Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes.

The changes in protein phosphorylation and cytoskeletal structure preceding the dramatic morphological changes in staurosporine-treated rat astrocytes were examined, and the dependence of these effects on protein kinase C (PKC) was studied. Fluorescence and photoelectron microscopy revealed that a 20-min exposure to the kinase inhibitor staurosporine at 100 nM substantially decreased the thickness and linear appearance of actin microfilament bundles (stress fibers) prior to major changes in cell shape, while 60 min of staurosporine depleted virtually all microfilament bundles and caused arborization and contraction of the cell body. The distribution of myosin light chain (MLC) labeling within the cytoplasm was also dramatically altered by staurosporine, progressing from a linear punctate pattern coincident with the linear pattern of filamentous actin to a diffuse pattern in cells in which microfilament dissolution was taking place. Two-dimensional gel analysis of astrocyte phosphoproteins demonstrated 50-80% reduction of 32P incorporation into four 20-kDa spots, one of which was recognized by an antibody to MLC, following a 15-min treatment with 100 nM staurosporine. Depletion of functinal PKC from astrocytes by a 24-h exposure to phorbol myristate acetate prior to staurosporine exposure did not reduce the extent of the cytoskeletal alterations or alter the decrease in protein phosphorylation. Two other protein kinase inhibitors which affect astrocyte morphology, H-7 and the MLC kinase inhibitor ML-9, were also observed to disrupt microfilament bundles with accompanying decreases in 32P incorporation into these same phosphoproteins, whereas the more selective PKC inhibitor Ro 31-8220 did not do either. The early onset of decreased phosphorylation of the 20-kDa proteins supports a direct relationship between the rapid dissociation of myosin light chain from actin microfilament bundles, the disruption of actin patterns, and the subsequent morphological alterations. These data also suggest that staurosporine and H-7 may exert their effects via a pathway involving inhibition of MLC kinase.

Transition metal chelator TPEN counteracts phorbol ester-induced actin cytoskeletal disruption in C6 rat glioma cells without inhibiting activation or translocation of protein kinase C.

Phorbol ester-induced reorganization of the actin cytoskeleton was investigated in C6 rat glioma cells. Observations by fluorescence microscopy and photoelectron microscopy indicated that pretreatment with the transition metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) for 1-2 h at 50 microM reduced the sensitivity of the actin cytoskeleton to disruption by the subsequent addition of 200 nM phorbol myristate acetate (PMA). The protective effect of TPEN was eliminated by adding back Zn2+ prior to PMA addition, implicating chelation of metal ions as the mechanism of action of TPEN. C6 cells exposed to PMA experience potent activation of protein kinase C (PKC) and substantial redistribution of the kinase from a soluble to a particulate cellular fraction (translocation). TPEN pretreatment did not block PKC translocation in PMA-exposed cells. By two-dimensional gel analysis, TPEN also did not reduce, but rather slightly increased, the PMA-stimulated phosphorylation of the acidic 80 kDa endogenous PKC substrate, as well as two other proteins at 18 kDa and 50 kDa. In contrast, TPEN significantly suppressed phosphorylation of a 20 kDa protein, both in cells treated with TPEN only and in TPEN-pretreated PMA-exposed cells. The results indicate that the ability of TPEN to protect against PKC-mediated actin cytoskeletal disruption is not due to either a block of PKC translocation or to general inhibition of PKC activity. Rather, the action of TPEN is more selective and probably involves chelation of Zn2+ at a critical Zn(2+)-dependent phosphorylation step downstream from the initial tumor promoter-induced effects on PKC.

Norepinephrine-mediated protein phosphorylation in astrocytes.

Studies were conducted to determine if norepinephrine activates both protein kinase C and the cyclic AMP-dependent protein kinase in cultured rat astrocytes using phosphoproteins as markers. Norepinephrine was found to decrease 32P incorporation into an acidic 80,000 M(R) protein. A similar response was observed with isoproterenol and cyclic AMP analogs. In contrast, phorbol myristate acetate (PMA) increased 32P incorporation into this protein. Further studies looked at phosphorylation sites on glial fibrillary acidic protein and vimentin using two-dimensional tryptic phosphopeptide maps. The pattern of phosphorylation of these two proteins by norepinephrine resembles that of 8-bromo cyclic AMP and isoproterenol, and not that of PMA. Additionally, the effect of norepinephrine on the phosphorylation of GFAP and vimentin was blocked by alprenolol. One difference noted between norepinephrine and isoproterenol was the phosphorylation of an 18,000 M(R) protein. Norepinephrine increased, and isoproterenol decreased, 32P incorporation into this protein; however, the mechanism which mediates the norepinephrine effect remains to be determined. Overall, these studies indicate that the most prominent phosphorylation events mediated by norepinephrine are the consequence of the activation of cyclic AMP-dependent protein kinase.

