Renal response to amino acid infusion in rats: effect of dopamine receptor antagonists and benserazide

Previously, we have found that feeding is a dominant factor controlling urinary dopamine excretion (U_DA) in conscious rats (Mühlbauer and Osswald 1992). Since the renal response to feeding is also characterized by an increase in glomerular filtration rate (GFR), we wanted to investigate in a first step whether the feeding-induced elevations of GFR and U_DA could be causally related phenomena. Therefore, we studied the influence of dopamine synthesis and dopamine receptor blockade on the renal response to amino acid infusion (AA) in thiopental anesthetized rats. AA infusion (n = 7) increased GFR by 33±7% (P<0.001) and U_DA by 87±19% (P<0.001). In the presence of benserazide (BZD, n = 5), an inhibitor of dopamine synthesis, infused i.v. at a dose of 30 g/min/kg, U_DA was suppressed to values below detection limit and the AA-induced GFR increase was abolished. Continuous intravenous infusion of the DA₁ receptor antagonist SCH 23390 (SCH, n = 7) in a dose of 4.0 g/kg/min did not prevent the AA-induced increase in GFR (33±3%, P<0.001) and U_DA (97±12%, P< 0.001). In contrast, S-sulpiride (SUL), a specific DA₂ receptor antagonist, infused continuously i.v. in a dose of 5 g/kg/min, completely abolished the AA-induced GFR increase, while U_DA was increased 1.6-fold (P<0.01). Like BZD, both dopamine receptor antagonists did not affect renal sodium excretion substantially.Our results suggest, that endogenous dopamine could act as a mediator in the renal response to amino acid infusion in the rat, most likely by activation of DA₂ receptors. Correspondence to:B. Mühlbauer at the above address     

Baclofen,a gamma-aminobutyric acid-b receptor agonist,delays diabetes onset in the non-obese diabetic mouse

Glutamic acid decarboxylase (GAD) is the enzyme responsible for the synthesis of gamma-aminobutyric acid (GABA). GAD has been identified as a 64-kDa antigen expressed in pancreatic beta-cells, to which autoantibodies are generated prior to the onset of type 1 (insulin-dependent) diabetes mellitus. GAD may therefore be an initiating factor in beta-cell destruction. We administered baclofen, a GABA-B receptor agonist, to non-obese diabetic (NOD) mice in an attempt to down-regulate GAD expression and thereby reduce the incidence of diabetes. Twenty-four female NOD mice were given baclofen in their drinking water at a final dose of 50 mg/kg body weight daily from weaning to 30 weeks of age. Twentyfour sex-and litter-matched mice were used as controls. At 30 weeks there was no difference in the incidence of diabetes in the treated group compared with the controls. However, there was a significant delay in the onset of diabetes in the treated group (P<0.001, parallelism test). The degree of insulitis and the GAD activity in the pancreas per mg of protein were unchanged by baclofen treatment with respect to controls. These results suggest that baclofen may be effective in delaying diabetes onset in NOD mice by stimulating GABA activity, as this neurotransmitter, localised in the islets, may modulate insulin secretion and the antigen expression associated with it.     

A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOA^R), on the average 60% of the 5-FOA^R colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5 end of the pyrG gene with vectors containing a mutation near the 3 end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.     

Molecular Human Reproduction: Evidence for {gamma}-aminobutyric acid specific binding sites on human spermatozoa

The possible presence of -aminobutyric acid (GABA) specificbinding sites on human spermatozoa was investigated. Swim-uppreparations of human spermatozoa were incubated with radiolabelledGABA in the presence of unlabelled GABA, alternatively displacersof GABA_A/B receptors and GABA transport proteins. The resultsindicate that GABA specific binding sites are present on thesurface of human spermatozoa, and that these binding sites possiblyindicate the presence of GABA transport proteins. Furthermore,GABA at different concentrations was added to swim-up preparationsof human spermatozoa. Possible effects of GABA on sperm motility,hyperactivation and acrosome reaction were explored. No significantdifferences were observed between treated groups and controlsconcerning motility parameters and hyperactivation. Incubationwith GABA did not cause any increase in spontaneous acrosomereaction. However, spermatozoa treated with the calcium ionophoreA-23187 showed a small but significantly increased ability toundergo the acrosome reaction following preincubation in 10–⁴M GABA (P < 0.05).     

