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Biomedical subjects

A Jakob

Publications and source records attributed to A Jakob.

At least 19 recordsLinked to original sources
PubMed1996

Sensitization of stress-induced feeding in rats repeatedly exposed to brief restraint: the role of corticosterone.

Groups of male Wistar rats lived in cages capable of monitoring feeding and drinking continuously at 0.1-s intervals, 24 h per day. Intact animals were subjected to 20 min of restraint stress or to brief handling (Brief Pick-Up), daily or every third day, 6 h after the beginning of the 12-h light period. In both studies, food-intake increased in the first hour after restraint, peaking between 15 and 45 min. Smaller increases were seen following Brief Pick-Up. More interestingly, the amount of food eaten increased across test sessions, indicating sensitization of the response to stress. Drinking also increased following stress, occurring before feeding and diminishing after the first 15 min. In adrenalectomized animals implanted with slow-release pellets to replace basal diurnal levels of corticosterone (ADX animals), sensitization of the feeding response to restraint stress developed across test sessions, although in these animals, the acute increase in food-intake following restraint stress was attenuated. ADX animals subjected only to Brief Pick-Up showed no increases in food-intake. Daily injections of 3.0 mg/kg corticosterone given to such ADX animals were unable to mimic the effects of restraint on either food-intake or drinking, nor did they augment the effects of restraint in ADX animals. We conclude that sensitization to the effects of brief restraint stress on food-intake can occur independently of a stress-induced rise in plasma corticosterone.

Adrenalectomy↗
PubMed1994

Sexual stimulation induces Fos immunoreactivity within GnRH neurons of the female rat preoptic area: interaction with steroid hormones.

We have shown previously that sexual stimulation (copulation with intromission or vaginocervical stimulation) induces c-fos mRNA and Fos-like immunoreactivity (IR) within estrogen-concentrating and nonconcentrating regions of the female rat forebrain, including regions that contain gonadotropin-releasing hormone (GnRH) neurons in septum and anterior preoptic area. The overall induction of Fos-like IR within these regions was specific to afferent sensory stimulation and did not require treatment with estrogen and progesterone. Because vaginocervical stimulation facilitates lordosis and increases the release of luteinizing hormone, the present study examined whether hormone treatment that promotes sexual receptivity, with or without sexual stimulation, increases Fos-like IR specifically within GnRH-containing neurons. Sexually experienced ovariectomized rats were administered estradiol benzoate (10 micrograms) 48 h and progesterone (500 micrograms) 4 h before either 1 h of paced copulation with a sexually vigorous male, 50 vaginocervical stimulations with a glass rod distributed over 1 h, or no stimulation. Control rats received injections of the oil vehicle. Fos-like IR was found within a significant number of GnRH-positive neurons in the anterior preoptic area caudal to the organum vasculosum following copulation with intromission or vaginocervical stimulation as compared with no stimulation. Although few GnRH cells coexpressed Fos following hormone treatment alone, this treatment enhanced the number of GnRH neurons that coexpressed Fos following vaginocervical stimulation as compared with the effect of vaginocervical stimulation in oil-treated rats. Together, these data indicate that estrogen and progesterone can augment the responsiveness of certain GnRH neurons to vaginocervical stimulation, consistent with the effects of sexual activity on GnRH release.

Animals↗
PubMed1993

Catalytic mechanism of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase. The use of synthetic analogues to probe the structure of the putative reaction intermediate.

The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate. Two 2-deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring-opening and possessing the stereochemistry of either the alpha-pyranase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 microM and 303 microM, respectively. Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki = 590 microM) suggests that both the alpha- and the beta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-anomer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable. More importantly, however, the isosteric phosphonate analogue 2,6-anhydro-3-deoxy-2 beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki value of 5 microM. Two hitherto unrecognized aspects of the mechanism of the synthase were identified. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open-chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme. Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenolpyruvate to the re face of the carbonyl of arabinose 5-phosphate, the present observation involving analogue 3 suggest that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the beta-configuration.

Aldehyde-Lyases↗
PubMed1993

The development of behavioral sensitization to apomorphine is blocked by MK-801.

