Activation of protein kinase in thyroid slices by thyroid-stimulating hormone.

Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone-induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G-100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NcCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [3H]cAMP binding. These results are compatible with the concept that the TSH-mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.     

Implication of microtubules and microfilaments in the response of the ovarian adenylate cyclase-cyclic AMP system to gonadotropins and prostaglandin E2.

Addition of anti-actin serum or cytochalasin B (3 μg/ml) to the medium abolished the stimulatory effect of LH and of choleragen, and inhibited the action of FSH, but not of PGE₂, on cyclic AMP production in cultured rat Graafian follicles. Colchicine and anti-sera to BSA, tubulin or smooth-muscle myosin, as well as anti-actin serum absorbed with actin, had no effect on the follicular response to LH, but anti-tubulin serum and colchicine inhibited the response to FSH and PGE₂. The inhibitory effect of cytochalasin B on LH-action was fully reversed 24 h after transfer of the follicles to drug-free medium. Neither anti-actin serum nor cytochalasin B had any effect on the binding of ¹²⁵I-hCG by the follicular cell membrane. The results suggest that microfilaments, but not microtubules, are intimately involved in the process of LH- and choleragen-stimulated ovarian adenylate cyclase activity. By contrast, the action of PGE₂ is dependent on microtubule assembly, while the action of FSH seems to depend on both these components of the cytoskeleton.     

Lipid peroxidation and antioxidant defense of erythrocytes during bubbling air ionization of donor packed red blood cells

It is shown that erythrocytes in packed red blood cell preparations can be maintained in native state for a long time using bubbling air ionization (BAI). The BAI procedure proposed to prolong the storage period of donor erythrocytes makes it possible to reduce the level of destructive processes in packed cells stored until use, as indicated by a decrease in the lipid peroxidation intensity and an increase in the antioxidant activity in them.     

Mechanism of hormonally induced refractoriness of ovarian adenylate cyclase to luteinizing hormone and prostaglandin E.

Stimulation by prostaglandinE2 (PGE2) or luteinizing hormone (LH) of cyclic AMP (cAMP) production by rat Graafian follicles was reduced when the follicles were cultured for 3-6 hours in PGE2 or 12-24 hours in cAMP. The follicles regained adenylcyclase response to PGE2 when held in a PG-free medium, but refractoriness to LH remained even after culture without LH for 8 hours or in anti-LH antiserum. Follicle desensitization to LH was not associated by a decrease in total number of LH-binding sites, nor by an altered activity of cAMP phosphodiesterase. Desensitized follicles responded fully to NaF, quanosine triphosphate (GTP), or guanylimidodiphosphate (Gpp(NH)p). Actinomycin D or cycloheximide prevented the development of refractoriness to PGE2 when added with PGE2. Actinomycin D also prevented desentization to LH. Therefore desensitization may involve synthesis of a protein that couples hormone reception to adenyl cyclase.