Ginkgo biloba extract protects rat kidney from diabetic and hypoxic damage.

Ginkgo biloba extract EGb 761 was studied for its nephroprotective effects in experimentally diabetic and hypoxic rats. Duration of streptozotocin-induced diabetes was 4 months, that of respiratoric hypoxia of the diabetic group 20 min. The daily dose of 100 mg EGb/kg bodyweight started 1 month after induction of the diabetes. EGb reduced diabetes-induced morphological alterations of the kidney such as increase in volume of glomeruli, capillary tufts, urinary space, and thickening of Bowman's capsule basement membrane. Diabetically increased immunostaining of interstitial collagenes of types I, III, and VI was diminished by the EGb extract. EGb reduced the relative total SOD activity from 163% in diabetic kidney to 46%. Additional hypoxia-induced ultrastructural damage was also diminished.

Ultrastructural, immunohistochemical and biochemical investigations of the rat liver exposed to experimental diabetes und acute hypoxia with and without application of Ginkgo extract.

The aim of this paper was to investigate the effect of streptozotocin-induced diabetes by i.p. bolus injection of streptozotocin at 60 mg per kg bodyweight over four months and additional acute respiratory hypoxia (20 min. duration, 5% oxygen v/v), and also the protective effect of Ginkgo biloba extract (EGb 761) on Wistar rat liver under these experimental conditions. Diabetic and additional hypoxic alterations in histology and ultrastructure were subjected to qualitative and quantitative analysis, collagen was investigated by immunohistochemistry, and some biochemical parameters of oxidative stress were determined. Diabetes caused an increase in the size of the hepatocytes and their nuclei with a decrease in nucleus-to-plasma ratio and glycogen content. Connective tissue was variably increased in individual cases as shown by routine histological staining. EGb did not influence these data. Ultrastructural morphometry revealed a significant reduction in rough endoplasmic reticulum (rER) and a significant increase in smooth endoplasmic reticulum (sER) through diabetes, an increase under EGb protection, with no significant alteration under hypoxia. The volume fraction of mitochondria was significantly increased after induction of diabetes but less increased in the protected group. Additional hypoxia reduced this parameter. The mean cross-section area of mitochondria was significantly elevated in all diabetic groups compared to controls. Volume density of mitochondrial cristae was significantly diminished in all diabetic groups; EGb could only improve this parameter in the diabetic-hypoxic group.

Identification of substrate specificity determinants in human cAMP-specific phosphodiesterase 4A by single-point mutagenesis.

To identify amino acids that might be involved in discriminating guanosine-3',5'-cyclic phosphate (cGMP) towards adenosine-3',5'-cyclic phosphate (cAMP) binding in the cAMP-specific phosphodiesterases, alignments of different human cyclic nucleotide phosphodiesterases (PDEs) were performed. Eight amino acid residues that are highly conserved in the cAMP-hydrolysing phosphodiesterases (PDE1, PDE3, PDE4, PDE7, PDE8) and that did not show any homologies to the cGMP-specific phosphodiesterases (PDE5, PDE6, PDE9) were selected from these alignments. Using the technique of site-directed mutagenesis, derivatives of PDE4A carrying single mutations at these conserved residues (amino acid positions are given according to the human PDE4A isoform HSPDE4A4B; accession number L20965) were generated and expressed in COS1 cells. The expression products were characterised with regard to cAMP and cGMP hydrolysis and sensitivity towards type-specific inhibitors. The mutation of Phe484 toward Tyr, Ala590 toward Cys, Leu391 and Val501 towards Ala had no significant influence on substrate affinity or specificity. However, the exchange of Trp375 and Trp605 for aliphatic residues abolished catalytic activity and the exchange of Pro595 for Ile led to sevenfold decrease of substrate affinity and an 14-fold decrease of the affinity towards the PDE4-specific inhibitor 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram). Both effects may provide evidence for a structural importance of Trp375, Trp605 and Pro595 for PDE function. By exchanging the aspartate residue for asparagine or alanine at position 440 of the human PDE4A4B isoform, the substrate specificity was altered from the highly specific cAMP hydrolysis to an equally efficient cAMP and cGMP binding and hydrolysis. In addition, the IC(50) values for common PDE4-specific inhibitors like rolipram, N-(3,5-dichlorpyrid-4-yl)-3-cyclopentyl-oxy-4-methoxy-benzamide (RPR-73401) and 8-methoxy-5-N-propyl-3-methyl-1-ethyl-imidazo[1,5-a]-pyrido[3,2-e]-pyrazinone (D-22888) were dramatically increased. These results demonstrate an important role of the aspartate at position 440 in determining substrate specificity and inhibitor susceptibility of PDE4A. The strong conservation of this residue suggests that Asp440 may play a similar role in other cAMP-PDEs.

