Development of saxitoxin-sensitive and insensitive sodium channels in cultured neonatal rat astrocytes

Voltage-sensitive Na channels were studied in cultures of neonatal rat cortical astrocytes. These channels were present at all times in culture as determined by tracer 22Na+ influx in the presence of batrachotoxin (BTX) and sea anemone polypeptide toxin (AxTx). The affinity of saxitoxin (STX) binding and sensitivity to STX inhibition of sodium influx were utilized to characterize these channels. Up to 7 d in culture, high-affinity 3H-STX binding (Kd of 0.2 nM at 4 degrees C) was very low, and 22Na+ influx was inhibited only by high concentrations (Ki = 170 nM) of STX. From 7 to 14 d, total specific binding of STX increased to a maximum of over 2 pmol/mg protein and remained constant for 28 d. By 14 d, inhibition of 22Na+ influx by STX was clearly biphasic, indicating the presence of 2 populations of channels with Ki's of 0.2 nM and 150 nM. At 14 d in culture, binding of 3H-STX to astrocyte membranes revealed the presence of 2 specific sites. During this second week, increasing numbers of high-affinity STX binding sites and increasing sensitivity to the inhibition of BTX + AxTx-stimulated 22Na+ influx by STX coincided with the change in morphology of primitive flat polygonal cells to highly branched stellate forms characteristic of mature astrocytes in vivo. Changes in culture conditions modified the time course of the onset of high STX affinity binding. Twenty-four hours after changing to serum-free G5 medium, there was both an 8-fold increase in STX binding sites and a change to a stellate shape in all cells. The results suggest that although low-affinity STX Na channels are always present in astrocytes, after 7 d in culture a different population of channels appears with the high affinity for STX characteristic of adult neuronal sodium channels. This spontaneous process is greatly accelerated by changing to a chemically defined medium.     

3H-batrachotoxinin-A benzoate binding to voltage-sensitive sodium channels: inhibition by the channel blockers tetrodotoxin and saxitoxin

The sodium channel blockers tetrodotoxin (TTX) and saxitoxin (STX) and the channel activator batrachotoxin (BTX) produce their effects by binding to separate and distinct sites on the channel protein. The fact that TTX- and STX-modified sodium channels are blocked to sodium flux has precluded drawing any direct conclusions regarding the effect of TTX/STX on BTX binding based on electrophysiological or 22Na flux measurements. Nevertheless, these sites have been presumed to be non-interacting. In this study, 3H-batrachotoxinin-A benzoate (BTX-B), a tritiated congener of BTX, has been used to provide a direct assessment of these binding interactions. Equilibrium specific binding of 3H-BTX-B to sodium channels in vesicular preparations of mouse brain in the presence of scorpion toxin was measured using a filtration assay procedure. At 25 degrees C both TTX and STX inhibit 3H-BTX-B binding in a concentration-dependent and noncompetitive manner. This inhibition is markedly temperature-dependent, being negligible at 37 degrees C and maximal at 18 degrees C, the lowest temperature investigated. Scatchard analysis of BTX-B binding isotherms at 25 degrees C in the presence and absence of 1 microM TTX revealed that inhibition is due to a 3-fold decrease in the affinity of BTX-B binding with no change in the number of binding sites (Bmax). The concentration dependence for TTX inhibition of both specific 3H-STX and 3H-BTX-B binding is identical, suggesting that inhibition of 3H-BTX-B binding is due to a direct effect of TTX/STX binding at their specific sodium channel site. The channel blockers did not alter the binding of scorpion toxin under these assay conditions, nor did BTX-B affect the binding of 3H-STX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple high affinity binding sites for 5-hydroxytryptamine: a new class of sites distinct from 5-HT1 and S2