The effects of the non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), on the development of sensitization to the mixed dopamine receptor agonist, apomorphine were examined. Rats pretreated with MK-801 (0.25 mg/kg) prior to 2.0 mg/kg apomorphine failed to show a progressive sensitization to the activating effect of apomorphine across six preexposure sessions, nor did they show enhanced activity relative to control rats in response to 0.5 mg/kg apomorphine, given alone, during a subsequent test for sensitization. These results support the view that MK-801 interferes with long-term neural changes underlying sensitization to stimulant drugs.

Animals↗
PubMed1992

Vasopressin stimulation of vanadate-sensitive Na+ transport by liver plasma membrane vesicles. Evidence for regulation via phospholipase C and protein kinase C activities.

The rate of vanadate-sensitive 22Na+ uptake by isolated liver membrane vesicles, reflecting transport by Na+/K(+)-ATPase, was measured to study the role played by phospholipase C and protein kinase C in the regulation of this process by vasopressin. Na+ uptake was enhanced 2-3-fold by 100 nM [Arg8]vasopressin and the hormone effect was mimicked by 0.1 microM inositol 1,4,5-trisphosphate as well as by 1.0 microM myo-inositol. The stimulation by vasopressin was potentiated by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis (5-10 mU/ml). No effect of the bacterial enzyme was observed in the absence of the hormone. Phorbol myristate acetate (0.5-1 microM) suppressed the stimulation by vasopressin but had no effect in the absence of the hormone. High concentrations of bacterial phosphatidylinositol-specific phospholipase C (50-100 mU/ml) also antagonized the hormone stimulation. Staurosporine (50-100 nM) prevented the antagonistic effect of bacterial phospholipase C (50 mU/ml) and EGTA (1 mM) partially protected the hormonal stimulation in the presence of phorbol myristate acetate. Our results suggest that the stimulatory effect of vasopressin on Na+ transport is mediated by phospholipase C and products derived from the inositol moiety of membrane phospholipids. Membrane-associated protein kinase C appears to be at least partially responsible for the desensitization to stimulation by vasopressin.

Animals↗
PubMed1992

Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli.

The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated. The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate. The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor. First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction. The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate). These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.

Aldehyde-Lyases↗
PubMed1991

Effect of hypothyroidism and thyroid hormone replacement on the level of protein kinase C and protein kinase A in rat liver.

We investigated the influence of the thyroid hormone status on the levels of protein kinases C (PKC) and A (PKA) in the soluble fraction of rat liver. The immunodetectable PKC level in hypothyroid liver was elevated 7.7-fold, whereas the phorbol-ester binding capacity and the immunodetectable alpha-PKC level were increased 2.4- and 2.6-fold, respectively. Conversely, in hypothyroid livers the abundance of the regulatory type I and the catalytic subunits of PKA were lowered to 42% of the euthyroid level as determined by immunoblotting and by measuring the substrate specific phosphorylation rate of PKA. These changes in the PKC and PKA levels were reversible upon treatment with 0.5 microgram T4/100 g body weight for 2-21 days. The thyroid state dependent alterations in hepatic PKC and PKA levels may be responsible for the known changes in the response of hepatocytes to other hormonal stimuli in hypothyroidism.

Animals↗
PubMed1991

Enzymatic properties of phosphatidylinositol-glycan-specific phospholipase C from rat liver and phosphatidylinositol-glycan-specific phospholipase D from rat serum.