Identification of inhibitor binding sites of the cAMP-specific phosphodiesterase 4.

Using the technique of site-directed mutagenesis, point mutants of human PDE4A have been developed in order to identify amino acids involved in inhibitor binding. Relevant amino acids were selected according to a peptidic binding site model for PDE4 inhibitors, which suggests interaction with two tryptophan residues, one histidine and one tyrosine residue, as well as one Zn(2+) ion. Mutations were directed at those tryptophan, histidine, and tyrosine residues, which are conserved among the PDE4 subtypes (PDE4A-D) and lie within the high-affinity 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) binding domain of human PDE4A (amino acids 276-681 according to the PDE4A sequence L20965). Truncations to this region do not alter enzyme activity or inhibitor sensitivity. The mutants were expressed in COS1 cells, and the recombinant cyclic nucleotide phosphodiesterase (PDE) forms have been characterized in terms of their catalytic activity and inhibitor sensitivities. Tyrosine residues 432 and 602, as well as histidine 588, were found to be involved in inhibitor binding, but no interaction was detected between tryptophan and PDE inhibitors tested. To test the possibility that other amino acids are of importance for hydrophobic interactions, selected phenylalanine residues were also mutated. We found phenylalanine 613 and 645 to influence inhibitor binding to PDE4. The significant differences in the inhibitor sensitivities of the mutants show that the various inhibitors have different enzyme binding sites. Based on the assumption that the known side effects of PDE4 inhibitors (like emesis and nausea) are caused directly by selective inhibition of different conformation states of PDE4, our results may be a hint to differ between PDE4 inhibitors, which have emetic side effects (like rolipram), and those that do not have side effects (like N-(3,5-dichlorpyrid-4-yl)-[1-(4-fluorbenzyl)-5-hydroxy-indol-3-yl]-glyoxylateamide [AWD12-281]) by the differences of their binding sites and in that context contribute to the development of novel drugs. Furthermore, the identification of amino acid interactions proposed by the peptidic binding site model, which was used for the mutant selection, verifies the PrGen modeling as a useful method for the prediction of inhibitor binding sites in cases where detailed knowledge of the protein structure is not available.

The influence of hypoxia on the myocardium of experimentally diabetic rats with and without protection by Ginkgo biloba extract. I. Ultrastructural and biochemical investigations on cardiomyocytes.

