Metabolism of [3H]Nipecotic Acid in the Rabbit Retina

Nipecotic acid has been demonstrated to block the gamma-aminobutyric acid transport systems. Previous studies have shown that the uptake system is the first transmitter-specific parameter to appear during the development of the rabbit retina. Use of these observations has been made to study the influence on the development of gamma-aminobutyric acid receptors of altering the uptake mechanism by treating newborn pups with nipecotic acid to block GABA transport. The present study of the in vivo metabolism of [3H]nipecotic acid in the CNS measured the changes in the levels of [3H]nipecotic acid in both adult and newborn rabbit retinas after injection of the label into the vitreal chamber. It was found that the effective half-life of [3H]nipecotic acid in the vitreous is about 5 h for adult tissue and 3 h for newborn. In contrast, all retinal fractions retained the label longer, the effective half-lives being about 60 h (adult) and 45 h (newborn). Further, no labeled metabolites of nipecotic acid were detected in either adult or newborn tissue. This study gives evidence that the degradation of nipecotic acid in nervous tissue is minimal and suggests that, although the rate of clearance is faster in neonates, the fate of nipecotic acid in vivo may be similar in both adult and newborn tissues.     

Characterization of Serotonin Transporter in Goldfish Retina by the Binding of [3H]Paroxetine and the Uptake of [3H]Serotonin: Modulation by Light

Abstract: The serotonin (5-HT) uptake system of goldfish retina was evaluated by the binding of [³H]paroxetine to membrane preparations and the uptake of [³H]5-HT into isolated cells from goldfish retina. The order of potency of inhibitors of [³H]paroxetine binding was imipramine > 5-methoxy- N,N -dimethyltryptamine > desipramine > fluoxetine > citalopram > 5-HT. The saturation experiments indicated a high-affinity binding site, and positive cooperativity with Hill coefficient higher than unity. The association reached equilibrium at about one hour of incubation and was efficiently displaced by imipramine. The equilibrium dissociation constants calculated by the antilog of the log concentration of ligand giving 50% of occupation, and by the ratio of dissociation/association constants, were similar: 5.84 and 2.34 n M , respectively. The binding was not significantly reduced by decreasing the temperature of incubation and was sodium dependent. The lesion with 5,7-dihydroxytryptamine reduced the binding to 60%. The uptake of [³H]5-HT into isolated cells also showed positive cooperativity. The order of potency of inhibitors was similar to the one obtained for the binding of [³H]paroxetine. Darkness increased the uptake of 5-HT. The allosteric regulation of the 5-HT transporter and the modulation by light could be related to the physiological role of the monoamine, as a neurotransmitter and as a precursor of melatonin synthesis in the retina.     

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

Uptake, Release, and Metabolism of D- and L-α-Aminoadipate by Rat Cerebral Cortex

Abstract: Accumulation of L-α-aminoadipate by rat cerebral cortical slices is a stereospecific and Na⁺-dependent process. The uptake of this compound is also temperature-dependent, with a K_m , of 1.6 × 10⁻⁴M for the high-affinity system. D-α-Aminoadipate has characteristics similar to those displayed by the L-isomer but to a lesser degree. L-Glutamate and L-aspartate inhibit the uptake of L-α-aminoadipate. D- and L-α-Aminoadipate are, respectively, weak uncompetitive and weak competitive inhibitors for the uptake of L-glutamate and L-aspartate. Both enantiomers inhibit GABA uptake but in quite different ways. The release of L-α-aminoadipate from the cerebral cortical slices is stimulated by a high concentration of K⁺ ions in the presence of Ca²⁺ in the perfusion buffer; the D-isomer displays this property to a lesser degree. The omission of Ca²⁺ markedly reduces the release of these two compounds. Less than 10% of the preloaded D- and L-α-aminoadipate are metabolized by the cerebral cortex during 40 min of superfusion. The possibility of L-α-aminoadipate as a neurotransmitter candidate is discussed.     

Presence of Radiolabelled Metabolites in Release Studies Using [3H]γ-Aminobutyric Acid

Abstract: Most studies on γ-aminobutyric acid (GABA) release from nervous tissue have been conducted using radiolabelled GABA in the presence of aminooxyacetic acid (AOAA) to inhibit GABA: 2-oxoglutarate aminotransferase (GABA-T) to prevent conversion of labelled GABA to labeled catabolites. Here we present data showing that even in the presence of 10 μM-AOAA the spontaneous release of tritium from rat cortical synaptosomes prelabelled with 2,3-[³H]GABA is mainly in the form of tritiated water but that the increase in tritium release in the presence of unlabelled GABA or high potassium-ion concentrations is in the form of authentic [³H]GABA. Interpretation of results should take these facts into account.     