Phosphorylation of glial fibrillary acidic protein and vimentin by cytoskeletal-associated intermediate filament protein kinase activity in astrocytes.

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of sphingosine on phorbol ester-mediated changes in astrocyte morphology and protein phosphorylation.

Previous studies indicate that phorbol myristate acetate (PMA) can induce morphological changes in astrocytes cultured from the rat neocortex. PMA also increased 32P incorporation into several proteins, including glial fibrillary acidic protein (GFAP), vimentin, and proteins with molecular weights of 80,000 (pI 4.5), 50,000 (pI 4.9), and 30,000 (pI 5.5). The present studies were conducted to determine if the morphological effect and the phosphorylation effect of PMA could be blocked by treatment with sphingosine, a protein kinase C inhibitor. Treatment with 15 microM sphingosine inhibited the effect of PMA on astrocyte morphology. This agent also inhibited the increase in phosphorylation mediated by PMA. The percent inhibition ranged from approximately 20% for the 30,000-Mr protein to 70% for GFAP. Analysis of phosphorylation sites on GFAP and vimentin using two-dimensional tryptic mapping techniques indicate that the partial inhibition of phosphorylation is likely the consequence of partial inhibition of protein kinase C rather than a selective inhibition at some phosphorylation sites and not others. In addition to increasing 32P incorporation into various proteins, PMA also decreased 32P incorporation in several 20,000-Mr proteins (pI values of 6.7, 6.4, 6.2, 4.9). However, this effect was not blocked by treatment with sphingosine. This suggests that the actions of PMA to increase and decrease 32P incorporation are mediated by different mechanisms.

Phorbol myristate acetate and 8-bromo-cyclic AMP-induced phosphorylation of glial fibrillary acidic protein and vimentin in astrocytes: comparison of phosphorylation sites.

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of microwave tissue fixation to demonstrate the in vivo phosphorylation of an acidic 80,000 molecular weight protein in the rat neocortex following treatment with soman.

Studies were conducted to determine if soman, a cholinesterase inhibitor, could activate the protein kinase C system in the rat neocortex. Using microwave radiation for rapid tissue fixation, it was demonstrated that treatment with soman increased 32P incorporation into an acidic 80,000 molecular weight, heat-stable protein in vivo. Based on relative molecular weight and isoelectric point this protein appears to be identical to a protein identified as a substrate for protein kinase C. Additionally, a protein of the same molecular weight and isoelectric point could be phosphorylated in tissue slices prepared from the neocortex by cholinergic dependent mechanisms. Also, treatment with soman decreased protein kinase C in the soluble fraction of this brain region; however, no corresponding increase was observed in the particulate fraction. These results suggest that soman can activate protein kinase C in vivo, and demonstrate the utility of using microwave tissue fixation to study protein phosphorylation events in vivo.

The cholinesterase inhibitor soman increases inositol trisphosphate in rat brain.

Studies were conducted to determine the effect of the cholinesterase inhibitor soman on the amount of inositol trisphosphate in the neocortex, striatum, cerebellum, and medulla-pons regions of rat brain in vivo. The studies indicate that treatment with soman increases inositol trisphosphate in the neocortex and striatum, but not in the cerebellum or medulla-pons region. In the neocortex the most pronounced increases were observed in animals with severe poisoning symptoms, however inositol trisphosphate was also found to be elevated in animals with only mild poisoning symptoms.

Phorbol ester-induced change in astrocyte morphology: correlation with protein kinase C activation and protein phosphorylation.

Treatment with 300 nM phorbol 12-myristate 13-acetate (PMA) transforms polygonal-shaped cultured astrocytes into process-bearing cells and produces a shift in protein kinase C (PK-C) from the cytosol to the membrane. Exposure to PMA also produces increases in the phosphorylation of several proteins including vimentin, glial fibrillary acidic protein (GFAP), an acidic 80,000 molecular weight protein, and two 30,000 molecular weight proteins (pI 5.5 and 5.7). The effects of PMA on the translocation of PK-C and on protein phosphorylation precede the PMA-induced changes in astrocyte morphology, and a close correlation exists between the concentration of PMA necessary to elicit half-maximal and maximal effects on the shift of PK-C to the membrane and on protein phosphorylation. In addition, the PMA-induced alterations in cell morphology are not permanent, and within 24 hr after PMA treatment the cells have reverted almost to their original morphology. A second exposure to PMA at this time fails to elicit further change in cell shape and is also incapable of producing increases in the phosphorylation of proteins. It was determined that there is little, if any, PK-C present in these PMA-pretreated cells. The morphological responsiveness to PMA gradually returns in 5 to 8 days after the initial treatment with PMA, and this is accompanied by the recovery of PK-C activity and the phosphorylation response. Therefore, these studies suggest that the effect of PMA on astrocyte morphology is mediated by the activation of PK-C and subsequent protein phosphorylation.

