cyclic AMP / Q000494:pharmacology; D004563:Electrochemistry; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D008066:Lipolysis; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D007546:Isoquinolines; D010879:Piperazines; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011493:Protein Kinase C; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D005947:Glucose