Inosine may be an endogenous ligand for benzodiazepine receptors on cultured spinal neurons.

Mouse spinal neurons grown in tissue culture were used to study the membrane effects of the benzodiazepine flurazepam and the naturally occurring purine nucleoside inosine, which competes for benzodiazepine receptor sites in the central nervous system. Application of inosine elicited two types of transmitter-like membrane effects: a rapidly desensitizing excitatory response and a nondesensitizing inhibitory response. Flurazepam produced a similar excitatory response which showed cross-desensitization with the purine excitation. Flurazepam also blocked the inhibitory inosine response. The results provide electrophysiological evidence that an endogenous purine can activate two different conductances on spinal neurons and that flurazepam can activate one of the conductances and antagonize the other.

Brain endolases as specific markers of neuronal and glial cells.

There are three distinct enolase isoenzymes in brain; neuron-specific enolase (NSE), formerly referred to as neuron-specific protein, which is specifically localized in neurons, a nonneuronal enolase (NNE), and a third hybrid form. Light microscopy with immunocytochemical techniques has permitted localization of non-neuronal enolase. The NNE is located in glial cells with no staining of endothelial cells or neurons. Thus, NSE and NNE can be used as specific metabolic markers for neurons and glial cells, respectively.

Interaction between purine and benzodiazepine: Inosine reverses diazepam-induced stimulation of mouse exploratory behavior.

Inosine, 2-deoxyinosine, and 2-deoxyguanosine completely reversed the increase in exploratory activity elicited in mice by diazepam. The inhibition of exploratory behavior by purines occurred at doses that when given alone have no effect on exploratory behavior. 7-Methylinosine, which does not bind to the brain benzodiazepine binding site in vitro, had no effect on the diazepam-induced increase in exploratory behavior. Behavioral effects produced by various combinations of inosine and diazepam indicate that the interaction between purine and benzodiazepine is antagonistic and support the hypothesis that the naturally occurring purines function in anxiety-related behaviors that respond to benzodiazepine treatment.

Differential expression of neural isozymes by human medulloblastomas and gliomas and neuroectodermal cell lines.

For the determination of their possible utility as tumors markers, 2 neural-associated isozymes, neuron-specific enolase [(NSE) EC 4.2.1.11] and creatine kinase BB [(CK-BB) EC 2.7.3.2], were quantitated by radioimmunoassay in human neuroectodermal-derived cell lines, primary brain tumors, and sera and cerebrospinal fluid (CSF) from brain tumor patients. The NSE content of neuroblastoma cell lines was more than sixfold that of the glioma and medulloblastoma lines; the CK-BB content of neuroblastoma and medulloblastoma lines was fourfold to nineteen-fold that of the glioma and other lines. Expression of NSE in neuroblastoma cell lines was not related to time in culture and was cell line specific. NSE in ex vivo medulloblastomas was raised six to ten times that in astrocytomas and gliomas, although no significant differences were noted for the CK-BB content. Serum and CSF NSE levels were markedly raised above control values in 10 of 29 and 6 of 10 cases of astrocytoma, respectively. Raised CK-BB levels in serum (greater than 10 ng/ml) and CSF (greater than 12 ng/ml) were found in 9 of 18 and 2 of 10 patients, respectively. These data suggest that NSE is preferentially expressed by neuroblastoma lines and medulloblastomas and that NSE and CK-BB may have clinical utility as markers for prognosis, diagnosis, and monitoring of response to therapy.

Immunoradiometric and immunohistochemical demonstration of neuron-specific enolase in experimental rat gliomas.

A number of neural and nonneural tumor cell lines of rat and human origin were assayed for neuron-specific enolase (NSE) by radioimmunoassay. Most neural tumor cell lines had appreciably higher levels of NSE than did the nonneural tumor cell lines, the highest levels being found in two anaplastic rat glioma lines ( F98 and T24). These two lines contained more than twice the amount of NSE found in a rat pheochromocytoma line (PC12) and in neuroblastoma lines derived from rats ( B35 and B50 ) or humans (IMR-32 and SHSY - 5Y ). Several of the rat glioma and schwannoma lines were inoculated intracerebrally into syngeneic rats. In the resulting tumors, NSE was demonstrable by immunohistochemistry only in those from the F98 and T24 cell lines. A number of ethylnitrosourea-induced rat tumors were also examined immunohistochemically for NSE: NSE was demonstrated in three anaplastic gliomas; three astrocytomas; and two mixed gliomas. Reactive astrocytes were also positive. Fibroadenomas of apocrine and mammary glands in rats were weakly positive, but other extraneural tumors tested were negative. Since normal neuronal elements, axonal swellings, and amine precursor uptake and decarboxylation cells are strongly positive for NSE, whereas glia and most other normal cells are negative, we hypothesize that the elevated metabolic demands imposed on neoplastic and reactive glial cells and on some extraneural tumors necessitate the opening up of metabolic pathways that are normally operative only in neurons and neuroendocrine cells, therefore resulting in the synthesis of the more stable neuron-specific form of enolase.