Comparison of the Binding of [3H]Spiperone and [3H]Domperidone in Homogenates of Mammalian Retina and Caudate Nucleus

Abstract— The specific binding of [³H]spiperone and [³H]domperidone, as defined by 1 μ m -(+)butaclamol, was compared in homogenates of bovine retina and caudate nucleus. Scatchard analyses of saturation data for [³H]spiperone binding yielded dissociation constants ( K _d) of 0.35 n m in the retina and 0.64 n m in the caudate nucleus. Comparison of the maximum number of binding sites (B_max) present in each tissue indicated that the density of sites in bovine caudate nucleus (270 fmol/mg protein) was approximately three times higher than in bovine retina (92 fmol/mg protein). This difference was even more marked in guinea pig tissues, with a ratio of 7:1 between corpus striatum and retina. The pharmacological analysis of [³H]spiperone binding in both the bovine retina and caudate nucleus indicated an interaction with dopaminergic rather than serotonergic sites. However, inhibition curves obtained to dopaminergic agonists in the bovine retina were significantly steeper than those observed in the bovine caudate nucleus, as reflected in the greater Hill coefficients obtained for these agents in the retina. Furthermore, only a small amount of specific [³H]domperidone binding was observed in either the bovine caudate nucleus or the guinea pig striatum, whilst no specific [³H]domperidone binding was detectable in homogenates of either bovine or guinea pig retina. These data suggest that the retina possesses only a small population of dopaminergic D2 sites and that these binding sites may differ from those present in the caudate nucleus.     

H-Pro-[3H]Leu-Gly-NH2: Uptake and Metabolism in Rat Brain

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.     

Clinical Concentrations of Volatile Anesthetics Reduce Depolarization-Evoked Release of [3H]Norepinephrine, but Not [3H]Acetylcholine, from Rat Cerebral Cortex

We compared the potencies of halothane, enflurane, and methoxyflurane in producing unconsciousness in vivo and in inhibiting the release of [3H]norepinephrine and [3H]acetylcholine in vitro. Rats were anesthetized with various concentrations of each anesthetic, and responsiveness was determined by a hemostat tail pinch. Slices of cerebral cortex were equilibrated with similar concentrations of each agent in vitro, and potassium-evoked release of [3H]norepinephrine and [3H]acetylcholine was determined. For both studies, brain concentrations of the anesthetics were determined by heptane extraction and gas chromatography. Using this method, we found that brain concentrations of all three agents which caused unconsciousness in vivo also reduced depolarization-evoked release of [3H]norepinephrine by approximately 30% in vitro. The release of [3H]acetylcholine was unaffected by similar concentrations of these anesthetics. Such selective interference with stimulus-secretion coupling in central noradrenergic, and possibly other, neurons might contribute to the depressant actions of volatile anesthetics. The differential effects on norepinephrine and acetylcholine release also suggest differences in the mechanisms by which these two transmitters are released.     

Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina

Abstract: The effect of melatonin on [³H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 n M ) significantly increased [³H]glutamate uptake, and this effect persisted in a Ca²⁺-free medium. On the other hand, melatonin significantly increased [³H]glutamate release in retinas excised at 2400 h, but this effect was Ca²⁺ sensitive. Melatonin significantly increased ⁴⁵Ca²⁺ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [³H]glutamate uptake, [³H]glutamate release, or ⁴⁵Ca²⁺ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'- O -dibutyrylguanosine 3',5'-cyclic monophosphate, significantly increased [³H]glutamate uptake, [³H]glutamate release, and ⁴⁵Ca²⁺ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.     