The influence of acute respiratoric hypoxia in streptozotocin-diabetic rats and protective effects of Ginkgo biloba extract (EGb 761)-pretreatment were investigated by the means of ultrastructural morphometry, biochemical parameters of oxidative stress and iNOS transcription and expression. Ultrastructural parameters revealed that acute hypoxia deteriorated the morphologic condition of the diabetic cardiomyocytes: volume fractions of sarcoplasm, t-tubules, mitochondria, cytoplasmic vacuoles, and degenerative intramitochondrial areas increased after hypoxia, those of myofibrils and mitochondrial cristae decreased. Since these alterations are more striking than after hypoxia of non-diabetic animals as demonstrated in preceding studies, we regard them as indicative for reduced hypoxia tolerance of the diabetic myocardium. EGb-treatment of the diabetic animals could improve the above mentioned parameters thus indicating a gradual improvement of the hypoxia tolerance. The biochemical parameters of oxidative stress (malondialdehyde, superoxide dismutase) were decreased after hypoxia in the diabetic myocardium but increased after EGb-pretreatment. The ultrastructural damage by hypoxia and its prevention by EGb should be regarded rather as a consequence of ATP--and energy deficiency and breakdown of membrane functions and--structure resp. as membrane stabilizing and enzyme-regulating effects of EGb than as radical-related events. The hypoxia-induced deprivation of creatine kinase activity of the diabetic myocardium was not prevented by EGb-treatment. Immunohistochemical demonstration of iNOS expression was strongest in the unprotected diabetic myocardium, absent after additional hypoxia and in the controls, and very weak in the protected hypoxic specimens. Transcription of iNOS as demonstrated by RT-PCR was present in few diabetic, some of the hypoxic diabetic, in most of the EGb-treated hypoxic diabetic, and in all control animals. EGb-treatment seems to improve the hypoxia tolerance of diabetic myocardium concerning ultratructural parameters. The partly conflicting immunohistochemical and biochemical results require further investigations.

Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli.

A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates.

Effects of dexamethosone and glucagon after long-term exposure on cyclic AMP phosphodiesterase 4 in cultured rat hepatocytes.

67% of total cAMP phosphodiesterase activity (PDE) in cultured rat hepatocytes could be detected in the cytosol, 15% in plasma membrane, 15% in 'dense vesicle,' and 3% in endoplasmatic reticulum fractions. Up to 84% of the PDE activity of the cytosol is represented by the rolipram-sensitive PDE 4. ICI 118233-inhibited PDE 3 was found predominantly in membranes. We were able to show that dexamethasone acts on the PDE 4 in cytosolic and plasma membrane fractions whereas glucagon effected the PDE 4 of the cytosol and the PDE 3 in 'dense vesicle' membranes. Primary culture of hepatocytes was used to study long-term effects of dexamethasone and glucagon on PDE 4 activity. Addition of dexamethasone (0.1 microM) at the beginning of cultivation leads to a decrease of total PDE 4 activity whereas after 24 h precultivation no dexamethasone effect could be observed. Glucagon effects on PDE 4 were investigated in 20 h precultured hepatocytes. Maximal stimulation was achieved after 2 h of exposure. PDE 4 subtypes A, B , D and, to a lesser degree, subtype C could be detected by RT-PCR analysis. The results of semiquantitative RT-PCR show that the presence of dexamethasone during the first 24 h of cultivation reduced selectively the transcription of PDE 4D, whereas glucagon was without any effect. Also the translation of PDE 4D was reduced as shown in the Western blot. We would like to discuss the way that dexamethasone influences PDE 4D expression-most likely in combination with other factors such as cytokines--during the time of cell plating, whereas glucagon actions are part of metabolic regulations via phosphorylation reactions.

Combined effects of insulin and dexamethasone on cyclic AMP phosphodiesterase 3 and glycogen metabolism in cultured rat hepatocytes.

Primary cultures of rat hepatocytes were used to study the combined effects of insulin and dexamethasone on cyclic AMP phosphodiesterase 3 (PDE 3) and glycogen metabolism. PDE activity was measured in extracts obtained by hypotonic shock treatment of the particulate fraction from cultured hepatocytes. PDE 3 was identified by inhibition with ICI 118233, Western blotting, immunoprecipitation of the activity with the use of a new PDE 3B-specific anti-peptide antibody and stimulation of the activity after adding insulin, glucagon and okadaic acid to the culture medium. Specific PDE inhibitors were always used to identify the measured PDE activities. Hypotonic extracts contained 30% PDE 3 and 50% PDE 4. Both PDE types show a nearly constant level during cultivation up to 48 h. Long-term exposure of dexamethasone alone has no effect on PDE 3 activity, whereas, in combination with insulin, the insulin stimulation of PDE 3 activity was found to be increased between 48 and 72 h of cultivation. Additionally, db-cAMP was able to stimulate PDE 3. A possible effect of insulin or db-cAMP on PDE 3B expression could not be found. On the other hand, activation of PDE 3B after 48 h of culturing decreased rapidly after removal of insulin or db-cAMP from the culture medium. Insulin-stimulated incorporation of 14C-glucose into glycogen was inhibited by PDE 3- and PDE 4-specific inhibitors as well as by the unspecific PDE inhibitor IMBX. Inhibitions by PDE 3- and PDE 4-specific inhibitors were found to be additive and reached the same extent as with IMBX. Summarising our results, we can conclude that PDE 3 and PDE 4 effectively control the hepatic glycogen metabolism. Insulin effects on PDE activity and glycogen metabolism require the presence of dexamethasone. Insulin-stimulated PDE seems to play an important role in realising insulin effects on hepatic glycogen metabolism.

