Partial characterization of cyclic AMP-dependent protein kinases in guinea-pig lung employing the synthetic heptapeptide substrate, kemptide. In vitro sensitivity of the soluble enzyme to isoprenaline, forskolin, methacholine and leukotriene D4.
This paper describes the partial characterization of soluble cyclic AMP-dependent protein kinase (A-kinase) in guinea-pig lung using Kemptide, a synthetic serine-containing heptapeptide, and examines the sensitivity of this enzyme to drugs which are reported to increase and to decrease the intracellular concentration of cyclic AMP. Differential centrifugation of lung homogenates revealed that 78% of A-kinase was present in the 31,000 gmax x 15 min supernatant fraction. Both basal and cyclic AMP-stimulated phosphotransferase activity of this 'soluble' enzyme were abolished by the heat-stable inhibitor of A-kinase. Soluble A-kinase was Mg2(+)-dependent (apparent Km and and Kact 8.6 and 2.6 mM, respectively) and was stimulated nine-fold by saturating concentrations of both cyclic AMP (Kact: 131 nM) and cyclic GMP (Kact: 28.7 microM) at a protein (enzyme) concentration of 1.3 micrograms. Kinetic analysis of the effect of Kemptide and ATP revealed linear, Hanes plots with Michealis constants of ca. 12 and 13 microM, respectively. Chromatography of the soluble enzyme over DEAE-cellulose resolved three peaks of catalytic activity when fractions were assayed in the presence of cyclic AMP (10 microM): (i) free catalytic subunits (5%), (ii) Type I isoenzyme (5%) and (iii) Type II isoenzyme (90%). The A-kinase activity ratio was markedly increased in lung pre-treated with the smooth muscle relaxants isoprenaline and forskolin. This biochemical effect was both time- and concentration-dependent and was temporally associated with the ability of these drugs to reduce lung parenchymal tone. In contrast, the contractile agonists, methacholine (Mch) and leukotriene (LT) D4 exerted opposite effects on A-kinase activity. Thus, Mch significantly reduced cyclic AMP levels and lowered basal A-kinase activity whilst the converse was true for LTD4. For both drugs this biochemical effect accompanied contraction of the lung. Pre-treatment of lung tissue with flurbiprofen, an irreversible inhibitor of cyclo-oxygenase in vitro, abolished the ability of LTD4 to increase the A-kinase activity ratio suggesting that this biochemical response was mediated indirectly through the stimulated biosynthesis and release of a prostanoid(s) able to activate adenylyl cyclase; the increase in tension induced by LTD4, however, was not significantly affected by flurbiprofen pre-treatment. Collectively, these data support the concept that soluble A-kinase activity in guinea-pig lung can be regulated by changes in intracellular cyclic AMP and that activation and/or inhibition of this biochemical cascade may influence alterations in lung contractility.(ABSTRACT TRUNCATED AT 400 WORDS)
Giembycz
M A
MA
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Diamond
J
J
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D006168:Guinea Pigs; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000502:physiology; D008297:Male; D016210:Methacholine Chloride; D008688:Methacholine Compounds / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013189:SRS-A / Q000494:pharmacology; D012995:Solubility
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008688:Methacholine Compounds; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D013189:SRS-A; C015482:protein kinase modulator; D016210:Methacholine Chloride; D005576:Colforsin; C037642:kemptide; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium; D007545:Isoproterenol