PKC regulation of microfilament network organization in keratinocytes defined by a pharmacological study with PKC activators and inhibitors.

The modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen was studied. Two actin microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inhibitors (0.3 mM genistein and 1 microM herbimycin A). By contrast, the cortical network occurred in quiescent cells, was unaffected by tyrosine kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 microM concentrations of calphostin C, chelerytrine chloride, and D-erythrosphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase function in vitro. Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA, which indicated that focal complex formation was highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics.

Evidence for the presence of prolyl oligopeptidase and its endogenous inhibitor in neonatal rat pancreatic beta-cells.

Prolyl oligopeptidase (PE), an enzyme that may be involved in the maturation and degradation of hormones and neuropeptides has been detected in neonatal rat pancreatic islet cell monolayer cultures. PE activity was not observed in islet cell homogenates but when cellular extracts were subjected to gel-filtration, a such activity with a molecular mass about 70 kDa can be detected. Gel-filtration experiment has led to the finding of a PE inhibitor in these extracts with an estimated molecular mass of 6.5 kDa. After separation of the endogenous inhibitor from PE enzyme by gel-filtration, PE inhibitor was partially purified in a single activity peak by reverse-phase high-performance liquid chromatography (HPLC). It inhibited the fluorogenic substrate Z-Gly-Pro-beta Na degradation by partially purified PE in a competitive manner. Inhibitor is shown to be specific for PE enzyme and it is not released by potassium depolarization of islet cell membrane. These findings indicated that inhibitor is localized in the cytosolic compartment as prolyl oligopeptidase. The specific activity of the inhibitor in beta-cell cultures derived from donor rats varying from 3-20 days of age was unchanged. In contrast, PE inhibitor can only be detected in pancreatic tissue from 3-day-old rats compared with tissue from 20-day-old and adult rats after gel filtration. This discrepancy can be relevant to the different endocrine/exocrine tissue ratios in the pancreas during developing rats. Furthermore, pancreatic tissue from streptozotocin-treated 3-day-old rats did not show PE inhibitory activity indicating that PE inhibitor was principally contained in beta-cells. Based on the biochemical characteristics of the beta-cell PE inhibitor, the enhancement of PE activity observed in neonatal pancreas of STZ-treated rat as previously described (P. Salers, Regul. Pept., 50 (1994) 101-111), appears to be due to the presence of the endogenous PE inhibitor in neonatal rat pancreatic beta-cells that disappears following STZ-treatment.

Enhancement by streptozotocin-induced diabetes of pancreatic prolyl endopeptidase activity in neonatal rats.

The effect of streptozotocin (STZ)-induced diabetes on the concentrations of deamidated TRH (TRH-OH), a metabolite of thyrotropin-releasing hormone (TRH) and prolyl endopeptidase (PE) activity were studied in the pancreas of neonatal rats to determine the contribution of beta-cells to PE activity and TRH-OH levels that we have previously found in this tissue. STZ treatment caused a significant reduction of immunoreactive TRH-OH levels on day-3 and -5 compared to untreated control rats. Reverse-phase high performance liquid chromatography of pooled extracts of 3-day-old normal rat pancreas revealed that about 50% of immunoreactive TRH-OH was found in the fractions representing authentic TRH-OH, whereas the remaining 50% eluted earlier. In STZ treated rats, all of the TRH-OH immunoreactive was associated with this early peak, no authentic TRH-OH could be detected. The specific activity of PE, on the other hand, rose 2.5-fold in diabetic 3-day-old pups (4.06 +/- 0.13 compared with 1.59 +/- 0.83 nmol/min/mg protein, P < 0.01, in controls). This increase declined with age (1.6- and 1.3-fold in 5 day- and 7-day-old pups, respectively). STZ treatment did not change pancreatic PE levels in 20-day-old rats control. Normalization of STZ induced hyperglycemia by sodium metavanadate treatment or by replacement of exogenous insulin did not restore pancreatic PE activity. The enhancement of PE activity following STZ treatment was specific for pancreas tissue. Furthermore, beta cytoxin drugs other than STZ that cause permanent diabetes such as alloxan enhanced PE activity to the same extend. Kinetic studies for PE activity show that Vmax is 3-fold higher in 3-day-old STZ-treated than in rat controls. In contrast, values for Km were comparable in rats of both groups (25 to 34 microM). We then tested whether the decrease of Vmax might have been caused by the presence of an PE inhibiting factor in these preparations. Gel-filtration experiments of pancreatic extracts revealed that the total apparent activity of eluted PE in 3-day-old control rats was 2-fold higher than in the original extract. In contrast, the recovery of eluted PE activity was not increased in the case of STZ-treated 3-day-old and untreated 20-day-old rats. These findings demonstrate that TRH-OH is identified in beta-cells and that an inhibiting factor(s) present in beta-cells appear(s) to be responsible for the unexpected enhancement of PE activity observed in STZ-treated neonatal rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Ontogeny of prolyl endopeptidase, pyroglutamyl peptidase I, TRH, and its metabolites in rat pancreas.