Two different classes of binding sites probably related to serotonergic receptors have already been reported: 5-HT1 binding sites recognize [3H]5-hydroxytryptamine with a high affinity (Kd = 3 nM) and S2 binding sites recognize [3H]spiroperidol and [3H]ketanserine. An additional population of sites has been observed in crude membrane preparations or fractions enriched with synaptosomal membranes obtained from rat brain cortex. This population was observed as a single class of sites in a synaptosomal fraction (L fraction--according to Laduron (1977)). It corresponded to a dissociation constant Kd = 13-15 nM, and Bmax = 0.80 +/- 0.15 pmol/mg protein. Displacement experiments showed that it recognized preferentially the 5-HT structure (bufotenin, 5-MeO-tryptamine). Tryptamine was a weak displacer and 5,7-dihydroxytryptamine totally inefficient. Neither 8-OH-DPAT, nor quipazine had any effect. Methiothepin, cinanserin and cyproheptadine displaced 5-HT from these sites whereas ergot derivatives did not. Contrary to 5-HT1 binding, this recently observed binding was not altered by GTP; alpha-MSH reduced the corresponding Bmax whereas Leu-enkephalin did not. The degenerative lesion of the serotonergic fibers led to a slight increase in the Bmax of the binding without altering the Kd which means that corresponding sites are not located on serotonergic fibers and might be postsynaptically located.     

Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site

Bovine and rat pineal benzodiazepine receptors were characterized using ligands with high affinities for either 'central-type' (CBR) or 'peripheral-type' (PBR) benzodiazepine receptors. The characteristics (Bmax = 83 +/- 10 fmol/mg protein, Kd = 3.88 +/- 0.46 nM) of benzodiazepine receptors in bovine pineal membranes measured with [3H]flunitrazepam (using flunitrazepam to define non-specific binding) were consistent with previously reported values. However, if non-specific binding was defined using Ro 15-1788 (a selective CBR ligand), the Bmax and Kd of [3H]flunitrazepam decreased 51 and 58%, respectively. In addition, when using PK 11195 to determine non-specific binding, the Bmax of [3H]flunitrazepam binding to bovine pineal decreased further (approximately 80%, Kd decreased approximately 39%). Together, these observations strongly suggested the presence of PBR in the bovine pineal. Bovine pineal PBR characterized with [3H]PK 11195 revealed a high density (relative to CBR) of high affinity binding sites (Kd = 1.08 +/- 0.30, Bmax = 776 +/- 33.0 fmol/mg protein). In contrast, when [3H]Ro 5-4864 (1-20 nM) was used to define PBR, no binding was detectable. These observations are in sharp contrast to the rat pineal gland, in which both [3H]Ro 5-4864 and [3H]PK 11195 bind to a large number of PBR with high affinity (Kd approximately equal to 1.9 nM, Bmax approximately equal to 26 pmol/mg protein). Bovine pineal PBR were further characterized with compounds structurally related to either Ro 5-4864 or PK 11195.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of 3H-spiperone to human lymphocytes: A biological marker in schizophrenia?

The specific binding of the dopamine antagonist 3H-spiperone to lymphocytes from a healthy control group (n = 40), a group of acute, unmedicated schizophrenic patients (n = 27), and a psychiatric control group (n = 16) was investigated. There were no differences in binding parameters between the healthy controls (Bmax 2.6 +/- 0.7 fmoles/10(6) cells; Kd 0.17 +/- 0.07 nM) and the psychiatric control group. Binding capacity of 3H-spiperone was significantly increased in lymphocytes from the schizophrenic patients (Bmax 14.4 +/- 9.3 fmoles/10(6) cells). Moreover, a slight decrease in affinity was observed (Kd 0.44 +/- 0.21 nM). Because the increase in binding appeared only in schizophrenic patients, this peripheral model could prove valuable as a diagnostic criterion.     

