Phosphorylation of myristoylated alanine-rich protein kinase C substrate by mitogen-activated protein kinase in cultured rat hippocampal neurons following stimulation of glutamate receptors

Glutamate-induced phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) was investigated in cultured rat hippocampal neurons. In 32P-labeled hippocampal neurons, exposure to 10 microM glutamate induced a long lasting increase in phosphorylation of MARCKS. The long lasting increase in MARCKS phosphorylation mainly required activation of the N-methyl-D-aspartate receptor. Unexpectatively, the MARCKS phosphorylation after the 10-min incubation with glutamate was not inhibited by treatment with calphostin C, a potent inhibitor for protein kinase C (PKC), or down-regulation of PKC but was largely prevented by PD098059, a selective inhibitor for mitogen-activated protein (MAP) kinase kinase. In contrast, the phosphorylation following the short exposure to glutamate was prevented by a combination of PD098059 and calphostin C. The phosphopeptide mapping and immunoblotting analyses confirmed that PKC-dependent phosphorylation of MARCKS was transient and the MAP kinase-dependent phosphorylation was relatively persistent. Investigations of the functional properties also showed that the MARCKS phosphorylation by MAP kinase regulates its calmodulin-binding ability and its interaction with F-actin as seen in the PKC-dependent phosphorylation. These results suggest that glutamate causes a long lasting increase in MARCKS phosphorylation through activation of the N-methyl-D-aspartate receptor and subsequent activation of MAP kinase in the hippocampal neurons.     

Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Phosphorylation and activation of cGMP-dependent protein kinase by Src

Naive CD8 T cells can be polarized into effectors producing the type 1 cytokines IFN-gamma and IL-2 or the type 2 cytokines IL-4, IL-5, and IL-10, respectively. To study whether the polarized cytokine phenotype of the effectors is stable, we generated highly cytotoxic hemagglutinin (HA) peptide-specific CD8 Tc1 and Tc2 (cytotoxic CD8 T cells producing type 1 or type 2 cytokines) effectors from Clone-4 TCR-transgenic mice, which were adoptively transferred into syngeneic adult thymectomized irradiated and bone marrow-reconstituted recipients. The highly activated blast-size, CD25+ Tc1 and Tc2 effectors gave rise to homogeneous resting CD25- CD44(high) Ly6C(high) Ag-specific populations, which persisted for at least 13 wk after adoptive transfer. These memory CD8 T cells, recovered 13 wk after transfer of Tc1 or Tc2 effectors, still produced either the type 1 or type 2 cytokines, i.e., IFN-gamma, or IL-4 and IL-5, respectively, upon restimulation with APCs loaded with the HA peptide, but not in the absence of Ag. The amounts of IL-2 detected in the supernatants of Tc1 and Tc2 memory populations were comparable to that in memory CD4 cells, and both Tc1 and Tc2 memory cells became cytotoxic upon restimulation. Thus, cytokine-polarized CD8 memory T cells are a source of a variety of cytokines, which were classically considered helper cytokines, opening new perspectives on their function as regulatory cells in an immune response.     

Independent and exclusive modulation of cardiac delayed rectifying K+ current by protein kinase C and protein kinase A

We have recently established that local exposure to a 929.2 MHz electromagnetic near-field, used for cellular phones, does not promote rat liver carcinogenesis in a medium-term bioassay system. In the present study, a 1.439 GHz electromagnetic near-field (EMF), another microwave band employed for cellular phones in Japan, was similarly investigated. Time division multiple access (TDMA) signals for the Personal Digital Cellular (PDC) Japanese cellular telephone standard system were directed to rats through a quarter-wavelength monopole antenna. Numerical dosimetry showed that the peak SARs within the liver were 1.91-0.937 W/kg, while the whole-body average specific absorption rates (SARs) were 0.680-0.453 W/kg, when the time-averaged antenna radiation power was 0.33 W. Exposure was for 90 min a day, 5 days a week, over 6 weeks, to male F344 rats given a single dose of diethylnitrosamine (200 mg/kg, i.p.) 2 weeks previously. At week 3, all rats were subjected to a two-thirds partial hepatectomy. At week 8, the experiment was terminated and the animals were killed. Carcinogenic potential was scored by comparing the numbers and areas of the induced glutathione S-transferase placental form (GST-P)-positive foci in the livers of exposed (48) and sham-exposed rats (48). Despite increased serum levels of corticosterone, adrenocorticotropic hormone (ACTH) and melatonin, the numbers and the areas of GST-P-positive foci were not significantly altered by the exposure. These findings clearly indicated that local body exposure to a 1.439 GHz EMF, as in the case of a 929.2 MHz field, has no promoting effect on rat liver carcinogenesis in the present model.     

