14-3-3 and Smad2/3 are crucial mediators of atypical-PKCs: Implications for neuroblastoma progression.

Neuroblastoma (NB) is a cancer that develops in the neuroblasts. It is the most common cancer in children under the age of 1 year, accounting for approximately 6% of all cancers. The prognosis of NB is linked to both age and degree of cell differentiation. This results in a range of survival rates for patients, with outcomes ranging from recurrence and mortality to high survival rates and tumor regression. Our previous work indicated that PKC-ι promotes cell proliferation in NB cells through the PKC-ι/Cdk7/Cdk2 cascade. We report on two atypical protein kinase inhibitors as potential therapeutic candidates against BE(2)-C and BE(2)-M17 cells: a PKC-ι-specific 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide and a PKC-ζ specific 8-hydroxy-1,3,6-naphthalenetrisulfonic acid. Both compounds induced apoptosis and retarded the epithelial-mesenchymal transition (EMT) of NB cells. Proteins 14-3-3 and Smad2/3 acted as central regulators of aPKC-driven progression in BE(2)-C and BE(2)-M17 cells in relation to the Akt1/NF-κB and TGF-ß pathways. Data indicates that aPKCs upregulate Akt1/NF-κB and TGF-ß pathways in NB cells through an association with 14-3-3 and Smad2/3 that can be diminished by aPKC inhibitors. In summary, both inhibitors appear to be promising potential neuroblastoma therapeutics and merit further research.

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

Role of protein kinase C-iota in transformed non-malignant RWPE-1 cells and androgen-independent prostate carcinoma DU-145 cells.

UNLABELLED: Prostate cancer is one of the leading causes of death among men in the USA. OBJECTIVE: In this study, we investigated the role of atypical protein kinase C-iota (PKC-iota) in androgen independent prostate DU-145 carcinoma cellscompared to transformed non-malignant prostate RWPE-1 cells. MATERIALS AND METHODS: Western blotting and immunoprecipitations demonstrated that PKC-iotaisassociated with cyclin-dependent kinase activating kinase (CAK/Cdk7) in RWPE-1 cells, but not in DU-145 cells. RESULTS: Treatment of prostate RWPE-1 cells with PKC-iota silencing RNA (siRNA) decreased cell viability,cell-cycle accumulation at G2/M phase, and phosphorylation of Cdk7 and Cdk2. In addition, PKC-iota siRNA treatment caused less phosphorylation ofBad at ser-155, ser-136, and greater Bad/Bcl-xL heterodimerization, leading to apoptosis. In DU-145 cells, PKC-iota was anti-apoptotic and was required for cell survival. Treatment with PKC-iota siRNA blocked increase in cell number, and inhibited G1/S transition by accumulation of cells in G0/G1phase. In addition to cell-cycle arrest, both RWPE-1 and DU-145 cells underwent apoptosis due to mitochondrial dysfunction and apoptosis cascades, such as release of cytochrome c,activation of caspase-7, and poly (ADP-ribose)polymerase (PARP) cleavage. CONCLUSION: Our results suggest that PKC-iota is required for cell survival in both transformed non-malignant prostate RWPE-1 cells and androgen-independent malignant prostate DU-145 cells, whereas suppressing PKC-iota lead to apoptosis in DU-145 prostate cells.

Involvement of PKC-iota in glioma proliferation.

UNLABELLED: Atypical protein kinase C-iota (PKC-iota) protects cells against apoptosis and may play a role in cell proliferation. However, in vivo, the status and function of PKC-iota in human normal brain tissue, gliomas, benign and malignant meningiomas as well as its in vitro status in proliferating and confluent glioma cells, remains unknown. OBJECTIVES: The objectives of our research were to determine whether expression of PKC-iota is altered either in gliomas or in benign and malignant meningiomas, compared to normal brain. In addition, we wished to establish the expression of PKC-iota in proliferating plus in cell cycle-arrested glioma cell lines, as well as the relationship between PKC-iota siRNA on PKC-iota protein content and cell proliferation. MATERIALS AND METHODS: Western blot analyses for PKC-iota were performed on 12 normal brain biopsies, 15 benign meningiomas, three malignant meningiomas and three gliomas. RESULTS: Results demonstrated no (n = 9) or very weak (n = 3) detection of PKC-iota in normal brain tissue. In comparison, PKC-iota was robustly present in the majority of the benign meningiomas. Similarly, PKC-iota was abundant in all malignant meningiomas and gliomas. Western blotting for PKC-iota in confluent or proliferating glioma cell lines depicted substantial quantities of PKC-iota in proliferating T98G and U-138MG glioma cells. In contrast, confluent cells had either 71% (T98G) or 21% (U-138MG) less PKC-iota than proliferating cells. T98 and U-138 MG glioma cells treated with 100 nm PKC-iota siRNA had lower levels of cell proliferation compared to control siRNA-A and complete down-regulation of PKC-iota protein content. CONCLUSION: These results support the concept that presence of PKC-iota may be required for cell proliferation to take place.