We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.

Peptidylglycine alpha-amidating monooxygenase activity and TRH and CRF biosynthesis. Role of copper.

Carboxy-terminal amidation of biologically active peptides, an important characteristic of more than half of these substances, occurs during the maturation process of peptide precursors. It is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme that is copper-dependent. We show here that alterations of copper stores in cultured cells from different origins (pancreas and hypothalamus) affect the immunoreactivity of thyrotropin-releasing hormone (TRH) and corticotropin-releasing factor (CRF) (two alpha-amidated peptides). This suggests that copper can affect neuropeptide biosynthesis and may play a role in the endocrine or central nervous system function.

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism.

Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of TRH release by the cultured neonate rat pancreas.

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic beta-cells.

Characterization of an alpha-amidating activity in a human pancreatic tumour secreting vasoactive intestinal peptide (VIP).

A case of watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome due to a pancreatic tumour and identified by VIP plasma level, VIP immunocytochemistry, and ultrastructural analysis of tumour sections, is reported. Since VIP is the mediator of the syndrome and is biologically active under its amidated form, the enzymatic alpha-amidating activity was investigated and characterized in tumour extract; using the synthetic substrate D-Tyr-Val-Gly, the enzyme displayed an optimal activity at pH 7.0, under aerobic conditions and with 35 microM CuSO4 and 3 mM ascorbate as co-factors. The Kmax and Vmax values of the enzymatic activity were 133.7 microM and 26.9 pmol/h/micrograms protein respectively. Its molecular weight, determined by molecular sieving, was close to 36 kDa. Other tumours of the human endocrine pancreas were also investigated for the enzymatic activity. The clinical interest of studying the regulation of the alpha-amidating activity in such tumours is discussed.

Ontogenetic expression of peptidyl-glycine alpha-amidating monooxygenase mRNA in the rat pancreas.

Qualitative and quantitative expression of m.RNA coding for Peptidyl-Glycine alpha-Amidating Monooxygenase (PAM) in the developing rat pancreas was investigated by Northern and dot blot hybridization, with a bovine PAM c.DNA probe (0.7 kb fragment). A specific hybridization signal was evidenced for a 3.7 kb m.RNA species. Measurement of PAM m. RNA rate during the rat pancreas ontogenesis revealed a biphasic profile which appeared corelated with that of gastrin and TRH m.RNA respectively. On the other hand, streptozotocin-treatment resulted in a 50% decrease of PAM m.RNA levels.

Thyrotropin releasing hormone in the pancreas of newborn rats from streptozotocin-treated mothers.

The effect of maternal diabetes (induced by i.p. injections of 40-50 mg/kg BW Streptozotocin on the day of mating) on TRH in the pancreas of newborn rats was studied. Determination of peptide alpha amidation activity and TRH precursor level on the day of birth revealed decreased biosynthesis of TRH resulting in profoundly (10 times) lower pancreatic TRH and TRH-OH concentrations in pups of diabetic rats. Pancreatic His-Pro-diketopiperazine (His-Pro-DKP) remained unaffected by maternal diabetes. The depression of pancreatic TRH was less profound 24 h later, and even elevated TRH was measured in the pancreas of pups of diabetic mothers on postnatal day 5. Short term postnatal starvation or nursing of intact pups by the diabetic foster mother did not affect pancreatic TRH. It could be postulated that postnatal TRH development in the rat pancreas is retarded by maternal diabetes, while His-Pro-DKP remains unaltered.