Evidence for multiple [3H]prazosin binding sites in canine brain membranes

Two classes of alpha 1 adrenoceptors were identified in canine brain and liver using conventional radioligand binding methods. Scatchard plots of specific [3H]prazosin binding to brain and liver membranes prepared from 100-150-day-old Doberman pinscher dogs were consistently curvilinear and best fit a two-site binding model (frontal cortex, Kd1 = 57.7 +/- 10.0 pM, Bmax1 = 64.6 +/- 17.1 fmol/mg protein, Kd2 = 1.5 +/- 0.5 nM, Bmax2 = 159 +/- 37.6 fmol/mg protein; liver, Kd1 = 82.6 +/- 36 pM, Bmax1 = 7.0 +/- 5.1 fmol/mg protein, Kd2 = 0.8 +/- 0.2 nM, Bmax2 = 62.1 +/- 8.7 fmol/mg protein). Kinetically derived affinity constants from association and dissociation experiments agreed with those obtained by Scatchard analyses of equilibrium binding data. Binding sites were saturable, heat labile, bound ligand reversibly, and appeared to be appropriately distributed in relation to endogenous catecholamine. [3H]Prazosin also bound with high affinity to two classes of binding site in porcine and bovine brain membrane but [3H]prazosin binding in monkey and rat brain was best described by a single-site binding model. Affinities obtained were in between values obtained for high and low affinity Kds in the other species. Competitions for [3H]prazosin binding sites in canine frontal cortex were conducted with the following antagonists: WB-4101, corynanthine, phentolamine, benoxathian, phenoxybenzamine, chlorethylclonidine, thymoxamine, prazosin, yohimbine and agonists: methoxamine, (-)-norepinephrine, and clonidine. All ligands but prazosin, norepinephrine and clonidine competed for specific [3H]prazosin binding in a statistically significant biphasic manner. Benoxathian and WB-4101 displayed the highest affinities (benoxathian: Ki1 = 0.26 nM, WB-4101: Ki1 = 0.20 nM) and selectivity (high affinity/low affinity: benoxathian = 1640, WB-4101 = 13204) for the high affinity [3H]prazosin binding site; chlorethylclonidine had highest affinity (Ki2 = 91 nM) and selectivity (low affinity/high affinity = 405) for the lower affinity [3H]prazosin binding site. As defined, the two sites were similar to the alpha 1a and alpha 1b recently described in the rat and rabbit. A noticeable difference was that the subtypes described in dog brain had a 30-fold difference in affinity for prazosin.     

Platelet alpha 2 adrenoceptors in human and canine narcolepsy.

We have recently established that canine narcolepsy (an autosomal recessive genetic model of the human disorder) is dramatically improved by treatment with alpha 2 antagonists such as yohimbine (Nishino et al: J Pharmacol Exp Ther 253:1145-1152, 1990). To further investigate the role of alpha 2 adrenoceptors in narcolepsy, receptors labeled with [3H] yohimbine were examined on platelets from human and canine narcoleptic subjects. Twenty-eight Doberman pinschers were studied, 7 controls (C), 7 heterozygous (Hz), and 14 narcoleptics (N) (age and sex matched), including eight animals born in a backcross setting (narcoleptic x heterozygous; 5 narcoleptics and 3 heterozygous). The Kd and Bmax of each group respectively, were as follows: C, Kd = 2.86 +/- 0.76 nmol/L, Bmax = 295.78 +/- 31.89 fmol/mg protein; Hz, Kd = 2.06 +/- 0.23 nmol/L, Bmax = 307.02 +/- 22.21 fmol/mg protein; and N, Kd = 2.72 +/- 0.45 nmol/L, Bmax = 267.52 +/- 19.47 fmol/mg protein. No statistical differences were found between groups using nonparametric (Kruskall-Wallis) statistical procedures, and there were no correlations between any binding parameter and symptom severity within the narcoleptic group. Platelet alpha 2 receptor affinity and density also did not differ between narcoleptic and heterozygous dogs in the backcross litter (N [n = 5], Kd = 1.94 +/- 0.59 nmol/L, Bmax = 290.6 +/- 64.7 fmol/mg protein; Hz [n = 3], Kd = 2.83 +/- 0.47 nmol/L, Bmax = 294.2 +/- 42.9 fmol/mg protein). Fourteen human subjects, seven control and seven narcoleptic patients (age and sex matched), were included in the study.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H-paroxetine binding in brains of alcoholics.