Effects of the PKC inhibitor PD 406976 on cell cycle progression, proliferation, PKC isozymes and apoptosis in glioma and SVG-transformed glial cells.

It is well established that protein kinase C (PKC) isozymes are involved in the proliferation of glioma cells. However, reports differ on which PKC isozymes are responsible for glioma proliferation. As a means to further elucidate this, the objectives of our research were to determine how inhibition of PKC-alpha, PKC-beta and PKCmu with PD 406976 regulates the cell cycle, cell proliferation and PKC during glioma growth and development. To establish the cell cycle effects of PD 406976 on brain cells (SVG, U-138MG and U-373MG glioma cells), specimens were treated with either dimethylsulfoxide (DMSO; control) or PD 406976 (2 microm). Results from flow cytometry demonstrated that PD 406976 delayed the entry DNA synthesis phase in SVG cells and delayed the number of cells entering and exiting the DNA synthesis phase in both U-138MG and U-373MG cells, indicating that PD 406976 may inhibit G(1)/S and S phase progression. Assessment of cell viability demonstrated a cytostatic effect of PD 406976 on SVG, U-138MG and U-373MG glioma cell proliferation. The PD 406976-induced decreased proliferation was sustained at 48-96 h. A PKC activity assay was quantified and demonstrated that exposure of SVG and U-373MG glioma cells to PD 406976 suppressed PKC activity. Western blotting demonstrated reduced PKC-beta1, PKC-gamma and PKC-tau protein content in cells treated with PD 406976. We determined that the growth inhibitory effect of PD 406976 was not as a result of apoptosis.

Human glioma PKC-iota and PKC-betaII phosphorylate cyclin-dependent kinase activating kinase during the cell cycle.

Cell cycle phase transition is regulated in part by the trimeric enzyme, cyclin-dependent kinase activating kinase (CAK) which phosphorylates and activates cyclin-dependent kinases (cdks). Protein kinase C (PKC) inhibitors prevent cell cycle phase transition, suggesting a fundamental role for PKCs in cell cycle regulation. We report that in glioma cells, CAK (cdk7) is constitutively associated with PKC-iota. In vitro phosphorylation, co-immunoprecipitation, and analysis of phosphorylated proteins by autoradiography indicate that CAK (cdk7) is a substrate for PKC-iota and PKC-betaII hyperphosphorylation. These results establish a role for PKC-iota and PKC-betaII in the activation of CAK during the glioma cell cycle.

Sensitivity of human glioma U-373MG cells to radiation and the protein kinase C inhibitor, calphostin C.

We assessed the radiosensitivity of the grade III human glioma cell line U-373MG by investigating the effects of radiation and the specific protein kinase C inhibitor, calphostin C on the cell cycle and cell proliferation. Irradiated glioma U-373MG cells progressed through G1-S and underwent an arrest in G2-M phase. The radiosensitivity of U-373MG cells to graded doses of either photons or electrons was determine by microculture tetrazolium assay. The data was fitted to the linear-quadratic model. The proliferation curves demonstrated that U-373MG cells appear to be highly radiation resistant since 8 Gy was required to achieve 50% cell mortality. Compared to radiation alone, exposure to calphostin C (250 nM) 1 h prior to radiation decreased the proliferation of U-373MG by 76% and calphostin C provoked a weakly synergistic effect in concert with radiation. Depending on the time of application following radiation, calphostin C produced an additive or less than additive effect on cell proliferation. We postulate that the enhanced radiosensitivity observed when cells are exposed to calphostin C prior to radiation may be due to direct or indirect inhibition of protein kinase C isozymes required for cell cycle progression.

Ultrastructural study of protein kinase C-betaII localization during the cell cycle of human glioma cells.

