Negative Modulatory Effect Research Articles

Allosteric modulation of [ 35S]TBPS-binding in the cerebral cortex of the rat during postnatal development

The ontogenesis of the GABA-gated Cl − channel was investigated in the cerebral cortex of the rat by monitoring the binding parameters of [ 35 S]t- butylbicyclophosphorothionate ([ 35S]TBPS) at intervals after birth (1–90 days). To investigate the influence of the developmental changes in the content of GABA on [ 35S]TBPS-binding, the assays were carried out in unwashed membranes, in which the concentration of GABA was dependent on its content in vivo, and in repeatedly washed membranes in the presence of defined concentrations of exogenous GABA. At birth, the density ( B max) of [ 35S]TBPS-binding sites in unwashed membranes was similar to that found in well-washed membranes. However, in unwashed membranes, the number of [ 35S]TBPS-binding sites increased by two-fold within 10 days after birth whereas in washed membranes it increased by four-fold during the same period. The higher density of [ 35S]TBPS-binding sites in washed membranes as compared with the unwashed counterparts persisted throughout development. In unwashed membranes, the apparent K d for [ 35S]TBPS-binding increased with age whereas in washed membranes the affinity of [ 35S]TBPS for its binding sites remained constant throughout development. The binding of [ 35S]TBPS to the GABA-gated Cl − channel is allosterically modulated by drugs acting on different sites of the GABA A receptor complex. Thus, GABA and diazepam decrease [ 35S]TBPS-binding whereas the GABA A receptor antagonist, bicuculline, and the inverse agonist for benzodiazepine receptors, 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid methyl ester, increase it. The magnitude of the effects of positive and negative modulators of the GABA A receptor on [ 35S]TBPS-binding did not change significantly from birth through adulthood, provided the concentration of GABA in the incubation medium was kept constant at 3 μM. These results reflect the early development of the functional interactions between the different components of the GABA A receptor.

Selective protection by adenosine receptor agonists against DMCM-induced seizures

The anticonvulsant actions of the adenosine receptor agonists, 1-phenylisopropyladenosine, 2-chloroadenosine and cyclohexyladenosine, against DMCM (methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate)-induced seizures in mice were studied with an infusion technique. 2-Chloroadenosine and cyclohexyladenosine were active at 1 mg/kg whereas l-phenyl-isopropyladenosine was active at 0.03 mg/kg given i.p. At 10 mg/kg, 1-phenylisopropyladenosine was only weakly active against pentylenetetrazol-induced seizures and not active against bicuculline-induced seizures. The selective effect of l-phenylisopropyladenosine against DMCM-induced seizures suggests that adenosine receptor agonists may allosterically counteract the negative modulating effect of DMCM on GABA coupling to the chloride channel. This indicates that adenosine receptors may have a physiological function within the GABA/benzodiazepine receptor complex in the brain.

Effects of calcium on the vasopressin-sensitive cAMP metabolism in medullary tubules

The modulatory effect of Ca on [Arg8]vasopressin-dependent (AVP) cAMP metabolism was studied in medullary collecting tubules (MCT) and medullary ascending limbs (MAL) microdissected from rat kidney. In MCT segments incubated in vitro with AVP, the accumulation of cAMP was enhanced (delta +59%) when Ca was omitted from the incubation medium compared with a medium with 2 mM of ionized calcium (Ca2+). Ionophore A23187 caused a decrease in AVP-stimulated cAMP accumulation in MCT in the presence of 2 mM Ca2+ but not in a Ca2+-free medium. Diltiazem and verapamil enhanced the AVP-stimulated cAMP accumulation in MCT; PTH had no detectable effect. A23187 caused a dose-dependent inhibition of cAMP accumulation stimulated by AVP with forskolin in both MCT and in MAL. However, in MAL the A23187 concentration needed for half-maximum inhibition (6.3 X 10(-6) M) was higher than for MCT (3.9 X 10(-7) M). The maximum inhibition in MAL (-65%) was less than in MCT (-97%). In the presence of 3-isobutyl-1-methylxanthine, AVP-stimulated cAMP accumulation was inhibited by A23187 in MCT (-45%) but not in MAL. Naproxen or ibuprofen did not relieve the inhibitory action of A23187 in MCT. Added Ca2+ inhibited the AVP-stimulated adenylate cyclase in MCT and MAL (half-maximum approximately equal to 5 X 10(-4) M Ca2+) and stimulated cAMP phosphodiesterase (cAMP-PDIE) in both MCT and in MAL (half-maximum approximately equal to 9 X 10(-5) M Ca2+). Incubation of MCT and MAL with A23187 decreased (-50%) the content of ATP. Results suggest that increased influx of extracellular Ca2+ inhibits the AVP-stimulated cAMP accumulation in MCT and to a much lesser degree in MAL. Deceased cAMP accumulation in MCT is probably due to both stimulation of cAMP-PDIE and the inhibition of adenylate cyclase, whereas in MAL it is due to stimulation of cAMP-PDIE. The results suggest that Ca2+ influx exhibits a negative modulatory effect on AVP-dependent cAMP metabolism mainly in MCT.

Evidence for a negative modulating effect of erucic acid on the peroxisomal β-oxidation enzyme system and biogenesis in rat liver

In male rats fed a 30 cal% high-erucic acid (22:1 (13) ( cis)) rapeseed oil diet for 4 weeks a transient, small increase in peroxisomal β-oxidation was observed in liver homogenates and isolated peroxisomes after approximately 1 week. Morphometric analysis revealed a progressive decrease in the average size of the liver peroxisomes (by approx. 20%), as well as their volume fraction (by more than 40%). A negative dose-response was observed when peroxisomal β-oxidation was determined in animals fed rapeseed oil diets with a variable content of erucic acid. When erucic acid was given as subcutaneous injections the peroxisomal β-oxidation decreased, in both liver homogenates and isolated peroxisomes. In contrast to recent proposals, our findings indicate that the amount of erucic acid cannot account for the small increase in peroxisomal β-oxidation observed in the liver of rats adapted to a diet containing 30 cal% rapeseed oil with a high content of this fatty acid. In fact, by the selected criteria erucic acid seems to have a negative modulating effect on both the peroxisomal β-oxidation enzyme system and biogenesis.