Using phosphatidylinositol-glycan (PtdIns-glycan) anchored acetylcholinesterase from bovine erythrocytes as substrate, we found PtdIns-glycan-anchor-degrading activity in rat liver and serum [corrected]. The hepatic enzyme was only soluble in detergents, whereas the serum enzyme occurs as soluble, slightly amphiphilic protein. Using 3-trifluoromethyl-3-(m- [125I]iodophenyl)diazirine-labelled acetylcholinesterase as substrate, we showed that the hepatic anchor-degrading enzyme had a cleavage specificity of a phospholipase C, whereas the serum enzyme was a phospholipase D. Both enzyme exhibited maximal activity in slightly acidic conditions and at low ionic strength. They had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 0.1 microM and 0.16 microM, respectively). Both hepatic PtdIns-glycan-specific phospholipase C and serum PtdIns-glycan-specific phospholipase D gave a large increase in activity between 0.1-10 microM Ca2+, indicating that PtdIns-glycan-specific phospholipases are only marginally active at physiological intracellular Ca2+ concentrations. The enzymes were inhibited by heavy metal chelating agents such as 1,10-phenanthroline and 2,2'-bipyridyl but not by the corresponding Fe2+ complexes or non-chelating analogues, indicating that they both require a heavy metal ion for the expression of catalytic activity in addition to Ca2+. Another interesting property of PtdIns-glycan-specific phospholipases is their inactivation by bicarbonate and cyanate. The inactivation was time- and pH-dependent and could be reversed by dialysis. These observations are in agreement with a covalent modification of the enzymes by carbamoylation.

Acetylcholinesterase↗
PubMed1990

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles.

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.

Adenosine Triphosphate↗
PubMed1989

Alpha 1-adrenergic stimulation causes Mg2+ release from perfused rat liver.

The possibility that Mg2+ mobilization is stimulated in perfused liver by alpha 1-adrenergic agonists was studied by measuring Mg2+ release in response to 0.5 and 20 microM phenylephrine. During preperfusion exogenous Mg2+ was added to the medium to give 1.2 mM. 5 min before starting the addition of phenylephrine the infusion of exogenous Mg2+ was stopped. Mg2+ in the perfusate leaving the liver was measured by atomic absorption spectroscopy. Analysis of the Mg2+ decay curves with two exponential models indicated that phenylephrine caused dose-dependent Mg2+ release from perfused rat livers.

Animals↗
PubMed1988

Enhancement of the antiproliferative effect of cis-diamminedichloroplatinum(II) and nitrogen mustard by inhibitors of protein kinase C.

Quercetin (3,3',4',5,7-pentahydroxyflavone) has been shown to inhibit a variety of enzymes including the calcium- and phospholipid-dependent protein kinase (protein kinase C) in vivo and in vitro. We show that this compound synergistically enhances the antiproliferative activity of cis-diamminedichloroplatinum(II) (cis-DDP) and nitrogen mustard. Quercetin does not affect the repair of DNA interstrand cross-links introduced by cis-DDP. Long-term exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA), which reduces total protein kinase C activity, also amplifies the growth-inhibitory effect of cis-DDP and acts synergistically with quercetin. A synergism is also observed if tamoxifen or staurosporine are combined with cis-DDP. For both drugs the dose-effect curves for the inhibition of protein kinase C closely resemble the dose-effect curves for the antiproliferative activities. Although alternative mechanisms cannot be definitively excluded, the effects of quercetin, TPA, tamoxifen and staurosporine may result from the inhibition of protein kinase C.

Alkaloids↗
PubMed1985

The hepatic response to Ca2+ is inhibited by Mg2+ and enhanced by phenylephrine or ouabain.

Net hepatic Ca2+ efflux, K+ uptake and glycogen breakdown in response to the alpha 1-adrenergic agonist phenylephrine were studied. Rat livers were perfused with CO2/bicarbonate-buffered solutions containing 10 microM Ca2+ and different amounts of Mg2+. K+-free medium and/or ouabain were used to block (Na+ + K+)-ATPase-dependent K+ uptake. In some experiments a sharp increase in extracellular Ca2+ concentrations was produced by infusing CaCl2 into the medium entering the liver. Perfusion with K+-free medium and ouabain enhanced the phenylephrine-induced Ca2+ efflux and diminished the glycogenolytic response, indicating a dissociation of Ca2+ release and glycogenolysis. Exogenous Ca2+ had practically no effect if livers were perfused with regular medium containing 1.2 mM Mg2+. In the presence of phenylephrine and if extracellular Mg2+ concentrations were lowered by omitting Mg2+ from the medium or by preperfusion with EGTA, exogenous Ca2+ was glycogenolytically effective and also produced a transient K+ uptake. Increased extracellular concentrations of Mg2+ inhibited the effects of exogenous Ca2+. In the presence of phenylephrine, higher concentrations of Mg2+ were needed than in the absence of alpha 1-adrenergic agonist to achieve a similar degree of inhibition. In one respect ouabain effects were comparable to those of phenylephrine: the glycoside also increased the metabolic response to exogenous Ca2+ and diminished the sensitivity towards Mg2+. Phenylephrine and ouabain may both enhance the permeability of plasma membranes for Ca2+.