Rapid appearance of β-amyloid precursor protein immunoreactivity in glial cells follwing excitotoxic brain injury

Clinical and experimental data have indicated an up-regulation of amyloid precursor protein (APP) after various types of CNS injury. In the present study the cellular source of lesion-induced APP has been investigated an a neurotoxic CNS model. Quinolinic acid injection into the striatum results in neuronal degeneration, while glial cells survive. APP immunoreactivity was detected in glial cells starting at postoperative day 3 and persisted until day 21, the last time point studied. Double immunocytochemistry identified the majority of APP-immunoreactive cells as glial fibrillary acidic protein-immunoreactive astrocytes. There was no evidence of amyloid fibril deposition during this time. It is concluded that following excitotoxic neuronal degneration APP is mainly produced by reactive astrocytes in the lesioned area.     

Inhibition of nitric oxide synthase dramatically potentiates seizures induced by kainic acid and pilocarpine in rats

We investigated whether the severity of convulsions evoked by kainic acid and pilocarpine is modified in nitric oxide synthase inhibitor-treated rats. We found that chronic treatment (4 days) withN_w-nitro-l-arginine greatly potentiates seizures induced by both convulsants suggesting a potential role for nitric oxide in mechanisms regulating seizure induction and propagation.     

Excitatory amino acid release from astrocytes during energy failure by reversal of sodium-dependent uptake

Non-synaptic release may be the major route of excitatory amino acid (EAA) efflux during cerebral ischemia. Possible routes of non-synaptic release include non-specific anion channels, reversal of Na⁺-, CI^?-, or Ca²⁺-dependent uptake, and cell lysis. In the present study we employ a novel approach to show reversal of Na⁺-dependent uptake as a major route of EAA efflux from astrocyte cultures under conditions of energy failure. Primary rat astrocyte cultures were subjected to combined blockade of glycolytic and oxidative metabolism after incubation with [³H]-D-aspartate (D-ASP). Energy failure produced an efflux of D-ASP that was maximal by 90 minutes. The efflux over this period was reduced by more than 50% in cells that had been pre-loaded with PDC (L-transpyrrolidine-2,4-dicarboxylic acid) or TBHA (threo-β-hydroxyaspartic acid), compounds that are competitive inhibitors of Na⁺-dependent glutamate uptake. The effect of pre-loading with the inhibitors was concentration dependent. No effect was seen if the inhibitors were added after induction of energy failure, suggesting that the attenuation of D-ASP efflux resulted from binding of the inhibitors to an intracellular site. These results provide strong evidence that EAA efflux from astrocytes under conditions of energy failure occurs largely through reversal of Na⁺-dependent uptake. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Synaptic organization of excitatory and inhibitory boutons associated with spinal neurons which project through the dorsal columns of the cat

The cell bodies and proximal dendrites of postsynaptic dorsal column neurons were examined for synaptic boutons which displayed immunoreactivity for the principal excitatory and inhibitory neurotransmitters, glutamate and GABA. The neurons were labelled by retrograde transport of horseradish peroxidase and GABA or glutamate-containing boutons were revealed by performing postembedding immunogold reactions on electron microscope sections. Five neurons were examined and all of them were postsynaptic to boutons which contained either GABA or glutamate. Quantitative analysis of two of the cells revealed that more than 90% of the synaptic profiles associated with them displayed immunogold reactions for these transmitters. Analysis of series of alternate sections, which were reacted for either GABA or glutamate, showed that there was no overlap in the populations of immunoreactive boutons. Furthermore, GABA and glutamate immunoreactions were associated with boutons which had different morphological characteristics. In addition, some large glutamate-enriched boutons were postsynaptic to small boutons which displayed immunogold reactions for GABA. This study demonstrates morphological bases for direct excitation, postsynaptic inhibition and presynaptic inhibition of postsynaptic dorsal column cells.     

Hydroxypropyl-β-Cyclodextrin Increases the Aqueous Solubility and Stability of Pilocarpine Prodrugs

Purpose. The effects of 2-hydroxypropyl--cyclodextrin (HP--CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. Methods. The solubility of prodrugs was studied by phase-solubility method (0–72.5 mM HP--CD). The stability of one of the prodrugs was investigated as a function of temperature (40°C–70°C) and HP--CD concentration (0–72.5 mM). The apparent rate constants (k ₁, k ₂) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K _1:1, K _l:2) were calculated by the curve-fitting method. Results. The phase-solubility diagrams were classified as A_p-type and the apparent stability constants (K _l:l, K _l:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143–815 M^–l and 29–825 M^–1, respectively. The stability of prodrug increased as a function of HP--CD concentration over the studied temperature range. The shelf-life (t _90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP--CD solution increased 5.1-fold and 6.1-fold at 25°C and 4°C, respectively. Conclusions. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the l:2-complex than in the l:l-complex and the relative amounts of complex species were found to be dependent on CD concentration.