Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Effect of diabetes mellitus induced by streptozotocin on renal Superoxide dismutases in the rat

Two forms of superoxide dismutase, CuZn-SOD and MnSOD, have been investigated in the kidneys of streptozotocin-induced diabetic rats using both radio-immunoassay and immunoenzyme staining. The rats were killed 2, 8 and 12 weeks after the induction of diabetes mellitus and the kidneys excised. Two weeks after the induction of diabetes, the kidneys were hypertrophied because of the proliferation of renal tubular epithelium. However, the total CuZnSOD content of the kidneys did not increase and, because of the epithelial proliferation, the CuZnSOD concentration in each proximal tubular cell was decreased. Armanni-Ebstein lesions were found in the distal tubules 8 and 12 weeks after the induction of diabetes. The cells in these lesions were intensely stained for CuZnSOD, suggesting an adaptive response to the enhanced oxidative stress. The MnSOD staining in the thick ascending limbs of Henle's loops was enhanced in the diabetic kidneys, while that in the cortical tubules was unaltered. MnSOD was assumed to increase in response to hypermetabolism associated with the proliferation of renal tubules. This was most marked in the cells which were rich in mitochondria, again suggesting an adaptive response to enhanced oxidative stress induced by diabetes mellitus. The glomeruli of both the diabetic and control groups were not stained for SODs, and no significant microscopic change was found even 12 weeks after the induction of diabetes mellitus.     

Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: Fluorescence microscopy image analysis

Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H₂O₂-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Piper longum Constituents Induce PANC-1 Human Pancreatic Cancer Cell Death under Nutrition Starvation

Pancreatic cancer is a highly aggressive form of cancer with a poor prognosis, partly due to ‘austerity’, a phenomenon of tolerance to nutrient deprivation and survival in its hypovascular tumor microenvironment. Anti-austerity agents which preferentially diminish the survival of cancer cells under nutrition starvation is regarded as new generation anti-cancer agents. This study investigated the potential of Piper longum constituents as anti-austerity agents. The ethanolic extract of Piper longum was found to have preferential cytotoxicity towards PANC-1 human pancreatic cancer cells in a nutrient-deprived medium (NDM). Further investigation led to the identification of pipernonaline ( 3 ) as the lead compound with the strongest anti-austerity activity, inducing cell death and inhibiting migration in a normal nutrient medium, as well as strongly inhibiting the Akt/mTOR/autophagy pathway. Therefore, pipernonaline ( 3 ) holds promise as a novel antiausterity agent for the treatment of pancreatic cancer.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites

Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed. Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity. These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites. Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity. Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.     

Identification of human Kir2.2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells and Xenopus oocytes

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.