Transmission electron microscopy and immunogold labeling were used to determine how PKC-betaII is localized at stages in the cell cycle of the human glioma cell line U-373MG. Results show that immunogold particles in both dimethylsulfoxide (DMSO) and calphostin C (0.5 microM)-treated cells were mainly located in the cytoplasm. The concentration of gold particles in the nucleus was relatively small and constant throughout the cell cycle of both DMSO and calphostin C treated cells. Micrographs revealed changes in PKC-betaII during the cell cycle. The concentration of gold particles in the DMSO-treated cells was constant until 8 h. Subsequently, cytoplasmic PKC-betaII oscillated with an increased at 10 h, a rapid decrease at 12 h, and a rise at 14 h. The concentration of the gold particles then gradually decreased. In contrast, immunogold labeling in calphostin C-treated cells increased gradually up to 10 h. Subsequently, the pattern of PKC-betaII labeling in calphostin C-treated cells recapitulated those of control cells as seen by the rapid decline of PKC-betaII labeling at 12 h and its re-accumulation at 14 h. Additionally, there was a rapid increase at 20 h. Western blots of PKC-betaII showed constant PKC-betaII immunoreactivity throughout the cell cycle. In comparison to Western blots, in-situ immunogold labeling revealed changes in PKC-II immunoreactivity at 10 h and 14 h. This technique may represent intracellular immunoreactivity of PKC-betaII. The results from the immunogold labeling technique suggest that binding of calphostin C to the regulatory domain of PKC-betaII provokes a conformation change in PKC-betaII, preventing its activation and degradation.

Acute glucose-induced downregulation of PKC-betaII accelerates cultured VSMC proliferation.

Accelerated vascular smooth muscle cell (VSMC) proliferation contributes to the formation of atherosclerotic lesions. To investigate protein kinase C (PKC)-betaII functions with regard to glucose-induced VSMC proliferation, human VSMC from aorta (AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cells overexpressing PKC-betaI (betaI-A10) and PKC-betaII (betaII-A10) were studied with the use of three techniques to evaluate glucose effects on aspects affecting proliferation. High glucose (25 mM) increased DNA synthesis and accelerated cell proliferation compared with normal glucose (5.5 mM) in AoSMC and A10 cells, but not in betaI-A10 and betaII-A10 cells. The PKC-betaII specific inhibitor CGP-53353 inhibited glucose-induced cell proliferation and DNA synthesis in AoSMC and A10 cells. In flow cytometry analysis, high glucose increased the percentage of A10 cells at 12 h after cell cycle initiation but did not increase the percentage of betaI-A10 or betaII-A10 cells entering S phase. PKC-betaII protein levels decreased before the peak of DNA synthesis, and high glucose further decreased PKC-betaII mRNA and protein levels in AoSMC and A10 cells. These results suggest that high glucose downregulates endogenous PKC-betaII, which then alters the normal inhibitory role of PKC-betaII in cell cycle progression, resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

Estrogen stimulation of ovarian surface epithelial cell proliferation.

Ovarian cancer is the leading cause of gynecological cancer mortality, and 85-90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown, but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17beta-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor alpha demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17beta-estradiol depends on the expression of the estrogen receptor alpha in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor alpha in OSE growth.

Ectopic expression of protein kinase CbetaII, -delta, and -epsilon, but not -betaI or -zeta, provide for insulin stimulation of glucose uptake in NIH-3T3 cells.

Insulin regulates a diverse array of signaling pathways involved in the control of growth, differentiation, proliferation, and metabolism. Insulin increases in glucose uptake via a protein kinase C-dependent pathway in target tissues such as fat and muscle are well documented. Insulin-regulated events, however, occur in all cells. The utilization of glucose as a preferred energy source is a ubiquitous event in eukaryotic cells. In NIH-3T3 fibroblasts, insulin treatment increased levels of the cPKC and nPKC activator, diacylglycerol. Insulin-responsive 2-[(3)H]deoxyglucose uptake was stimulated in a dose-dependent manner. The overexpression of protein kinase C (PKC)betaI, -betaII, -delta, -epsilon, and -zeta was used to investigate the specificity of PKC isozymes for insulin-sensitive glucose uptake. The stable overexpression of PKCbetaII, -delta, and -epsilon resulted in increases in insulin-stimulated 2-[(3)H]deoxyglucose uptake compared to vector control cells, while basal 2-deoxyglucose uptake levels were not elevated. Overexpression of PKCbetaI and PKCzeta isozymes had no further effect on basal or insulin-stimulated 2-deoxyglucose uptake. The PKC-specific inhibitor, CGP41251, blocked insulin effects on 2-deoxyglucose uptake but not its effects on tyrosine phosphorylation of cellular substrates. Insulin-stimulated 3-O-methylglucose uptake was also greater in cells overexpressing PKCbetaII, -delta, and -epsilon, compared to control cells. The increased responsiveness was not accompanied by conversion of 3T3 cells to the adipocyte phenotype or the increased expression of insulin receptors or glucose transporters (GLUT1-type). Insulin-stimulated recruitment of GLUT1 to plasma membranes of cells overexpressing PKCbetaII, -delta, and -epsilon, was greater than that in control cells. The data suggest that more than one PKC isozyme is involved in insulin signaling pathways in fibroblasts, resulting in increased GLUT1 transporter recruitment to cell membranes.