Phorbol ester effects on hormonal responses in freshly isolated short-term incubated and cultured hepatocytes.

Beta-adrenergic, alpha-1-adrenergic and glucagon stimulation of glucose release were compared between hepatocytes which were freshly isolated, incubated for 3 h in suspension or cultivated for 4 or 24 h in plastic culture flasks in the presence and absence of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast to the absence of an isoproterenol effect in freshly isolated hepatocytes, an increased sensitivity of glucose liberation towards isoproterenol could be observed 4 h after the start of culture, whereas the beta-receptor number was not found to be increased before 24 h. TPA has no effect on isoproterenol-stimulated glucose release at all investigated conditions. The alpha-1-adrenergic responses tested by using the alpha-1-adrenergic agonist phenylephrine is blocked completely in freshly isolated hepatocytes preincubated with 10(-6) M TPA. However, after 3 h incubation of hepatocytes in suspension or in primary culture, TPA had no effect on phenylephrine-stimulated glucose release. The effect of 10(-9) M glucagon on glucose release from freshly isolated hepatocytes was not influenced by TPA, whereas after 90 and 180 min incubation a significant decrease could be observed. On the other hand, TPA inhibited stimulation of adenylate cyclase activity by glucagon concentrations of 10(-5) M in freshly isolated hepatocytes, but no effect was found in hepatocytes incubated for 3 h in suspension or maintained for 24 h in primary culture. The different TPA effects may be an expression of changes of the accessibility of protein kinase C to TPA caused by translocation and/or intracellular activation of this enzyme at the tested experimental conditions.

Enzyme activities, isoenzyme pattern and alpha-1-adrenergic receptor number in primary cultured hepatocytes.

Changes in the activities of pyruvate kinase, tyrosine aminotransferase and adenylate cyclase as well as in the number of alpha-1-adrenergic receptors of hepatocytes maintained in primary culture were investigated. During the culture in the presence of insulin and dexamethasone the activity of tyrosine aminotransferase (TAT) increased. The increase was suppressed by 12-O-tetradecanoylphorbol-13-acetate (TPA). The basic activity of adenylate cyclase increased; however, a weaker stimulation of the enzyme by glucagon was found. A loss of stimulation of pyruvate kinase by fructose-1,6-bisphosphate may result from phosphorylation of the enzyme. The number of alpha-1-adrenergic receptors decreased during culture, an event not influenced by TPA.

Differences in the accessibility of the beta-adrenergic receptor in isolated hepatocytes from foetal and adult rats.