Protein phosphorylation in astrocytes mediated by protein kinase C: comparison with phosphorylation by cyclic AMP-dependent protein kinase.

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.

Astrocyte morphology altered by 1-(5-isoquinolinylsulfonyl) 2-methyl piperazine (H-7) and other protein kinase inhibitors.

Studies were conducted to determine if the protein kinase C inhibitor H-7 could block the effects of phorbol-12-myristate-13-acetate (PMA) on astrocyte morphology. Contrary to expectation, H-7 alone was found to induce morphological changes very similar to those elicited by PMA. This effect was shared by two other inhibitors of protein kinase C, H-8 and staurosporine, but not by the cyclic nucleotide-dependent protein kinase inhibitor HA-1004 or the calcium/calmodulin dependent protein kinase inhibitor W-7. Although the morphological effects observed with H-7 resemble those induced by PMA, H-7 did not promote the redistribution of protein kinase C to the membrane or induce the phosphorylation of endogenous proteins like PMA. In addition, the effects of H-7 were still observed in cells depleted of protein kinase C activity which were no longer responsive to treatment with PMA. Cytoskeletal elements appear to be involved in the effect of H-7 on cell shape since this effect is blocked by treatment with colchicine. Activators of the cyclic AMP-dependent protein kinase also alter astrocyte shape, however, while H-7 did cause a slight increase in cyclic AMP levels, it was unlikely that this action is responsible for its effect on morphology. One common action of both H-7 and PMA was to decrease the 32P content of several 20,000 Da proteins. While the mechanism by which H-7 exerts its influence on astrocyte morphology remains to be clarified, be it by the inhibition of protein kinase C or some other mechanism, the results suggest that caution must be used when interpreting the effects of activators and inhibitors of this kinase.

Pharmacological identification of the alpha-adrenergic receptor type which inhibits the beta-adrenergic activated adenylate cyclase system in cultured astrocytes.

The adrenergic agonist norepinephrine can exert its influence on cell function by activating both alpha- and beta-adrenergic receptors. In astrocytes, the alpha-adrenergic receptor activity of norepinephrine is known to inhibit the cyclic AMP response elicited by its action at beta-adrenergic receptors. Pharmacological studies were conducted to identify the subtype of alpha-adrenergic receptor which mediates this inhibitory action. The alpha 2-adrenergic antagonist yohimbine potentiated the cyclic AMP response elicited by norepinephrine, whereas the alpha 1-adrenergic antagonist prazosin did not affect the response. The alpha 2-adrenergic agonist clonidine inhibited the cyclic AMP response elicited by the beta-adrenergic agonist isoproterenol and this inhibition could be blocked by yohimbine but not by prazosin. In contrast, the alpha 1-adrenergic agonist phenylephrine did not inhibit the cyclic AMP response to isoproterenol. These studies indicate that the inhibitory action of norepinephrine is mediated by its action at alpha 2-adrenergic receptors.

Clonidine inhibits the isoproterenol-induced desensitization of the beta noradrenergic activated adenylate cyclase system in astrocytes.

Studies were conducted to determine if alpha agonists could influence the desensitization of the beta-noradrenergic receptor activated adenylate cyclase system. Pretreatment of astrocyte cultures with isoproterenol results in a rapid decrease in the cyclic AMP response to subsequent re-exposure to this agonist. The cyclic AMP response to isoproterenol in astrocytes pre-exposed for 2 h to isoproterenol plus clonidine was 2-3 times higher than in cells pre-exposed to isoproterenol alone. The response in astrocytes pretreated with phenylephrine plus isoproterenol was not different from that in cells pretreated with isoproterenol alone. Cyclic AMP may be involved in the effects elicited by clonidine, since this agonist can inhibit the accumulation of cyclic AMP in these cells. Also pretreatment with isobutylmethylxanthine decreases the cyclic AMP response to isoproterenol. The results suggest that clonidine can effect the desensitization of the noradrenergic receptor coupled adenylate cyclase system independent of effects on neurotransmitter release.