The roles of protein kinase C beta I and beta II in vascular smooth muscle cell proliferation.

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-beta I and -beta II isozymes. PKC-beta I and -beta II are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-beta I and PKC-beta II (beta I-A10 and beta II-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in beta I-A10 cells decreased by 12 h, and, in contrast, the doubling time of beta II-A10 cells increased by 12 h compared to A10 cells. The increase of [3H]thymidine (TdR) incorporation was accelerated and increased in beta I-A10 cells, but slowed and diminished in beta II-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of beta I-A10 cells showed an acceleration of S phase entry while beta II-A10 cells slowed S phase entry. These results suggest that PKC-beta I and PKC-beta II regulate cell proliferation bidirectionally and that PKC-beta I and PKC-beta II may have distinct and opposing functions as cell cycle check point mediators during late G1 phase and may regulate S phase entry in A10 VSMC.

Effects of interferon and PKC modulators on human glioma protein kinase C, cell proliferation, and cell cycle.

The in-vitro effects of human interferon alpha-2b (HuIFN alpha-2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373 MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN alpha-2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC50 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN alpha-2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN alpha-2b is similar to TPA (potent PKC activation and S phase arrest).

In situ effects of interferon on human glioma protein kinase C-alpha and -beta ultrastructural localization.

Transmission electron microscopy was used to determine how immunogold labeling of PKC-alpha or -beta is modulated by the antitumor drug IFN (HuIFN alpha-2b) in the cytoplasm, membrane structures, and nucleus of rapidly dividing and confluent human glioma U-373 cells. Results showed that except for nuclear localization, there were no specific cytoplasmic organelles that PKC-alpha or -beta translocated to following HuIFN alpha-2b treatment. Electron micrographs of PKC-beta in proliferating cells depicted 1.34-fold more PKC-beta in the nucleus than in the cytoplasm and a 1-min HuIFN alpha-2b (500 units/ml) treatment transiently increased PKC-beta immunoreactivity in the cytoplasm (1.95-fold) and nucleus (1.97-fold). In confluent cells, incubation with HuIFN alpha-2b for 2 min significantly decreased cytoplasmic PKC-beta immunoreactivity by 37%, and no change was observed in nuclear PKC-beta labeling. PKC-alpha labeling in proliferating cells showed similar immunoreactivity in both control cytoplasm and nucleus. Treatment of proliferating cells with HuIFN alpha-2b for 2 min decreased PKC-alpha in the cytoplasm (59%) and nucleus (44%). In confluent cells, cytoplasmic PKC-alpha labeling decreased 59% at 1 min, 61% at 2 min, and 76% at 10 min of HuIFN alpha-2b treatment. Nuclear PKC-alpha decreased by 65% at 1 min, 80% at 2 min, and 62% at 10 min after HuIFN alpha-2b treatment. Western blots of total PKC-alpha in proliferating and confluent cells and PKC-beta in confluent cells showed similar results. However, Western blots of total PKC-alpha and -beta in proliferating cells did not demonstrate any significant changes in either PKC-alpha or -beta immunoreactivity following 1-min HuIFN alpha-2b treatment. These results suggest that treatment of proliferating U-373 cells with HuIFN alpha-2b for 1 min unfolds and exposes PKC-beta antigenic sites (hinge region) and increases in situ PKC-beta immunogold labeling.

Heterogeneity of protein kinase C activity in human U-373 and G-26 mice glioma cells.

