Characterization of extracellular binding sites for [3H]-staurosporine on capillary endothelial cells.

[3H]-staurosporine, a non-specific protein kinase inhibitor, bound with high affinity and in a reversible manner to specific and saturable binding sites in cultured bovine cerebral cortex capillary endothelial cells. Scatchard analysis revealed the presence of one class of non-interacting binding sites with an equilibrium dissociation constant (KD) of 9.2 nM and Bmax of 19.3 fmol/10(5) cells. The binding of [3H]-staurosporine was fully displaced by unlabelled staurosporine, H-7 and ATP with IC50 values of 6.9 nM, 3 microM and 0.4 microM respectively. Mild trypsinization of cells after [3H]-staurosporine binding revealed the presence of membrane-associated, extracellular binding sites which could be an ecto-protein kinase.     

Human cytomegalovirus stimulates arachidonic acid metabolism through pathways that are affected by inhibitors of phospholipase A2 and protein kinase C

Inhibitors of phospholipase A2, tetracaine and quinacrine, inhibitors of protein kinases, H-7 and H-8, and a diacylglycerol lipase inhibitor reduced the level of CMV-induced [3H]AA release. A combination of H-7 and quinacrine inhibited stimulation of [3H]AA by about 80%. LU cells chronically treated with TPA and infected with CMV, had a reduced level of CMV-induced [3H]AA release and in the presence of quinacrine it was completely inhibited. These results suggest that CMV-induced stimulation of AA metabolism is mediated by pathways which are associated with activation of PLA2 and protein kinase C.     

Phorbol ester-mediated desensitization of histamine H1 receptors on a cultured smooth muscle cell line

The present study was undertaken in order to examine the effect of protein kinase C (PKC) on histamine H1 receptors (H1R) present on the smooth muscle cell line, DDT1MF-2. [3H]-pyrilamine binding revealed that specific [3H]-pyrilamine binding sites were reduced by pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, but not the Kd. The TPA analogue, 4 alpha phorbol 12,13-didecanoate, which does not activate PKC, failed to induce down-regulation of H1R. TPA-induced down-regulation of H1R was inhibited by pretreatment with 1-(5-Isoquinilinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, in a dose dependent manner. The H-7 analogue, H-8, which is a less potent inhibitor of PKC, but a potent inhibitor of cyclic nucleotide dependent protein kinase, had no effect on H1R. Moreover, treatment with TPA inhibited histamine-induced increases in [Ca2+]i in cells loaded with the fluorescent indicator, indo-1. These data suggest that H1R in DDT1MF-2 cells are functionally regulated by PKC.     

H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices but does not affect the phosphorylation of glucocorticoid receptor.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, on the nuclear binding of [3H]dexamethasone and on the phosphorylation of glucocorticoid receptor was studied in rat liver slices to ascertain the role of protein kinase C in the expression of glucocorticoid action. H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices. It does not affect the extent of phosphorylation of glucocorticoid receptor both in the absence or in the presence of glucocorticoid. These findings indicate that protein kinase C may be involved in the nuclear binding of glucocorticoid receptor but does not directly influence the receptor phosphorylation.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1

Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates [1] [2] [3] [4]. The MAP kinase-activated protein (MAPKAP) kinase p90 rsk contains two kinase domains [5]: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes [6] [7] [8], where inactive p90(rsk) is bound to the inactive form of the Erk2- like MAP kinase p42(mpk1). Here, we identify a new MAP kinase docking site located at the carboxyl terminus of p90(rsk). This docking site was required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and was also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site was conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases.     

Activation of cyclic AMP-dependent protein kinase isoenzymes: studies using specific antisera

A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nM for protein kinase I and 80 nM for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [gamma-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.     

Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase

The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved.     

pH induced increase in cyclic GMP reactivity with cyclic AMP-dependent protein kinases

Cyclic AMP-dependent protein kinases have been found which exhibit an enhanced capacity to bind cyclic GMP at acidic values of pH. The binding of cyclic GMP to a protein kinase from skeletal muscle, eluted as a single peak from DEAE cellulose columns, is inversely proportional to pH between the values of 7 to 4; the enzyme exhibits a 5 fold greater ability to bind cyclic [³H]-GMP (10^?8M) at pH 4.0 than 7.0. Protein kinases prepared from skeletal or uterine muscle, eluted as the first of two peaks from DEAE cellulose, exhibited similar pH dependent changes in specificity for cyclic GMP as determined by inhibition of cyclic [³H]-AMP binding. Acidic pH did not appreciably enhance the binding of cyclic [³H]-AMP to kinases prepared from aged skeletal muscle or kinase eluted as the second peak from DEAE cellulose.     