Animals↗
PubMed1982

alpha-Adrenergic stimulation of glycolysis and Na+, K+-transport in perfused rat liver.

Interactions between alpha-adrenergic-induced lactate production, respiration and K+ uptake of perfused rat livers were investigated. Inhibition of mitochondrial electron transport by cyanide had no effect on K+ uptake, decreased phenylephrine-induced lactate production by 60% and suppressed extra O2 consumption by more than 90%. Ouabain did not affect alpha-adrenergic-induced respiration but inhibited K+ uptake and extra lactate production. Effects of ouabain and K+-free medium on lactate production were additive and independent of cyanide, indicating that they were mediated by extramitochondrial mechanisms. Extra ATP utilized for K+ uptake in response to phenylephrine was apparently of glycolytic rather than mitochondrial origin. We propose that alpha-adrenergic-induced lactate production is secondary to an enhanced operation of the plasma membrane (Na+,K+)-ATPase.

Animals↗
PubMed1982

Effects of ethacrynic acid on the alpha-adrenergic control ov hepatic glycogenolysis.

Ethacrynic acid (ECA) was used to study the relationship between ion transport and alpha-adrenergic activation of glycogenolysis in perfused rat livers. ECA alone enhanced glycogenolysis and produced a massive loss of K+ from the liver. These effects were partially blocked by dithioerythritol. ECA suppressed the metabolic responses to phenylephrine and the alpha-adrenergic redistribution of ions. Ouabain-sensitive and ouabain-insensitive ATPase of isolated liver plasma membranes were inhibited and binding of [3H]epinephrine to alpha-adrenergic sites was decreased by ECA. It is concluded that in liver ECA acts at three different sites by blocking SH groups: active ion transport across the plasma membrane, alpha-adrenoreceptors, and phosphorylase phosphatase.

Animals↗
PubMed1980

Ca2+, K+ redistributions and alpha-adrenergic activation of glycogenolysis in perfused rat livers.

1. The alpha-adrenergic activation of glycogenolysis was investigated in isolated rat livers perfused in a non-recirculating system. Net uptake and/or release of Ca2+, K+ and H+ by the liver (measured by ion-selective electrodes) were correlated with the glycogenolytic effects of phenylephrine. Uptake and retention of 45Ca by the mitochondria of perfused livers were studied to obtain information on the role played by exchangeable mitochondrial calcium in alpha-adrenergic activation of glycogenolysis. 2. Between 1 and 5 min after starting the addition of phenylephrine a net release of Ca2+ was observed, this was paralleled by an uptake of K+. Production rates of glucose and lactate from endogenous glycogen started to increase at the same time. During the following minutes K+ was released. 2 mM EGTA and a high concentration of Mg2+ strongly diminished the ionic and metabolic responses to phenylephrine, 0.2 mM EGTA was less effective. 3. High concentrations of K+ prevented the metabolic response to phenylephrine but had no effect on the release of Ca2+ into the extracellular medium. Tetracaine activated glycogenolysis and suppressed all the effects of the alpha-adrenergic agonist. 4. Experiments with 45Ca provided no evidence for an alpha-adrenergic release of Ca2+ from the exchangeable mitochondrial pool. Incorporation of 45Ca into the mitochondria of perfused livers was enhanced by phenylephrine. 5. We propose that the alpha-adrenergic release of Ca2+ from a pool located close to the surface of the cell is capable of triggering the glycogenolytic response.

Adrenergic alpha-Agonists↗