Abstract
Evidence has been obtained for a specific protein receptor for prostacyclin on cells of the NCB-20 somatic hybrid. A new stable prostacyclin analog, 5-[(E)-(1S,5S,6R,7R) - 7 - hydroxy-6-[(E) - (3S,4RS) -3-hydroxy-4-methyl-1 -octen-6-inyl]bicyclo[3.3.0]-octan-3-ylidene]pentanoic acid (Iloprost, ZK36374) activates adenylate cyclase of NCB-20 cell membranes to an extent similar to prostacyclin and with a comparable high affinity. The binding of [3H]Iloprost to NCB-20 membranes was rapid with an association rate constant (k+1) of 2.01 X 10(5) M-1 s-1 at 20 degrees C. The rate constant for the dissociation of the ligand-receptor complex (k-1) was 1.19 X 10(-3) s-1, giving a dissociation constant (k-1/k+1) of 5.9 nM. The equilibrium dissociation constant was 29.9 nM, and the membranes had a maximum binding capacity of 347 fmol mg-1 protein. Radiation inactivation has been employed to determine the molecular weights of the functional prostacyclin receptor and components of the adenylate cyclase system in the plasma membrane of the NCB-20 cells. Cell membranes were lyophilized prior to irradiation, which lead to the formation of high-molecular-weight aggregates. The aggregation was avoided, however, when membranes were prepared in an isotonic Tris-HCl buffer containing sucrose. Molecular weight values of 111,000 for the catalytic subunit of adenylate cyclase, 89,000 for the regulatory subunit, and 83,000 for the prostacyclin receptor were obtained. Loss of [3H]Iloprost binding capacity after irradiation of lyophilized membranes yielded a molecular weight value (mean +/- S.E.) for the prostacyclin receptor of 82,800 +/- 12,900 (n = 3).
Highlights
3-hydroxy-4-methyl- l-octen-6-inyl]bicyclo[3.3.0]- without loss of the capacity of the receptor to bind PGIZ
Cell Culture-Cells of the NCB-20 neuronal hybrid cell line were cultured in Dulbecco'smodifiedEagle'smedium (Gibco Europe) containing either 10% (v/v) newborn calf serum (Gibco Europe) or Prostacyclin(epoprostenol) is an unstable metabolite of PG' endoperoxide [1]that activates adenylate cyclase (ATP pyrophosphate lyase EC 4.6.1.1) in platelets [2, 3] and vascular smooth muscle [4]
Activation of Adenylate Cyclase-A saturating concentration of Iloprost activated adenylate cyclase to an extent similar to PGIz and its stable analog carbacyclin (Fig. 1).Each of the prostaglandins yielded linear Eadie-Hofstee plots (Fig. 1B) suggesting that activation of adenylate cyclase by these prostaglandins is mediated by single receptor populations
Summary
Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) activity was measured in NCB20 membranes which had been prepared and lyophilized in 25 mM Tris-HC1 buffer, pH 8.5, containing 0.29 M sucrose. N-Acetyl-8-D-glueosaminidase Actiuity-N-Acetyl-8-D-glucosaminidase activity was measured [29] by the addition of 50 pl of the sample (reconstituted in water) to 450 pl of 4 mM p-nitropheny1-Nacetyl-B-D-glucosaminide (Sigma London Chemical Co. Ltd.) in 40 mM citrate buffer, pH 4.5, containing 5% methanol. Ltd.) plus 1.79 mM sodium bicarbonate in 100 mM Tris-HC1 buffer, pH 7.0 To this was added 6 pl of mM acetylthiocholine iodide (Sigma London Chemical Co. Ltd.), and the increase in absorbance at 412 nm was measured using a Perkin-Elmer 55s spectrophotometer.
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