Identification and characterization of leukotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

We studied the characteristics of the leukotriene (LT) C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [3H]LTC4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 +/- 2.0 nM (n = 9). The association and dissociation of [3H]LTC4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [3H]LTC4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC4 to compete for the specific binding of [3H]LTC4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [3H]LTC4 binding was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712. The maximum number of binding sites (Bmax) of [3H]LTD4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [3H]LTC4. The calculated values of Kd and Bmax of [3H]LTD4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg protein, respectively (n = 3). The rank order of potency or the unlabeled competitors for competing specific [3H]LTD4 binding was LTD4 = LTE4 greater than FPL-55712 much greater than LTC4. These findings demonstrate that BC3H-1 cell line possess both LTC4 and LTD4 receptors with a predominance of LTC4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC4 and LTD4 receptors.     

Interleukin-1 stimulation of arachidonic acid release from human synovial fibroblasts; blockade by inhibitors of protein kinases and protein synthesis

Addition of IL-1 (interleukin-1) to human synovial fibroblasts radiolabelled with [3H]arachidonic acid caused a linear dose-dependent increase in arachidonic acid release and a transient rise in labelled diacylglycerol. Protein kinase C activators PMA 4-phorbol 12-myristate 13-acetate and DiC8 (1,2-dioctanoyl-sn-glycerol) also increased arachidonic acid release, but the time course observed with PMA was different from that of IL-1. When cultures were treated with PMA for 16-24 h to down regulate protein kinase C, the ability of IL-1 to increase arachidonic acid release persisted to the same extent as in nontreated cultures. In contrast, PMA pretreatment prevented the eight-fold stimulation of arachidonic acid release in response to PMA observed in cultures not previously exposed to PMA. To examine the role of other kinases in IL-1 stimulated arachidonic acid release, cultures were treated with H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dichloride), H-8 (N-[2-(methylamino) ethyl]-5-isoquinolinesulphonamide dichloride), HA1004 (N-(2-guanidoinoethyl)-5-isoquinolinesulphonamide hydrochloride), and staurosporine. IL-1 stimulation of arachidonic acid release was blocked by H-7, H-8 and staurosporine. H-7 was a more potent inhibitor than H-8, suggesting that cAMP dependent kinase did not mediate IL-1 action. Addition of H-7 at various times following IL-1 decreased IL-1 stimulated arachidonic acid release, suggesting that continued protein kinase activity was necessary for IL-1 action. Cycloheximide and actinomycin D inhibited the stimulation of arachidonic acid release by IL-1, PMA or DiC8. The addition of cycloheximide or actinomycin D 15-45 min after IL-1 also inhibited IL-1 stimulated arachidonic acid release, indicating that continued protein synthesis was required for IL-1 action. These results suggest that IL-1 stimulation of acylhydrolyase activity in human synovial cells occurs by a mechanism requiring continued protein synthesis and protein kinase activity and that neither protein kinase C nor cAMP dependent protein kinase is involved.     

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Phorbol ester-induced expression of the common, low-affinity binding site for primary prostanoids in vascular smooth muscle cells

