Dennis E. Rhoads

Chemoreception in Hydra vulgaris (attenuata): initial characterization of two distinct binding sites for l-glutamic acid

To elucidate the relationship between L-glutamic acid and the putative chemoreceptor for glutathione, binding of L-[3H]glutamate to a crude membrane fraction from Hydra vulgaris (attenuata) has been characterized. The binding of L-[3H]glutamate was rapid, reversible and saturable. A Scatchard analysis of the specific binding revealed values of 10 microM for the dissociation constant (Kd) and 170 pmol/mg for the maximal capacity of binding sites (Bmax). A maximum of 65% of the specific L-[3H]glutamate binding was inhibited by the chemostimulatory peptide, glutathione. This glutathione-sensitive glutamate binding presumably represents the association of glutamate with a putative chemoreceptor which modulates feeding behavior in hydra. The remaining 35% of the specific L-[3H]glutamate binding may be due to a second class of glutamate binding sites which is insensitive to glutathione. The identification of glutathione-insensitive glutamate binding is the first indication of a putative glutamate receptor, which may mediate an action independent of the glutathione-induced feeding response. The glutathione-insensitive and glutathione-sensitive sites must have similar affinities for glutamate since these sites were indistinguishable by Scatchard analysis. A preliminary characterization of the glutathione-insensitive site, performed in the presence of saturating levels of glutathione, revealed inhibition of glutathione-insensitive glutamate binding by kainate and quisqualate, but not by N-methyl-D-aspartate. A glutathione-insensitive L-[3H]glutamate binding suggests that kainate and alpha-aminoadipate may be selective ligands for the glutathione-insensitive and glutathione-sensitive glutamate binding sites, respectively.

Release of Immunoreactive Insulin from Rat Brain Synaptosomes Under Depolarizing Conditions

Abstract: Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization‐induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3–7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization‐independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.

Chemoreception in hydra: specific binding of glutathione to a membrane fraction

Specific binding of reduced [35S]glutathione (GSH) was measured using a crude membrane fraction of Hydra vulgaris (attenuata). The specific binding shows both rapid displaceable and nondisplaceable components. Rapid displaceable binding accounted for 20% of the total specific binding. Data from saturation binding experiments indicates half maximal total specific binding occurs at 2 microM GSH which is similar to reported EC50 values from behavioral experiments. Calcium is required for displaceable binding of GSH to the putative receptor. Oxidized glutathione (GSSG), an antagonist of the GSH-activated feeding response, and S-methylglutathione (GSM), an agonist of the feeding response, inhibit the binding of radiolabeled GSH to the putative receptor. Glutamate, a putative competitive antagonist of the GSH-activated feeding response in hydra, does not inhibit the specific binding of radiolabeled GSH to the receptor and must therefore block the feeding response by a mechanism other than competitive inhibition.

Severity of alcohol withdrawal symptoms depends on developmental stage of Long-Evans rats

To investigate alcohol dependency and the potential role of age of initial alcohol consumption, Long-Evans (LE) rats were fed an ethanol-containing liquid diet starting at postnatal (P) ages (days): P23-27 (juvenile), P35-45 (adolescent) or P65-97 (young adult). Severity of subsequent withdrawal symptoms was dependent on age when consumption began and on duration of alcohol consumption. Frequency of withdrawal seizures was highest for rats starting consumption as juveniles, intermediate for adolescents and lowest for adults. Normalized to body weight, alcohol consumption was significantly higher for adolescent and juvenile rats than for adults. Sprague-Dawley rats that began alcohol consumption as adolescents (P35) had a level of alcohol consumption identical to that of the adolescent LE rats but showed much lower frequency of withdrawal seizures when tested after 2, 3 and 5 weeks of alcohol consumption. Based on several indicators, the capacity of the juveniles to metabolize ethanol is equal to or exceeds that of adults. Recoveries from a single dose of ethanol (2.5 g ethanol/kg body weight) were faster for juvenile LE rats than adults. The rate of decline in blood ethanol concentration was identical for juvenile and adult rats while the corrected ethanol elimination rate was higher for juveniles. The primary isozyme of alcohol dehydrogenase (ADH) in rat liver, ADH-3, had a similar Km and higher activity in liver preparations from juveniles. In conclusion, LE rats beginning alcohol consumption as juveniles or adolescents develop a severe alcohol withdrawal syndrome that may not be attributed entirely to higher levels of consumption and was not explained by any obvious deficiencies in metabolism.

Effects of ethanol on Na+-dependent amino acid uptake: dependence on rat age and Na+, K+-ATPase activity

Acute effects of ethanol on Na(+)-dependent transport of gamma-aminobutyric acid (GABA) and glutamic acid (GLU) were investigated in crude synaptosomal preparations from rat cerebral cortex. In experiments with 30-40-day-old (peripubertal) rats, the overall dose responses of the GABA and GLU transport systems to ethanol were biphasic. Stimulation was observed at ethanol concentrations (40-160 mM) relevant to intoxication. Inhibition was observed at higher concentrations of ethanol. The stimulatory phase of the dose response was not observed in 60-100-day-old (adult) rats. In preparations from peripubertal rats, other alcohols also had biphasic dose response curves with stimulation at low alcohol concentrations. The relative efficacy of the different alcohols appeared to correlate with the relative membrane-buffer partition coefficient. In synaptosomal membrane vesicles, where artificial ion concentration gradients rather than Na+,K(+)-ATPase activity provide the driving force for uptake, ethanol did not stimulate GABA uptake. In direct measures of Na+,K(+)-ATPase activity, both Rb+ uptake and ATP hydrolysis were enhanced by 80 mM ethanol. We conclude that stimulation of Na(+)-dependent uptake of amino acids by ethanol was secondary to enhanced Na+,K(+)-ATPase activity and may be associated with a specific developmental stage in the rat.

