[Role of nitric oxide synthase III and guanosine 3':5'- cyclic monophosphate in the protection exerted by nitric oxide on hepatic ischemia-reperfusion injury]. / Rôle de la monoxyde d'azote synthase-III et du guanosine 3':5'-monophosphate cyclique dans la protection exercée par le monoxyde d'azote lors d'une ischémie-reperfusion hépatique.

Nitric oxide (NO) exerts cytoprotective effects against hepatic ischemia-reperfusion damage. This study was designed to evaluate which isoform of NO synthase (NOS) is implicated in the generation of cytoprotective NO and to investigate whether NO effects are mediated by cyclic GMP (cGMP). After partial ischemia for 45 min, liver damage was estimated by the release into plasma of cytolytic enzymes. Ischemia-reperfusion induced marked increases in plasma creatine kinase and lactate dehydrogenase after 1 h of reperfusion and of aminotransferases after 6 h of reperfusion. The pretreatment of ischemic rats with 8-bromo-cGMP (16 mg/kg i.v. 30 min before ischemia) or with L-arginine (the endogenous precursor of NO, 100 mg/kg i.v.) significantly diminished the ischemia-reperfusion-induced release of all these enzymes. This demonstrates that cGMP possesses hepatoprotective properties. By immunohistochemistry, we observed, after 6 h of reperfusion, an increase in endothelial NOS-III immunoreactivity, particularly in the small arteries and sinusoids. This NOS-III accumulation in endothelial cells could protect the liver against ischemia-reperfusion by the local generation of NO probably via cGMP.

Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi-endosomal fraction.

The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.

[Therapeutic management of traumatic avulsion of permanent teeth]. / Prise en charge thérapeutique des expulsions traumatiques des dents définitives.

Management of avulsed permanent teeth requires perfect knowledge of the different parameters influencing the short and mid-term and above all long-term prognosis. Based on a review of the literature and the analysis of 50 cases cared for in our unit enables us to propose, in accordance with the extra oral delay and the degree of dental maturation, a protocol for the different emergency situations. This clarification emphasizes the contribution of new preserving solutions which allow reimplantation delays up to 24 hours without effect on prognosis.

Increased expression of liver PKC alpha in hypoinsulinemic diabetic rats: a post-translational effect.

Ca2+-dependent protein kinase C (cPKC) activity and expression have been studied in livers from hypoinsulinemic streptozotocin (STZ)-induced diabetic and untreated control rats. In diabetic rats, cPKC activity was slightly decreased in liver total particulate and nuclear fractions but was unchanged in mitochondrial-lysosomal, microsomal and cytosolic fractions. On Western immunoblot analysis, PKC alpha was identified as two distinct proteins of 90 and 81 kDa. In diabetic rats, the abundance of the 90 kDa protein was increased in most subcellular fractions with a maximum in the cytosolic and microsomal fractions (180%) but that of the 81 kDa protein was unchanged. PKC beta2 was detected as a single 81 kDa protein in cytosolic and microsomal fractions with unchanged levels in diabetic rats. Liver PKC alpha mRNA levels as measured by reverse transcription and competitive PCR amplification were similar in diabetic and control rats. The increased expression of PKC alpha protein in diabetic rats was reversed by insulin but not by phlorizin, suggesting that it did not result from hyperglycemia. We conclude that STZ-induced diabetes induces the expression of a biologically inactive form of PKC alpha which differs from active PKC alpha by an undefined post-translational modification, possibly an increase in phosphorylation state.

1 alpha,25-dihydroxyvitamin D3 stimulated alkaline phosphatase activity in cultured pig kidney epithelial LLC-PK1 cells.