Ontogenesis of TRH mRNA in the rat pancreas.

The rapid changes in TRH levels in the rat pancreas during the neonatal period make this organ an interesting model for the study of the regulation of TRH biosynthesis. Pancreatic RNAs were isolated by the guanidinium thiocyanate method and layered onto CsCl cushion. Northern blot preparations were hybridized with 32P labeled TRH cDNA probe. Pancreatic TRH mRNA was first detected in 19-day old fetuses and reached the highest level on day 0, then decreased, being barely detectable 14 days after birth. The neonatal injection of streptozotocin induced a dramatic drop of TRH mRNA levels 24 hours later. This result suggests that the peculiar evolution of TRH level in pancreas is partly due to the evolution of the expression of the TRH gene.

Evidence for high peptide alpha-amidating activity in the pancrease from neonatal rats.

A high peptidylglycine alpha-amidating mono-oxygenase (PAMase) activity has been measured in the pancreas of neonatal rats. A significant fraction of this activity is contained in the beta cells of the islets of Langerhans and is colocalized with thyrotropin-releasing hormone (TRH) and its precursor in secretory granules. The ontogenetic variation of PAMase activity in the pancrease parallels that of TRH concentrations, suggesting that this enzymatic activity is directly related to TRH biosynthesis. In addition, PAMase activity is able to generate TRH when incubated with less than Glu-His-Pro-Gly, a tetrapeptide present as a repetitive sequence in the TRH precursor. The perinatal evolution of the TRH precursor levels in the pancreas is similar to that of PAMase activity (unpublished results). Thus, the neonatal rat pancreas offers an endocrine model in which the levels of a neuropeptide precursor and an enzyme activity, involved in the posttranslational modification of this precursor, are similarly regulated. Our results suggest also that a fraction of PAMase activity may be produced outside of the beta cells and related to the biosynthesis of COOH-terminally amidated peptide(s) other than TRH. The ontogenetic changes in PAMase activity imply that the synthesis of this peptide(s) is high during the neonatal period, decreasing thereafter.

Evidence for high peptide alpha-amidation activity in the neonatal rat pancreas.

A high peptide alpha-amidating activity is present in a mitochondrial/secretory granules preparation from 3-day old rat pancreas. It is dependent on copper, ascorbate and molecular oxygen. This preparation is able to generate TRH when incubated with Pyroglu-His-Pro-Gly, a sequence present in the TRH precursor molecular. The peptide alpha-amidating activity may be involved in the high rate of TRH biosynthesis in the pancreas during the neonatal period. In the pancreas of adult rats which contain low levels of TRH, the peptide alpha-amidating activity is barely detectable.

Effects of apoproteins C on lipoprotein lipase activity bound to rat fat cells.

During short-term incubation, a suspension of fat cells isolated from female rats hydrolyzed tri[3H]oleyl[14C]glycerol added as substrate and incorporated an average of 42% of the amounts of [3H]oleic acid released. Lipolysis was catalyzed by cell-bound lipoprotein lipase (LPL), whose activity was influenced by apoproteins (apo) C purified from human plasma. ApoC-II enhanced 12-fold the hydrolysis rates with cells from fed rats versus 3,4-fold with cells from fasted rats. In both cases, the stimulation by apoC-II was greater than that by whole serums from led or fasted rats. Maximal LPL activity at the cell surface depended more on the preexisting nutritional state of the cell than on apoC-II or serums added extracellular. ApoC-I, apoC-III1 and apoC-III2 had no or moderate direct effects on LPL activity, whereas apoC-III1 and apoC-III2 reduced markedly the stimulating effect of apoC-II. At all levels of LPL activity, the amounts of [3H]oleic acid liberated by hydrolysis and incorporated as [3H]acylglycerol in cell lipids were highly correlated with LPL activity, suggesting that the uptake process was directly related to enzyme action.

Estrogen hormones and lipid metabolism. Effect of ethynyl-estradiol on liver lipases.

Lipase activities assayed in liver subcellular fractions from rats given ethynyl-estradiol are lowered, while triglycerides increase and cholesterol decreases in blood. Triester lipase in microsomes is the most altered activity. Such enzymatic changes might play a role in the development of lipid abnormalities in blood of women taking estrogens as oral contraceptives.