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

Abstract: The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I‐insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin‐receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5‐1,000 nM, revealed binding site heterogeneity consistent with a two‐site model for insulin binding to a high‐affinity (KD= 6 nM), low‐capacity (Bmax= 110 fmol/mg of protein) site and a low‐affinity (KD= 190 nM), high‐capacity (Bmax= 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.

l-Alanine binding sites and Na+, K+-ATPase in cilia and other membrane fractions from olfactory rosettes of Atlantic salmon

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.

High-affinity Ca2+,Mg2+-ATPase in plasma membrane-rich preparations from olfactory epithelium of Atlantic salmon

High-affinity Ca2+,Mg(2+)-ATPase was identified in a plasma membrane-rich fraction of olfactory epithelium from Atlantic salmon (Salmo salar). The enzyme required both Ca2+ and Mg2+ for activation. The apparent Km for Ca2+ was 9.5 nM and Vmax was 0.85 mumol Pi/mg of protein per min. Stimulation by Ca2+ was optimal at 5-100 microM MgCl2. Bovine brain calmodulin had no effect on Ca2+,Mg(2+)-ATPase, even after multiple washes of the membrane preparation with EDTA or EGTA. Endogenous calmodulin was somewhat resistant to removal and could be detected with immunoblotting after multiple washes of the membrane preparation with EDTA or EGTA. This endogenous calmodulin may regulate Ca2+,Mg(2+)-ATPase activity because the activity was inhibited by calmidazolium. Vanadate inhibited Ca2+,Mg(2)-ATPase activity and thapsigargin, a specific inhibitor for Ca2+,Mg(2+)-ATPase of endoplasmic reticulum, had no effect on the enzyme activity. High affinity Ca2+,Mg(2+)-ATPase exists in both ciliary and nonciliary membranes with a similar Km for Ca2+. Ca2+,Mg(2+)-ATPase activity is greater in cilia preparations than in membranes from the deciliated olfactory epithelium. As a putative plasma membrane Ca2+ pump, this high-affinity Ca2+,Mg(2+)-ATPase may play an important role in the regulation of intracellular Ca2+ in olfactory epithelia. In particular, the ciliary membrane may play a prominent role in the removal of Ca2+ from ciliated olfactory receptor cells after odorant stimulation.

Stimulation of synaptosomal Na+,K+-ATPase by ethanol: possible involvement of an isozyme-specific inhibitor of Na+,K+-ATPase

In synaptosomal preparations from rat cerebral cortex, ouabain-sensitive Rb+ uptake was stimulated by ethanol (20-80 mM). Based on differential sensitivity to ouabain, 80% of this Na+,K(+)-ATPase activity represented activity of the alpha 1 isozyme while 20% was due to the alpha 2 and/or alpha 3 isozymes (alpha 2/ alpha 3). Stimulation of Na+,K(+)-ATPase was selective for the activity of alpha 2/alpha 3 which was increased by 167% in the presence of 80 mM ethanol. In this concentration range, ethanol had no effect on alpha 1 activity. Exposure of synaptosomal preparations to EGTA increased basal (no ethanol) alpha 2/alpha 3 activity with no effect on alpha 1 activity. Further, ethanol no longer stimulated alpha 2/alpha 3 activity after EGTA treatment. An EGTA extract was concentrated and desalted to yield a fraction that selectively inhibited alpha 2/alpha 3 activity when reconstituted with EGTA-treated synaptosomal preparations. This inhibition was trypsin-sensitive, suggesting protein involvement, and was prevented by 80 mM ethanol. In the presence of the inhibitory protein fraction, ethanol stimulated Na+, K(+)-ATPase activity in EGTA-treated membranes with a dose-response like that observed with the crude (no EGTA) synaptosomes. We propose that the alpha 2/alpha 3 activity of Na+,K(+)-ATPase is subject to inhibitory regulation and that ethanol stimulates this activity by releasing it from inhibition, an effect that may mimic in vivo deregulation of the enzyme by ethanol.

AFFINITY PURIFICATION OF HYDRA GLUTATHIONE BINDING PROTEINS

The association of glutathione (GSH) with putative external chemoreceptors elicits feeding behavior in Hydra. In the present study, solubilized membrane proteins were chromatographed on an affinity column of immobilized GSH in order to isolate GSH‐binding proteins that may represent the Hydra GSH chemoreceptor. The most abundant of the affinity‐purified proteins was a triplet of peptides ranging in molecular weight from 24.5–26 kDa. Antiserum generated against the 24.5–26 kDa triplet peptides inhibited GSH‐stimulated feeding behavior by 47%, implicating a role for one or more of these peptides in Hydra chemoreception.