The effects of 1 alpha,25-dihydroxyvitamin D3 (1,25 (OH)2D3) and human growth hormone (hGH) on the activity of alkaline phosphatase (ALP) were evaluated in pig kidney LLC-PK1 cultured cells. 1,25(OH)2D3 (10(-9)-10(-6) mol L-1) and hGH (10(-7) mol L-1) increased ALP activity in these cells while no hormonal effect was detected on two other enzyme activities: gamma-glutamyl transferase and acid phosphatase. ALP activity was maximally increased after 4.5-6 h incubation with both hormones. The hormonal induction of ALP activity was prevented by a pretreatment of cells with actinomycin D. Thus, 1,25 (OH)2D3 and may be hGH could stimulate ALP activity via a transcription of some gene.

Sensitive northern blot hybridization using digoxigenin RNA probes for the mRNA detection of two glucose transporter isoforms.

Diseases involving glucose metabolism disorders are more and more prevalent. Therefore the question of glucose transporter gene expression is being addressed in experimental and clinical studies. Radioactive probes are generally used to assess glucose transporter mRNA levels, but these probes are short-lived, costly and harmful to the environment. Alternative methods that do not present these disadvantages, for example digoxigenin (DIG) labelled probes, might prove to be very interesting for the study of glucose transporter mRNA. The aim of the present work was to compare DIG-labelled cRNA probes to 32P-labelled cRNA probes in order to see whether or not the non-radioactive method can be used to assess glucose transporter gene expression. This work shows that DIG-labelled glucose transporter (GLUT1 and GLUT4) cRNAs are suitable probes for the assessment of these gene expressions. We have found that the DIG system offers a much higher sensitivity than the 32P system for both GLUT1 and GLUT4 mRNA detection. This represents a decisive advantage in human studies where tissue quantity is a limiting factor. In addition, stability, safety, time saving and cost reduction are other considerations that make DIG-labelled GLUT1 and GLUT4 cRNAs very attractive.

Evidence that growth hormone stimulates protein kinase C activity in isolated rat hepatocytes.

The mechanism of action of growth hormone (GH) is not known, although indirect evidence suggests that protein kinase C (PKC) might play an important role in the insulin-like actions of GH. In this investigation, we directly examined the effects of GH relative to those of insulin on PKC activity in isolated rat hepatocytes. Human GH (10(-7) mol/L) significantly increased the activity of PKC in both cytosolic and particulate fractions. The effect was maximal at 1 minute, disappeared at 5 minutes, and then increased again at 30 minutes in both fractions. At 1 minute, maximal and half-maximal stimulation of PKC activity occurred at hGH concentrations of 10(-7) and 5 x 10(-9) mol/L, respectively. Insulin (10(-7) mol/L) also induced a significant and transient increase in enzyme activity at 2 minutes in cytosolic and particulate fractions; at 30 minutes, PKC activity was decreased in the soluble fraction (-17%) and increased in the particulate fraction (+65%). Measurement of specific [3H]-phorbol dibutyrate (PDBu) binding suggested translocation of PKC from the cytosol to the membrane fraction after 30 minutes of incubation, only after insulin treatment. The early effects of GH and insulin on PKC activity were additive in both the particulate and cytosolic fractions. Although the later effects of GH and insulin on PKC were quite different, both hormones rapidly activated PKC in isolated hepatocytes, suggesting that PKC might be involved in triggering the insulin-like actions of GH.

Effects of hormones on SBP mRNA levels in human cancer cells.

The human plasma sex steroid binding protein (SBP) has been previously shown to be synthesized in liver cells. The hormonal regulation studies of hepatic SBP mRNA demonstrate that it is controlled by estradiol, antiestrogen tamoxifen, dihydrotestosterone, triiodothyronine and insulin in a similar way as secreted SBP. The metabolic inhibitor cycloheximide was unable to prevent the estrogen or thyroid hormone induced increase in SBP mRNA. The slight stimulation of SBP synthesis by estradiol suggests that non-steroidal factors may be involved in its regulation and that the estrogen regulatory mechanism could also be partly post-transcriptional. In endometrial (Ishikawa cells) and prostatic (LNCaP cells) carcinoma cells, SBP mRNA has been detected suggesting that SBP may play a role in the uptake and intracellular mechanism of action of sex steroid in target cells.