We showed in an earlier study (Hanasaki, K., and Arita, H. (1989) Biochim. Biophys. Acta 1013, 28-35) that there is a common, low-affinity binding site for primary prostanoids in cultured vascular smooth muscle cells (VSMC). This site, called the "primary prostaglandin (PG) site," can be evaluated by radioreceptor assay using [3H]PGF2 alpha and [3H]PGE1. Comparison of the capacity of several PGF2 alpha analogs to displace both radioligand bindings indicated strict requirements of the 15-hydroxy group as well as the 13,14-double bond in the omega-side chain of prostaglandins for recognition of this site. Treatment of VSMC with phorbol 12-myristate 13-acetate (PMA), a known protein kinase C activator, led to concentration- and time-dependent increases in the binding activities of [3H] PGF2 alpha as well as [3H]PGE1, which could be completely suppressed by the addition of protein kinase C inhibitor, H-7. The PMA effects could be mimicked by phorbol 12,13-dibutylate, but not by inactive phorbol ester. Scatchard analyses revealed an approximately 8-fold increase in the binding density with unaltered binding affinity after PMA treatment. This expression of the primary PG site was blocked by the addition of cycloheximide and actinomycin D. In contrast, PMA did not affect the binding activity for the thromboxane A2/prostaglandin H2 receptor in VSMC. These results suggest that the expression of the primary PG site is regulated by a protein kinase C-dependent mechanism in VSMC.     

Interconvertible cGMP-free and cGMP-bound forms of cGMP-dependent protein kinase in mammalian tissues

Several vascular and nonvascular mammalian tissue extracts exhibited variable amounts of two peaks (peaks I and II) of cGMP-dependent protein kinase by NaCl elution of DEAE columns. When [3H]cGMP was added to the extracts before chromatography, a peak of protein-bound [3H]cGMP coeluted with peak II. [3H]cGMP was added to purified bovine lung cyclic nucleotide-free enzyme followed by chromatography on high performance liquid chromatography-DEAE. Two kinase peaks, the first of which represented mainly cGMP-free enzyme and the second of which represented cGMP-bound enzyme, eluted at the same positions as peaks I and II, respectively, of the crude extracts. The relative amount of peak II increased as a function of increasing the [3H]cGMP added before chromatography, and peak II could be converted partially to peak I by rechromatography. The holoenzyme is known to contain two slowly exchanging cGMP binding sites (sites 1) and two rapidly exchanging sites (sites 2). Some protein-bound [3H] cGMP found entirely in site 1 coeluted with peak I, although most of the enzyme in that peak was cGMP-free. When low [3H]cGMP was used for the initial incubation, relatively more of the protein-bound [3H] cGMP appeared in peak I and could represent binding of [3H]cGMP to only one of the two sites 1 of the kinase. The [3H]cGMP bound to the peak II enzyme completely filled both sites 1. Cyclic GMP binding to these sites caused the apparent conformational change which shifted the DEAE elution position of the enzyme. The peak II kinase was partially active and had a higher sensitivity to further cGMP activation of kinase than did the cGMP-free enzyme, suggesting that activation of kinase by binding of cGMP to site 2 was facilitated by prior binding at site 1. In fractions of the trailing edge of peak II, the kinase activity was virtually cGMP-independent, and both sites 1 and 2 were almost saturated with [3H]cGMP. These results suggested a further conformational change and direct increase in activity by binding of cGMP at site 2.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca2+ ionophores, antigen and diacylglycerol: essential role for protein kinase C and prevention by glucocorticosteroids

The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 micrograms/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2(+)-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.     

Characterization of cyclic GMP-binding sites of cyclic GMP-dependent protein kinase by rapid filtration assay.

The kinetics of cyclic [3H]GMP binding to the purified cyclic GMP-dependent protein kinase (cG kinase) were studied by using the rapid filtration assay method with polyethyleneimine-treated glass filters (method A), and the data were compared with those of the (NH4)2SO4 precipitation procedure (method B), which has been used for many previous studies on cyclic GMP binding to cG kinase. Each method gave a similar stoichiometry of approx. 2 mol of cyclic GMP/mol of cG kinase subunit; however, other binding kinetics obtained with these two methods were different. The dissociation of bound cyclic [3H]GMP from the kinase showed a single slow component when method A was used, whereas rapid and slow dissociation components were observed with method B. The Scatchard plot of cyclic [3H]GMP binding with method A was linear with a Kd value of 11 +/- 2 nM, suggesting that the two intrachain binding sites have similar high affinity for cyclic GMP. Results obtained on cyclic nucleotide analogue specificity of the two intrachain cyclic GMP-binding sites were also different between these two methods. These findings suggest that cG kinase has two high-affinity cyclic GMP-binding sites per subunit in the native state, and that when (NH4)2SO4 is added, ostensibly to stop the binding reaction, one low-affinity site is created from one high-affinity site.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.