High affinity 3H-paroxetine binding was studied in human frontal cortex and hippocampus obtained from normal controls and alcoholics. On the basis of Scatchard analyses, a significant decrease in the maximal number of binding sites (Bmax) was found in the hippocampus of alcoholics (n = 8) as compared with that of controls (n = 10) (mean +/- SD = 63 +/- 35 vs. 114 +/- 70 fmoles/mg protein). There was no significant difference in the dissociation constants (Kd) between the two groups. The presumed effect of chronic alcohol abuse on 3H-paroxetine binding may be region-specific since no significant difference in either Bmax or Kd for 3H-paroxetine binding was found in the frontal cortex between normal controls and alcoholics. No significant correlation of 3H-paroxetine binding with age or postmortem interval was observed. The decrease in 3H-paroxetine binding in the hippocampus of alcoholics is probably indicative of reduced density of serotonergic nerve terminals either as a preexisting condition or as a result of neuronal damage caused by ethanol or the sequelae of alcoholism, such as nutritional deficiencies.     

Platelet 5-HT2 serotonin receptor binding sites in autistic children and their first-degree relatives

We examined platelet serotonin2 [5-hydroxytryptamine2 (5-HT2)] receptor binding sites, whole blood serotonin (5-HT), and plasma norepinephrine (NE) in male autistic children and their first-degree relatives. Saturation studies utilizing 125I-spiroperidol labeled the 5-HT2 sites with an affinity of 224.6 +/- 84.4 pmol/L (Kd). No group differences, i.e., autistic (n = 12), siblings (n = 6), parents (n = 22), control (adult; n = 7: child; n = 10), were seen for either the Kd or the total number of sites (Bmax: 14.3 +/- 10.9 fmol/mg protein). No correlations were found in any group between binding parameters (Kd or Bmax) and whole blood 5-HT. For the parental group, inverse correlations were found between NE and Bmax (standing NE, rs = -0.67, n = 21, p = 0.001; supine NE, rs = -0.49, n = 22, p = 0.021). In the autistic group, no correlation was seen between plasma NE and Bmax. A correlation between the autistic boys' Bmax and their fathers' Bmax was observed (rs = 0.79, n = 11, p = 0.004). These findings suggest (1) circulating NE may be involved in heterologous regulation of 5-HT2 receptors in the platelet and (2) genetic (paternal-filial) factors may play a role in the expression of 5-HT2 binding sites in the platelet. These preliminary findings are discussed in relation to heterologous receptor regulation. The relationships between these findings and either the pathophysiology of autism or hyperserotonemia in autism are unknown.     

3,5,3'-Triiodothyronine binding sites in synaptosomes from brain of chick embryo. Properties and ontogeny.

In this study we have demonstrated the presence of specific 3,5,3'-L-triiodothyronine (T3) binding sites in the synaptosomes of chick embryo cerebral cortex and described their ontogeny. Scatchard analyses of binding data obtained with synaptosomal preparations from 17-day-old embryos revealed two T3 binding sites. The first site (N1) had a high affinity and low capacity since its dissociation constant (Kd) was 68 +/- 1.3 nM T3 (mean +/- S.D.; n = 3-5) and its maximal binding capacity (Bmax) was 8.63 +/- 1.59 ng T3/mg of protein, whereas the second site (N2) had a higher Kd of 5.04 +/- 0.5 microM T3 and a larger Bmax of 405 +/- 49 ng T3/mg of protein. The relative affinity of the synaptosomal fraction for T3 and other analogs was the following: T3 greater than T4 (thyroxine) greater than D-T3 (3,5,3'-D-triiodothyronine) = TRIAC (triiodothyroacetic acid) greater than rT3 (reverse T3). Gel chromatography of the [125I]T3 labeled fraction revealed a partially saturable peak with an estimated MW of more than 100 kDa. The ontogenic pattern showed a progressive increase of Kd and Bmax of N1, occurring mainly between the 12 and 19 days of incubation, and a marked fall, particularly of the Bmax, after hatching. The second site did not show any important variation during the embryogenesis. These data indicate the existence of specific T3 binding sites in synaptosomes from cerebral cortex of chick embryo, whose properties and ontogeny are completely different from those of the nuclear receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic muscarinic receptors in rat cochlea

