Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Myocardial cell vulnerability to exogenous phospholipase attack

Myocardial cell vulnerability to phospholipase C (PC-PLC) attack was investigated in three different preparations of rat myocardial cells: triacylglycerol (TG)-loaded, hypothermic/rewarmed and energy depleted myocytes. The attack by PC-PLC was evaluated as PC-PLC induced glycerol output due to the combined action of phospholipase C and intracellular lipases. PC-PLC induced glycerol output was significantly higher (p < 0.05) in all three myocyte preparations, compared to their respective controls. Cell morphology (% rod shaped myocytes) of TG-loaded or hypothermic/rewarmed myocytes was not different from their controls, whereas energy depleted myocytes almost exclusively were rounded up, due to hypercontraction of the myofilaments. Hypothermic/rewarmed and energy depleted myocytes showed a significantly higher release of lactate dehydrogenase (LDH), compared to their controls although the difference was much more pronounced in the latter. Finally, the cellular contents of ATP were maintained both in TG-loaded and hypothermic rewarmed myocytes, while energy depleted myocytes contained only about 25% of the normal ATP level. These results demonstrate that attack from exogenously added phospholipases can occur, not only in seriously damaged cardiac myocytes, but in myocytes with a more subtle damage as well. (Mol Cell Biochem 116: 47–52, 1992)     

Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

Karyosystematic studies on threeCrepis species (Asteraceae) endemic to Greece

This work examines the cytogeographical distribution, the morphological characters, and the karyotypes of threeCrepis species endemic to Greece (C. sibthorpiana, C. incana, andC. heldreichiana). C. sibthorpiana is diploid (2n = 2x = 8),C. incana is diploid (2n = 2x = 8) and tetraploid (2n = 4x = 16, 17), andC. heldreichiana is always dekaploid (2n = 10x = 40). The Giemsa positive bands, usually pairs of dots, are mainly centromeric inC. incana, while they are terminal inC. sibthorpiana (on the short arm of all chromosomes) and inC. heldreichiana (on both arms of all chromosomes). Intercalary C-bands are scarce and usually variable within karyotypes, individuals, and species. The most variable karyotype both in Feulgen and Giemsa preparations is that ofC. incana, in which also supernumerary chromosomes were observed, which are polysomic to standard set members. On the basis of morphological and karyological data the evolutionary relationships among the threeCrepis taxa are discussed.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle.

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.     

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XII. Enantioselectivity and temperature dependence in microsomes and reconstituted cytochrome P-450 systems from rat liver.

Formation of metabolic intermediate (MI) complexes was studied with the enantiomers of amphetamine, 1-phenyl-2-pentanamine, N-hydroxyamphetamine, and 2-nitroso-1-phenylpropane (the C-nitroso analogue of amphetamine). Three different enzyme systems were used; liver microsomes from phenobarbital pretreated rats and two reconstituted systems containing the P450 2B1 and P450 2C11 forms of cytochrome P-450. Enantioselective complex formation in microsomes was shown for the amines and the nitroso compound, but not for the hydroxylamine. The highly purified P450 2B1 system formed the MI complex with all substrates tested, and the enantioselectivity observed with the microsomal system was reproduced. In the P450 2C11 system the nitroso compounds were completely inactive, whereas the enantiomers of N-hydroxyamphetamine still produced the complex at a high rate. Changes in temperature were shown to affect (R)-2-nitroso-1-phenylpropane more than its enantiomer. Both enantiomers showed biphasic Arrhenius plots for MI complex formation in microsomes (breaks around 22 degrees C), but the activation energies of the (R)-isomer were about five times higher than those of the (S)-isomer. A theory is presented which suggests different modes of interaction with the active site of P-450 to account for the different behaviour of the various substrates.     

4',6-Dichloroflavan (BW683C): Enantiomeric separation by hplc/dad and antirhinovirus activity

Racemic 4',6-dichloroflavan (BW683C), a highly effective inhibitor of rhinovirus serotype 1B in vitro, was resolved by high-performance liquid chromatography on a chiral stationary phase. The enantiomers were separately collected and circular dichroism curves were obtained, in order to determine the absolute configuration of the two enantiomers. The activity of the isomers was studied on human rhinovirus serotype 1B multiplication in HeLa cell cultures, by means of the plaque reduction assay. Both enantiomers were potent inhibitors of virus replication; by comparing the IC₅₀ values, the S form was 3.5 times more effective than the R form.