Protein kinase C in astrocytes: a determinant of cell morphology.

Protein kinase C-like activity was found to be present in astrocytes prepared from rat neocortex and maintained in culture. Exposure to phorbol 12-myristate 13-acetate (PMA) caused a redistribution of this kinase from the cytosol to the membrane fraction of these cells. Also PMA was found to cause a profound change in astrocyte morphology; cells were converted from flat, polygonal, undifferentiated cells to process-bearing cells.

Norepinephrine-sensitive adenylate cyclase system in rat brain: role of adrenal corticosteroids.

Two weeks after bilateral adrenalectomy, the responsiveness of the norepinephrine (NE)-sensitive adenylate cyclase system in the rat frontal cortex was increased. This effect was restricted to the non-beta-component of the system as no change was observed in the cyclic AMP response elicited by isoproterenol after bilateral adrenalectomy, thus indicating that subpopulations of cortical NE receptor systems are under separate endocrine control. The effect of adrenalectomy on the NE-sensitive adenylate cyclase system could be completely reversed by administering corticosterone for 3 days. No changes in the cyclic AMP response to NE were observed 2 weeks after bilateral medullectomy. Furthermore, an increase in the responsiveness of the system was also observed 2 weeks after hypophysectomy. These results suggest that the effects observed in the NE-sensitive adenylate cyclase system after adrenalectomy are mediated by the loss of adrenal corticosteroids. Adrenalectomy did not alter the activities of either adenylate cyclase or phosphodiesterase. No apparent changes were observed in the maximum binding or dissociation constant values of either beta or alpha adrenoceptors as assessed with [3H]alprenolol, [3H]WB-4101 and [ [3H]clonidine. Furthermore, the effects of adrenalectomy cannot be accounted for by a shift in the diurnal variation of the system as the cyclic AMP response to NE in tissue from adrenalectomized animals was higher than that in tissue from shamoperated rats throughout a 24-hr period.

Affinity of 10-(4-methylpiperazino)dibenz[b,f]oxepins for clozapine and spiroperidol binding sites in rat brain.

10-(4-Methylpiperazino)dibenz[b,f]oxepins were prepared and evaluated as potential antipsychotic agents using specific clozapine [8-chloro-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]diazepine] binding sites in rat forebrain that are noncholinergic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. [3H]Clozapine binding in the presence of atropine represents nonmuscarinic binding, while binding in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity for dopamine binding sites was determined by displacement of [3H]spiroperidol from binding sites in rat caudate nuclei. Thus, clozapine, its 2-chloro isomer, its dechloro analogue, and their 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepine analogues have about the same relative affinity for the nonmuscarinic clozapine binding sites. At the spiroperidol (dopaminergic) sites, both the nature of the tricyclic system and the presence of a chlorine atom on the tricyclic system have a substantial effect on the binding affinity. Within each series, shift or a chlorine atom from the position distal to the piperazino group to the proximal position increases the binding affinity by a factor of about nine, but removal of the chlorine atom substantially decreases the binding affinity. Nevertheless, 10-(4-methylpiperazino)dibenz[b,f]oxepin has a threefold greater affinity for the dopaminergic binding sites than does clozapine itself.

Synthesis of clozapine analogues and their affinity for clozapine and spiroperidol binding sites in rat brain.

Analogues of clozapine, some prepared by a novel, shorter synthesis than those described previously, were evaluated as potential antipsychotic agents using clozapine binding sites in rat forebrain that are nonmuscarinic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. The binding of clozapine to muscarinic sites is inhibited in the presence of atropine. Displacement of [3H]clozapine by an analogue of clozapine in the presence of atropine represents nonmuscarinic binding, while displacement in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity of the analogues for dopamine binding sites was determined by their ability to displace [3H]spiroperidol from binding sites in rat caudate nuclei. To the extent which binding affinity for nonmuscarinic clozapine sites in rat forebrain reflects the antipsychotic potential of a particular drug, dibenzo-5H-cycloheptene analogues of clozapine are as effective as clozapine itself. Strong binding to nonmuscarinic clozapine sites is not dependent on the presence of a chlorine atom on th tricyclic system. One or both of the nitrogen atoms in the dibenzo-5H-[1,4]diazepine ring of clozapine appear to be necessary for the strong inhibition of clozapine binding to spiroperidol sites in rat caudate nuclei. Anticholinergic activity is substantially higher for clozapine and its dibenz[1,4]oxazepine analogue than for its benzo-5H-cycloheptene analogue.