Opposing effects on modulation of angiogenesis by protein kinase C and cAMP-mediated pathways

The role of cAMP-mediated pathway in modulating angiogenesis was investigated. We have shown previously that activators of protein kinase C (PKC) caused a marked increase in angiogenesis, while the specific inhibitor of PKC, Ro 318220 suppressed angiogenesis. Here we show that forskolin, which activates adenylate cyclase and elevates the intracellular levels of cAMP, and the Sp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Sp-cAMPS), caused a dose-dependent suppression of collagenous protein biosynthesis and angiogenesis in the chick chorioallantoic membrane system (CAM). The opposite modulation of angiogenesis by activators of PKC and elevated cAMP levels was further confirmed by the suppression of 4 beta-phorbol-12-myristate-13-acetate (4 beta-PMA)-stimulated angiogenesis by either forskolin or Sp-cAMPS. On the contrary, the Rp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Rp-cAMPS), which antagonises endogenous cAMP biochemical actions, had no effect on angiogenesis alone and did not suppress 4 beta-PMA stimulated angiogenesis. However, Rp-cAMPS antagonised the effect of forskolin and Sp-cAMPS on 4 beta-PMA induced angiogenesis. Similar results were obtained in the human umbilical vein endothelial cell tube formation assay. In this system, the PKC inhibitor, Ro 318220, caused a dose-dependent inhibition of 4 beta-PMA reversed this effect. Also, forskolin and Sp-cAMPS caused an inhibition in tube formation. These results indicate that increased levels of intracellular cAMP have a negative effect in normal angiogenesis and cause a large reduction of the promotion of angiogenesis resulting from PKC activation.     

Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin

Vitronectin, found in the extracellular matrix and in circulating blood, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiological stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2-3 mol covalently bound phosphate, it was reasonable to assume that other protein kinases might phosphorylate vitronectin at other sites in the protein. We have reported earlier that human serum contains at least three protein kinases, one of which was found to be cAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Since there are now several examples of proteins with extracellular functions that are phosphorylated by PKC, we undertook to study the phosphorylation of vitronectin by PKC. Here, we show that vitronectin is a substrate for PKC, and characterize the kinetic parameters of this phosphorylation (Km approximately tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic event. We also identify Ser362 as the major PKC phosphorylation site in vitronectin, and confirm this localization by means of synthetic peptides derived from the cluster of basic amino acids in vitronectin surrounding Ser362. We show that the PKC phosphorylation at Ser362 alters the functional properties of vitronectin, attenuating its cleavage by plasmin at Arg361-Ser362. This phosphorylation has the potential to regulate plasmin production from plasminogen by a feedback mechanism involving the above-mentioned plasmin cleavage, a loosening of the vitronectin grip on inhibitor 1 of plasminogen activators, and a subsequent latency of this regulatory inhibitor.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

The potential for isoenzyme-selective modulation of protein kinase C

Protein kinase C (PKC) is a phospholipid-dependent serine/threonine kinase family consisting of at least 11 closely related isoenzymes. The different PKC isoenzymes play important roles in signal transduction pathways. The exact significance of each isoenzyme is not known at present; therefore, the elucidation of the roles of the various PKC isoenzymes is important. To explain the function of distinct PKC isoenzymes, the availability of isoenzyme-specific inhibibitors or activators would be an advantage. PKC inhibitors have been known for some time, but these compounds are not isoenzyme-specific and also inhibit other kinases. Recently, an inhibitor selective for PKC alpha and another one selective for PKCbetaI and betaII were described. Both compounds compete with the ATP binding sites that exhibit high homologies among the different PKC isoenzymes. Among others, the phosporyl transfer region, the pseudosubstrate domain, the phorbolester binding sequences, and the phosphorylation sites may also be targets for modulation of isoenzyme-specific PKC activity. The question is whether the differences in these domains and the substrate specificity of the PKC isoenzymes will allow isoenzyme-specific inhibition. In this review the human sequences of these sites, isoenzyme-specific substrates, inhibitory compounds, and inhibitory peptides are summarized.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.     

Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C

Phospholipase A2 (PLA2) is the enzyme regulating the release of arachidonic acid in most cell types. A high molecular mass, 85-kDa soluble form of PLA2 (cPLA2) has recently been identified, the activity of which is stably increased by stimulation of cells with hormones and growth factors. Growth factor stimulation of cells has been reported to result in increased phosphorylation of cPLA2 on serine residues, but the kinases mediating this effect have not been identified. We report here that human cPLA2 is phosphorylated in vitro by two growth factor-stimulated serine/threonine-specific kinases, p42 MAP kinase and protein kinase C (PKC). Phosphorylation of the cPLA2 enzyme by either kinase results in an increase in catalytic cPLA2-specific activity. Domains of the cPLA2 molecule have been expressed in Escherichia coli, and the fusion proteins purified. PKC and p42 MAP kinase give different patterns of phosphorylation of the recombinantly expressed cPLA2 fragments. p42 MAP kinase selectively phosphorylates the domain of cPLA2 containing a MAP kinase consensus sequence, whereas PKC phosphorylates sites in all three recombinantly expressed domains of the enzyme. Peptide mapping indicates that the site phosphorylated by p42 MAP kinase is different from those phosphorylated by PKC. The combined action of both of these kinases is likely to mediate the effects of growth factor stimulation on arachidonic acid release through the activation of cPLA2.     

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase A

The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in Vmax without alterations in the Km for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.     

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.     

Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C betaII, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increased the protein's thermal stability and decreased its Km for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.     

Astrocytic dye coupling in rat hippocampus: topography, developmental onset, and modulation by protein kinase C

Previous studies have shown that astrocytes constitute a functional syncytium whereas the cytoplasmata of individual cells are connected via gap junctions. Many studies have used cultured astrocytes and have examined electrical coupling with the help of double electrode techniques. Another approach has been the immunohistochemical detection of gap junction proteins in sections of brain tissue. From the results of these experiments it is difficult to infer the extent of astrocytic coupling in situ. To get an impression of the distribution of coupled astrocytes we took advantage of the hippocampal slice preparation which leaves the topography of neurons and astrocytes intact. We performed injections of low molecular weight dyes into single electrophysiologically identified astrocytes. As these dyes can pass through gap junctions this leads to the staining of all connected cells in a certain area, limited by the diffusional spread of the dye. The results show that there is virtually no border to astrocytic coupling between the diverse hippocampal subdivisions. This widespread coupling could already be detected at postnatal day 4, the earliest age tested. Activation of protein kinase C with phorbol esters showed that it is possible to reduce gap junctional communication by some extent. We conclude that although no compartmentalization was seen with respect to astrocytic coupling, the intercellular communication of these glial cells has the capability of being regulated for example by neuronal signals which activate the phospholipase C pathway in astrocytes.     

Protein kinase C involvement in maintenance and modulation of noradrenaline release in the mouse brain cortex

1. The role of protein kinase C in the modulation of noradrenaline release was investigated in mouse cortical slices which were pre-incubated with [3H]-noradrenaline. The aim was to investigate the hypothesis that protein kinase C is activated during high levels of transmitter release to maintain transmitter output. 2. The protein kinase C activators, phorbol myristate acetate (0.01-0.3 microM) and to a greater extent 4 beta-phorbol 12,13-dibutyrate (0.01-0.3 microM) significantly enhanced stimulation-induced noradrenaline release whereas 4 alpha-phorbol 12,13-dibutyrate (0.1 microM) which does not activate protein kinase C was without effect. The effect of the protein kinase C activator, phorbol myristate acetate, on noradrenaline release was attenuated by the protein kinase C inhibitor, polymyxin B (21 microM) which by itself inhibited stimulation-induced noradrenaline release. 3. Protein kinase C was down-regulated by 10 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta-phorbol 12,13-dibutyrate (0.1 microM) on noradrenaline release was abolished as was the inhibitory effect produced by polymyxin B. This indicates that polymyxin B was acting selectively at protein kinase C. 4. The inhibitory effect of polymyxin B on noradrenaline release, when expressed as a percentage of the appropriate frequency control, was constant at 1, 5 and 10 Hz. Furthermore, the ratio of release at 5 Hz to that at 10 Hz was not altered by protein kinase C down-regulation, indicating that there is no additional effect of protein kinase C at higher stimulation frequencies. 5. When transmitter release was elevated by blocking alpha 2-adrenoceptor auto-inhibition with idazoxan (0.1 microM) or K+ channels with tetraethylammonium (300 microM), the elevation in transmitter release was significantly attenuated by protein kinase C down-regulation, suggesting an involvement of protein kinase C. 6. We conclude that protein kinase C is involved in the modulation of noradrenaline release over a wide range of stimulation frequencies, in addition to a role when noradrenaline release is elevated by presynaptic mechanisms.     

Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C

Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5'-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5'-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5'- and 8-kb 3'-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.     

Irreversible inactivation of protein kinase C by glutathione

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca2+- and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca2+- and PS-dependent activity of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca2+- and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-alpha remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-alpha was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-alpha and the tripeptide, but it was associated with an increase in the susceptibility of PKC-alpha to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca2+- and PS- dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.