Protein kinase C (PKC) enzyme activity in a mouse glioma cell line G-26 and a human glioma cell line U-373 were compared at similar cell confluency in-vitro to establish if a G-26 in-vivo mouse model would be useful to examine the role of PKC inhibitors in controlling human glioma growth in-vivo. Original crude cytosolic and membrane PKC fractions of both mouse glioma G-26 and human glioma U-373 cells did not display significant PKC activity compared to partially purified PKC. Partial purification of mouse glioma G-26 and U-373 cytosolic and membrane fractions showed different cytosolic and membrane PKC activity profiles. Total PKC activity was higher (rho = 0.0001) in human glioma U-373 (7840 picomoles/mg/min) than in mouse glioma G-26 cells (2890 picomoles/mg/min). Thus, results from trials using nude mice human glioma xenografts may be more valid than those obtained from a G-26 in-vivo mouse model for studying the effects of therapeutic drugs on PKC isozymes.

Treatment of primary cultures of calf adrenal glomerulosa cells with adrenocorticotropin (ACTH) and phorbol esters: a comparative study of the effects on aldosterone production and ACTH signaling system.

We studied the mechanism that underlies the desensitization of calf adrenal glomerulosa cells induced by 4 h of ACTH treatment. In control cells, acute ACTH treatment provoked sizeable increases in aldosterone, cAMP, and diacylglycerol, and translocated protein kinase-C from cytosol to membrane. In desensitized cells, acute ACTH effects on aldosterone and cAMP decreased by 25-60%, and diacylglycerol levels and protein kinase-C translocation were persistently stimulated and not substantially affected by further acute ACTH treatment. After 4 h of treatment with 1 microM phorbol 12-myristate 13-acetate (PMA) there were no acute effects of ACTH on the production of aldosterone, cAMP, or diacylglycerol or on protein kinase-C, which was already strongly translocated. These results suggest that ACTH-mediated desensitization of calf adrenal glomerulosa cells may be at least partially mimicked by long term treatment with phorbol esters and could be due to ACTH-induced increases in diacylglycerol-protein kinase-C signaling.

ACTH increases de novo synthesis of diacylglycerol and translocates protein kinase C in primary cultures of calf adrenal glomerulosa cells.

Effects of ACTH on the production of diacylglycerol (DAG) and translocation of protein kinase C were studied in primary cultures of calf adrenal glomerulosa cells. To study DAG production two different labeling protocols were used: (a) cells were prelabeled for 3 days with [2-3H]glycerol before ACTH addition; (b) ACTH and [2-3H]glycerol were added simultaneously to cells. In both cases, ACTH provoked rapid increases in the labeling of DAG which were maximal in 2 min, dose-dependent, and paralleled by increases in DAG mass. ACTH also increased the labeling of total glycerolipids including phosphatidic acid (PA), phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and triacylglycerol. In both labeling protocols, the rates of increase in the labeling of DAG and PA were greater than those of other glycerolipids. Our results indicate that ACTH rapidly increases DAG, at least partly by stimulating the de novo synthesis of PA. In addition, we found that ACTH, like phorbol esters, stimulated the apparent translocation of immunoreactive protein kinase C from the cytosol to the membrane fraction.

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes.

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.

Immunological evidence that insulin activates protein kinase C in BC3H-1 myocytes.

Effects of insulin on immunoreactive protein kinase C were examined in BC3H-1 myocytes. Insulin provoked rapid dose-dependent decreases in cytosolic enzyme, and transient increases and subsequent decreases in membrane-associated enzyme. Phorbol esters provoked similar changes. Our findings suggest that insulin provokes the translocative activation of protein kinase C.

Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263).

We have measured the exchange of (18)O between CO(2) and H(2)O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of (18)O from CO(2) in the fluid outside the cells provides a method to study CO(2) and HCO(3) (-) kinetics in suspensions of algae that contain carbonic anhydrase since (18)O loss to H(2)O is catalyzed inside the cells but not in the external fluid. Low-CO(2) cells of Chlorella vulgaris (grown with air) were added to a solution containing (18)O enriched CO(2) and HCO(3) (-) with 2 to 15 millimolar total inorganic carbon. The observed depletion of (18)O from CO(2) was biphasic and the resulting (18)C content of CO(2) was much less than the (18)O content of HCO(3) (-) in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO(3) (-) into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 x 10(-4) s(-1) due to our experimental errors. The Fick's law rate constant for entry of CO(2) to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO(2) flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO(2) entry into the cell or both. The CO(2) hydration activity inside the cells was 160 times the uncatalyzed CO(2) hydration rate.