Specific 3H-1-quinuclidinylbenzilate (3H-1-QNB) binding to rat cochlea homogenates occurs to a homogeneous class of binding sites with Kd = 0.13 +/- 0.01 nM and Bmax = 0.57 +/- 0.07 fmol per cochlea. Binding is stereoselectively inhibited by benzetimide enantiomers. Dexetimide was more effective than levetimide in displacing 3H-1-QNB from its binding sites (Ki = 4 x 10(-10) M and 6.5 x 10(-6) M, respectively). Pirenzepine inhibits 3H-1-QNB binding with low affinity (Ki = 2 x 10(-6) M), classifying the binding sites as muscarinic M2 receptors.     

GABAA and GABAB sites in bovine adrenal medulla membranes

The effect of several ligands and Ca2+ ions on [3H]GABA binding to bovine adrenal medulla membranes was investigated. Without any blockade, the [3H]GABA binding showed two components, one of low affinity (Kd = 139 +/- 22 nM and Bmax = 3.2 +/- 0.4 pmol/mg protein) and the other of high affinity (Kd = 41 +/- 6 nM and Bmax = 0.35 +/- 0.26 pmol/mg protein). Muscimol specifically blocked low-affinity sites, and (-)baclofen blocked high-affinity components. Ca2+ ions were strictly necessary for maximum binding to high-affinity sites, whereas they did not significantly affect sites of the lower affinity. These results show that the bovine adrenal medulla has a GABAA receptor population of low affinity together with a GABAB receptor of high affinity.     

A developmental study of adenohypophyseal dopaminergic receptors and of haloperidol-induced prolactin release in rats

The ontogenesis of adenohypophyseal dopamine receptors, assessed by haloperidol-displaceable [3H]didydroergocryptine (DHE) binding of 1-, 12-, 20-, 28-day-old female rats was studied in correlation with the prolactin releasing effect of haloperidol (1 mg/kg), a dopaminergic antagonist. A specific dopaminergic receptor could be quantified at the time of birth (Bmax = 2.5 +/- 0.5 fmol/mg; Kd = 1.5 +/- 0.2 microM), anterior pituitary receptor density (fmol bound/mg) increased non-significantly henceforth and a slight ontogenic increase of Kd values was also observed. Haloperidol failed to increase prolactin in newborn female rats; at 4 days, a significant increase was evidenced, and from then onwards the response rose markedly with age. As sex differences in the dopaminergic modulation of prolactin release have been documented, the hyperprolactinemic effect of haloperidol in correlation with [3H]DHE binding in anterior pituitary of 28-day-old female and male rats was studied. Though the prolactinemic increment achieved by haloperidol was significantly higher in female than in male rats, [3H]DHE binding was not statistically different between sexes. These data indicate: (a) a specific binding site for [3H]DHE in anterior pituitary of female rats is present from the first postnatal days. From then onwards, a gradual but slight increment in both, Bmax and Kd for the dopaminergic agonist is observed until puberty; (b) at 28 days, no clear difference in Bmax and Kd is present in [3H]DHE binding between male and female rats; (c) by contrast, haloperidol shows a prolactin releasing effect that increases markedly with age in the female.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet imipramine binding in depressed children and adolescents.