Beta-receptor number (measured by [3H]-CGP 12 177 binding) and beta-adrenergic response (measured by isoproterenol stimulated glucose liberation and isoproterenol stimulated adenylate cyclase activity) were compared in hepatocytes isolated from foetal (on day 22 of gestation), adult female and adult male rats. Beta-receptor numbers in crude membrane preparations of hepatocytes from adult female and adult male rats were found to be nearly equal (15.5 and 15.1 fmol/mg), but in crude membrane preparations of foetal rats beta-adrenergic receptor number was significantly higher (34.3 fmol/mg). Determination of number of beta-adrenergic surface receptors of intact hepatocytes showed relative high values in foetal rats (about 22,000/cell) and adult female rats (about 20,000/cell), but in male rats the number was less (about 6500/cell). Glucose liberation was stimulated by isoproterenol to the same extent in hepatocytes isolated from adult female and foetal rats (about 150% over basal), whereas no effect was found in hepatocytes isolated from adult male rats. Dose-response curves showed that in foetal rat hepatocytes glucose release was already increased by 10(-8) M isoproterenol, whereas in female rat hepatocytes at least 10(-6) M isoproterenol was required. Adenylate cyclase was stimulated by isoproterenol in lysates of hepatocytes from adult female rats by about 180% and from foetal rats by about 250%. No effects were observed using lysates of hepatocytes from adult male rats. We interpret the observed differences of beta-adrenergic responses between adult female and male rats as being primarily caused by different accessibility of the beta-receptor to the beta-agonist isoproterenol in intact hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent effects of phorbol ester on adenylate cyclase stimulation by glucagon in liver of female rats.

The stimulation of adenylate cyclase by glucagon and isoproterenol in cell lysates of hepatocytes isolated from fetal and adult female rats were measured after pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Glucagon stimulation of adenylate cyclase activation was found to decrease in the hepatocyte lysate of adult female rats. Application of guanyl-5'-yl-imidodiphosphate (GppNHp) and forskolin showed this effect to be localized on the receptor level. However, glucagon stimulation of glucose liberation from phorbol ester-treated hepatocytes from adult female rats was not influenced. Maximum effects of glucose liberation were observed at glucagon concentrations which did not stimulate adenylate cyclase. The results are in agreement with the proposed existence of a low and high affinity glucagon receptor coupled to two different transducing systems. It is concluded that TPA uncouples in the liver of adult female rats--most likely by phosphorylation--the low affinity receptor from the adenylate cyclase system, whereas the high affinity receptor and phospholipase C/inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) signalling systems do not seem to be affected. Such TPA effects could not be found in the liver of fetal rats.

Effect of hexadecane-induced vesiculation on the outer membrane of Acinetobacter calcoaceticus.

Lipopolysaccharide-rich vesicles were released from Acinetobacter calcoaceticus 69V during growth on hexadecane. Vesicle formation occurred over the whole surface of the cell as demonstrated by scanning electron microscopy. In contrast, the surface of acetate-grown cells, for which little lipopolysaccharide was found in the growth medium, appeared smooth. The overall chemical composition as well as the protein and phospholipid composition of the outer membranes of both cell types was very similar. In the vesicles all outer membrane proteins were found with the exception of an Mr 10,000 polypeptide corresponding to Braun's lipoprotein. Compared with the outer membrane, the vesicles contained more phosphatidylethanolamine. Hexadecane-grown cells were susceptible to exogenously added phospholipase. Nevertheless the barrier function towards lysozyme was retained.

Developmental changes of the alpha-1-adrenergic receptor system in rat liver.

The alpha-1-adrenergic receptor system in liver plasma membranes purified from rats of different ages were quantified by means of the [3H]prazosin binding assay. Maximum binding was found to increase with age. The binding constant, however, does not change significantly. Coupling of the receptor to the nucleotide-binding protein was studied by competitive displacement analysis of [3H]prazosin binding with norepinephrine (NE) in the presence and in the absence of guanyl-5'-yl-imidodiphosphate (GppNHp). GppNHp induced a shift in the NE-displacement curves to lower affinities in membranes of adult rat livers, but not in liver membranes prepared from 12-day-old rats. Computer analysis showed that in the adult liver the displacement curves fit a two site binding model in the absence of GppNHp and in its presence a one site model. In liver membranes of adult rats more than 70% of the alpha-1-receptor population are apparently in the high affinity form, whereas in 12-days old rats the percentage is only 30%. Protease inhibitors were found without effect on the displacement curves. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) blocks the stimulation of glycogenolysis by alpha-1-agonists in hepatocytes of adult rats without changing their binding capacity for [3H]prazosin. The liver of newborn rats strongly responds towards exogenous stimulation by alpha-1-agonists.