Portacaval Shunting and Hyperammonemia Stimulate the Uptake of l-[3H]Arginine but Not of l-[3H]Nitroarginine into Rat Brain Synaptosomes

Abstract: Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval-shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As l -arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of l -[³H]-arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that l -arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham-operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of l -[³H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity ( V _max) without any change in affinity ( K _m). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose-dependent stimulation of l -[³H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of N ^G-nitro- l -[³H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of l -arginine resulting from a direct stimulatory effect of ammonia on l -arginine transport.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

Perinatal Hypoxia-Ischemia Disrupts Striatal High-Affinity [3H]Glutamate Uptake into Synaptosomes

We examined the impact of hypoxia-ischemia on high-affinity [3H]glutamate uptake into a synaptosomal fraction prepared from immature rat corpus striatum. In 7-day-old pups the right carotid artery was ligated, and pups were exposed to 8% oxygen for 0, 0.5, 1, or 2.5 h, and allowed to recover for up to 24 h before they were killed. High-affinity glutamate uptakes in striatal synaptosomes derived from tissue ipsilateral and contralateral to ligation were compared. After 1 h of hypoxia plus ischemia, high-affinity glutamate uptake in the striatum was reduced by 54 +/- 13% compared with values from the opposite (nonischemic) side of the brain (p less than 0.01, t test versus ligates not exposed to hypoxia). There were similar declines after 2.5 h of hypoxia-ischemia. Activity remained low after a 1 h recovery period in room air, but after 24 h of recovery, high-affinity glutamate uptake was equal bilaterally. Kinetic analysis revealed that loss of activity could be attributed primarily to a 40% reduction in the number of uptake sites. Hypoxia alone had no effect on high-affinity glutamate uptake although it reduced synaptosomal uptake of [3H]3,4-dihydroxyphenylethylamine. Addition of 1 mg/ml of bovine serum albumin to the incubation medium preferentially stimulated high-affinity glutamate uptake in hypoxic-ischemic brain compared with its effects in normal tissue. These studies demonstrate that hypoxia-ischemia reversibly inhibits high-affinity glutamate uptake and this occurs earlier than the time required to produce neuronal damage in the model.     

Solubilization of Quisqualate-Sensitive [3H]Glutamate Binding Activity from Rat Retina

Binding activity of a putative central neurotransmitter, L-glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P-40. [3H]Glutamate binding activity increased linearly with increasing concentrations of the solubilized proteins up to 15 micrograms. The binding activity reached an equilibrium within 10 min at 2 degrees C, while increasing with incubation time up to 60 min at 30 degrees C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30 degrees C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 microM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to L-glutamate, and was abolished by quisqualate, L-glutamate diethyl ester, and DL-2-amino-3-phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation-exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30 degrees C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate-sensitive membranous enzyme with a stereospecific and structure-selective high affinity for the central neurotransmitter.     

Stimulation of [3H]GABA and β-[3H]Alanine Release from Rat Brain Slices by cis-4-Aminocrotonic Acid

Abstract: cis -4-Aminocrotonic acid (CACA; 100 µ M ), an analogue of GABA in a folded conformation, stimulated the passive release of [³H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β-[³H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 µ M ) did not stimulate the passive release of [³H]taurine from slices of cerebellum and spinal cord or of d -[³H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [³H]taurine from the cerebellum and spinal cord and d -[³H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β-alanine release are due to CACA acting as a substrate for a β-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of β-[³H]alanine into slices of rat cerebellum and cerebral cortex. The observed K _i for CACA against β-[³H]alanine uptake in the cerebellum was 750 ± 60 µ M . CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABA_c receptor. The effects of CACA on GABA and β-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, β-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.     

5-Hydroxytryptamine Stimulates [3H]Dopamine Release from the Fish Retina

Abstract: Serotonin (5-hydroxytryptamine, 5-HT; 0.5 μM and above) stimulated the release of [³H]dopamine ([³H]DA) from particulate fractions of the carp ( Cyprinus carpio ) retina. The 5-HT effect was dose- and Ca²⁺-dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 5-hydroxytrypto-phan, or 5-hydroxyindoleacetic acid) examined. An increase in [³H]DA release was elicited by addition of 5-HT agonists (5-methoxytryptamine, 5-methoxy- N,N- dimethyltryptamine, and tryptamine), but not antagonized by three 5-HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 m M ) produced a large increase in [³H]DA release from the particulate fractions, but this action was Ca²⁺-independent. Further, no significant release of [³H]γ-aminobutyric acid could be evoked by 5-HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5-HT stimulates [³H]DA release from the fish retina through a specific receptor-mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.     