Kinetic constants of platelet imipramine binding were determined in youths with major depression, and a contrast group. Subjects actively depressed (N = 10) had significantly fewer imipramine binding sites (Bmax) (877 +/- 148 fmol/mg protein) than recovering depressives (N = 12) (1220 +/- 428 fmol/mg protein) and contrasts (N = 10) (1270 +/- 230 fmol/mg protein). Affinity constants (Kd) (1.14 +/- 0.36 nM, 0.97 +/- 0.31 nM, and 1.17 +/- 0.39 nM, respectively) were similar among the groups. Actively depressed males but not females had fewer imipramine binding sites than both their sex-matched comparison groups. Although actively depressed females' Bmax was significantly lower than recovering depressed and nondepressed males, neither age, Tannner stage, nor circannual rhythms influenced Bmax, but suicidality may be associated with low Bmax. A decrease in Bmax may be a state-specific marker of major depression in boys or associated with a depressive disorder with a suicidal history.     

Platelet imipramine binding in autistic subjects

Previous reports of elevated platelet serotonin (5-HT) concentrations in autistic subjects suggest that platelet 5-HT uptake might be altered in autism. Parameters of 3H-imipramine (IMI) binding were measured in 11 drug-free autistic subjects and 10 normal volunteers. Similar means (+/- SD) for Bmax (autistics, 1350 +/- fmole/mg protein; normals, 1590 +/- 206 fmole/mg protein) and Kd (autistics, 0.98 +/- 0.10 nM; normals, 0.94 +/- 0.13 nM) were found in the two groups. The normal number (Bmax) and affinity (Kd) of the IMI binding site in autistic subjects suggest that the regulation of 5-HT uptake is not different in autism.     

Ontogenesis of adenosine receptors in the central nervous system of the rat

The ontogeny of adenosine receptors was studied in rat brain and spinal cord using the specific ligand [3H]cyclohexyladenosine [( 3H]CHA). The [3H]CHA affinity constant (Kd) and the maximum receptor binding capacity (Bmax) were analyzed at all ages and in all CNS regions studied. Throughout development the Kd of [3H]CHA binding remained relatively stable and for cortex, cerebellum, subcortex, midbrain, brainstem and spinal cord ranged from 2.2 +/- 0.2 to 5.5 +/- 0.6 nM (mean +/- S.E.M.). In contrast, the Bmax values from 1- and 90-day animals increased by as little as 2-fold in subcortical regions and by as much as 9- and 16-fold in cortex and cerebellum, respectively. The highest density of binding sites was observed in subcortical structures and the lowest in brainstem and midbrain. In cortex, a steady increase in receptor number began at day 1 and stopped at the adult level by 21 days. In cerebellum, maximum receptor proliferation began at about 14 days and continued to adulthood. Other CNS regions showed intermediate rates of receptor development. These differences may reflect both the pattern of postnatal neurogenesis in the rat CNS and the maturation of those neural elements containing adenosine receptors.     

Properties of dopamine agonist and antagonist binding sites in mammalian retina

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.     

Expression of mineralocorticoid type I and glucocorticoid type II receptors in astrocyte glia as a function of time in culture.

In the present study we have examined the expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia maintained in culture for different periods of time. Cytosolic mineralocorticoid Type I receptors were labeled with [3H]aldosterone (ALDO) in the presence of a 500-fold molar excess of the potent Type II receptor ligand RU 28362. [3H]Dexamethasone (DEX) was used to label cytosolic Type II receptors. Both Type I and Type II receptor binding was saturable in astrocyte glia that had been maintained in culture for 20 and 30 days following final plating (i.e. 20- and 30-day-old cultures). Scatchard analysis of [3H]ALDO binding revealed a single class of Type I receptors, with dissociation constants (Kd) of 0.45 +/- 0.13 nM and 0.53 +/- 0.07 nM, respectively, in 20- and 30-day-old cultures. The number of Type I receptors in 30-day-old cultures was nearly half that found in 20-day-old cultures (22.06 vs 42.64 fmol/mg protein). Linear Scatchard plots were also obtained for [3H]DEX binding to cytosol prepared from 20- and 30-day-old cultures. There were no significant differences in the Kd or Bmax values for [3H]DEX binding in 20- or 30-day-old cultures, i.e. 2.06 +/- 0.15 nM and 247.36 +/- 18.16 fmol/mg protein for 20-day-old cells and 2.3 +/- 0.74 nM and 261.02 +/- 3.08 fmol/mg protein for 30-day-old cells. These Bmax values are more than double the Bmax value for [3H]DEX binding observed in our previous studies in 10-day-old astrocyte glial cultures. Switching cultured astrocyte glial from serum-supplemented to serum-free medium had no significant effects on the Kd values of Type I or Type II receptors in all the cultures tested. However, treatment with serum-free medium increased the number of Type I receptors in 30-day-old cultures to a level similar to that found in 20-day-old cultures. Taken together, these binding data suggest that Type I and Type II receptors are expressed differently in astrocyte glia as a function of time in culture.     

Lymphocyte beta-adrenoreceptor density in patients with unipolar depression and normal controls

Values of binding maximum (Bmax) and dissociation constant (Kd) of (-)3-[125I]iodocyanopindolol (ICYP) were determined in beta-adrenergic receptors of membranes of peripheral lymphocytes in 32 patients with unipolar depression (DSM-III-R) and 31 normal controls. Results were analyzed by a two-way Analysis of Covariance method. A significant difference was noted for group assignment (patient versus control, p less than 0.05). Mean Bmax (fmol ICYP bound/mg lymphocyte membrane fraction total protein) of patients was 31.9 +/- 3.84 (SE) and controls 46.3 +/- 3.92 (SE). A significant interaction was found between group membership and gender (p less than 0.05). In the female patient group (n = 14), mean Bmax was 30.5 +/- 5.79 (SE); in female controls, mean Bmax was 56.0 +/- 5.15 (SE). Differences between male patients and male controls were not significant. Mean values of Kd (pmol/liter) showed a trend for patient values to be lower than control values [69.0 +/- 13.66 (SE) versus 108.5 +/- 14.42 (SE), respectively]. A significant inverse relationship was noted between lymphocyte beta-receptor Bmax and frequency of panic attacks during the depressive episode in 18 patients (p = 0.05). No relationship was found between values of Kd and frequency of panic attacks in these patients. Thus, preliminary evidence is provided for relationships among altered beta-adrenergic receptor binding, gender, and indices of panic-anxiety in unipolar depressed patients.     

Characterization and localization of glutathione binding sites on cultured astrocytes.

Glutathione (GSH) binding sites found in brain white matter in a previous study using biotinylated GSH (Third IBRO World Congress Neurosci. Abstr., 1991, P59.17) suggested that there might GSH receptors on glial cells. In the present study, radioligand receptor assays were performed on cultured astrocytes using [35S]GSH. Scatchard analyses of saturation binding of [35S]GSH revealed two binding sites: Kd1 = 2.0 +/- 0.1 nM, Bmax1 = 89.5 +/- 1.5 fmole/2.2 x 10(5) cells and Kd2 = 12.8 +/- 0.4 nM, Bmax2 = 187.7 +/- 2.4 fmol/2.2 x 10(5) cells. The saturable and displacible high affinity [35S]GSH binding we have observed suggests that this binding is not due to GSH sequestration by uptake sites or to the association of GSH with GSH S-transferases or GSH peroxidases which have Kds in the microM range. Colloidal gold and immunofluorescence double labelling were used to visualize the binding sites at the cellular level. Positive colloidal gold decoration further suggests that these labelled binding sites are membrane receptors on astrocytes.