Intrasynaptosomal Sequestration of [3H]Amphetamine and [3H]Methylenedioxyamphetamine: Characterization Suggests the Presence of a Factor Responsible for Maintaining Sequestration

We examined the incorporation of [3H]methylenedioxyamphetamine ([3H]MDA) and [3H]amphetamine into rat brain synaptosomes. Saturation studies, using increasing concentrations of nonradioactive ligand, revealed that [3H]-MDA interacted with two saturable sites that were sensitive to boiling of the tissue. Eadee-Scatchard plots of [3H]MDA saturation data were curvilinear; nonlinear curve-fitting analysis of these data suggested the presence of high- and low-affinity [3H]MDA sites of association: KD high = 295 nM, Bmax high = 32 pmol/mg of protein; KD low = 45 microM, Bmax low = 5.2 nmol/mg of protein. Association of [3H]MDA to the low-affinity site was dependent on the presence of isotonic sucrose in the incubation medium. The high capacities of these sites argue against a bimolecular interaction of [3H]MDA with monovalent protein binding sites. [3H]MDA incorporation was reduced under conditions that disrupt the integrity of plasma membranes, such as sonication, incubation in hypotonic media, and incubation in the presence of the detergent digitonin. These data indicate that [3H]MDA incorporation into synaptosomes may represent an internalization and sequestration phenomenon. [3H]MDA incorporation was also reduced by preincubation of the synaptosomal preparation at 37 degrees C or in hypotonic buffer at 4 degrees C, a result suggesting that this sequestration is maintained by an intrasynaptosomal component that is lost under the preincubation conditions described above. [3H]MDA incorporation was pH dependent (maximal at pH 7.5) and temperature sensitive (maximal incorporation occurred at 21 degrees C and was substantially reduced at 37 degrees C). [3H]Amphetamine was also incorporated into synaptosomes, and this incorporation was sensitive to the same physical manipulations of the tissue preparation as [3H]MDA incorporation. The synaptosomal sequestration of both [3H]MDA and [3H]amphetamine was inhibited by permeant cations, such as sodium and potassium, a result suggesting that the proposed intrasynaptosomal component that maintains the sequestration is anionic. Preliminary pharmacological profiles of [3H]MDA and [3H]amphetamine sequestration were identical. The rank order of inhibitor potencies for the incorporation of both ligands was desipramine greater than amphetamine greater than MDA greater than methylphenidate. This order of potency does not correspond to the lipophilicity of the test drugs. The synaptosomal incorporation and sequestration of [3H]MDA, [3H]methylenedioxymethamphetamine, and [3H]amphetamine described in the present report may be important in the molecular mechanism of action of monoamine release induced by the amphetamines.     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

Binding of [3H]Serotonin to Skeletal Muscle Actin

We previously observed that the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) binds with high- and low-affinity interactions to an actin-like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G-actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 X 10(-5) M and 4 X 10(-3) M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 microM) to G-actin was increased as much as 26-fold by divalent cations. Potassium iodine (KI) increased the affinity of G-actin for serotonin, KD values for this binding being 3 X 10(-7) M and X 10(-5) M. Serotonin bound with even higher affinity to polymerized F-actin, with KD values of 2 X 10(-8) M and 2 X 10(-5) M. However, the total number of binding sites on F-actin was only about 4% of the number of G-actin. The binding of serotonin (0.1 microM) to G-actin could be inhibited by phenothiazines (1 microM) or reserpine (10 microM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.     

Uptake and Metabolism of [3H] Adenosine by Aplysia Ganglia and by Individual Neurons

[3H]Adenosine was taken up and metabolized by isolated ganglia of the marine mollusc Aplysia californica. After 2 h, most of the radioactivity was recovered as metabolites, including ATP, ADP, and AMP, as well as the deaminated products, inosine, hypoxanthine, and uric acid. Little remained in the form of adenosine. These pathways were not uniformly distributed among various tissue elements. In most individual neurons, inosine and its breakdown products were the principal metabolites of [3H]adenosine, whereas ATP and other nucleotides predominated in the connective tissue sheath. Endogenous levels of ATP, ADP, AMP, and adenosine in ganglia, sheath, and individual neurons were also determined using a fluorimetric-HPLC assay. The concentrations of the nucleotides were quite uniform in sheath and among the individual neurons assayed (1-5 pmol/microgram of protein); however, concentrations of adenosine were considerably higher in neurons than in the sheath.