Establishment and identification of small cell lung cancer cell lines having classic and variant features.

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.

Potential utility of serum neuron-specific enolase levels in small cell carcinoma of the lung.

To assess the value of neuron-specific enolase (NSE) as a possible biomarker of small cell lung cancer, serum levels were determined by radioimmunoassay in 93 newly diagnosed untreated patients and were compared to the NSE levels of 20 healthy adult controls [9.6 +/- 0.7 (S.E.) ng/ml]. Serum NSE was elevated (greater than 20 ng/ml) in 73% of all patients including 23 of 39 (59%) with limited-stage disease and 45 of 54 (83%) with extensive-stage disease. The mean serum NSE was significantly higher in extensive-stage disease (94.5 +/- 13.8 ng/ml) compared to the mean value for limited-stage disease (33.7 +/- 4.7 ng/ml) (p less than 0.001). NSE was elevated in all patients with three or more sites of metastatic disease. Serial NSE determinations were obtained on 57 small cell lung cancer patients. NSE levels fell in 40 of 50 (80%) of patients responding to treatment, increased in 5 of 7 (71%) of patients with progressive disease, and increased in 30 of 35 (86%) of patients who relapsed. A persistent rise in serum NSE occurred as many as 12 weeks before the clinical recognition of relapse in 15 of 23 (65%) of patients for whom adequate serial NSE data were available. These findings indicate that serum NSE may be a useful marker for staging, monitoring treatment, and predicting relapse in patients with small cell lung cancer.

Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes.

5-Amino-4-imidazolecarboxamide (AICA) riboside, the nucleoside corresponding to AICA ribotide (AICAR or ZMP), an intermediate of the de novo pathway of purine biosynthesis, was found to exert a dose-dependent inhibition on gluconeogenesis in isolated rat hepatocytes. Production of glucose from lactate-pyruvate mixtures was half-maximally inhibited by approximately 100 microM and completely suppressed by 500 microM AICA riboside. AICA riboside also inhibited the production of glucose from all other gluconeogenic precursors investigated, i.e., fructose, dihydroxyacetone, and L-proline. Measurements of intermediates of the glycolytic-gluconeogenic pathway showed that AICA riboside provoked elevations of triose phosphates and fructose-1,6-bisphosphate and decreases in fructose-6-phosphate and glucose-6-phosphate. The effects of AICA riboside persisted when the cells were washed 10 min after its addition but were suppressed by 5-iodotubercidin, an inhibitor of adenosine kinase. AICA riboside provoked a dose-dependent buildup of normally undetectable Z nucleotides. After 20 min of incubation with 500 microM AICA riboside, ZMP, ZTP, and ZDP reached 3, 0.3, and 0.1 mumol/g cells, respectively. Concentrations of ATP were not significantly modified by addition of up to 500 microM AICA riboside when the cells were incubated with lactate-pyruvate but decreased with fructose or dihydroxyacetone. The activity of rat liver fructose-1,6-bisphosphatase was inhibited by ZMP with an apparent Ki of 370 microM. It is concluded that AICA riboside exerts a suppressive effect on gluconeogenesis because it provokes an accumulation of ZMP, which inhibits fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic and clinical aspects of adenosinergic neuromodulation.

Adenosine and the methylxanthines have marked and opposite effects on behavior both of which are now thought to be mediated by cell surface adenosine receptors present in brain. These receptor sites have now been characterized using simple radioreceptor ligand binding techniques. Pharmacologic, autoradiographic and behavioral studies involving adenosine and the methylxanthines strongly suggest a neuromodulatory role for adenosine and indicate that adenosinergic neurons constitute an important central nervous system depressant system. A key component of the adenosinergic system is the adenosine uptake site which represents the inactivation mechanism for receptor mediated adenosine action. The adenosine uptake site can be identified as distinct from the adenosine receptor using a specific ligand. The two key components of the adenosine system, i.e., the receptor and uptake site, can therefore be studied using simple binding techniques. This should facilitate the development of new drugs specific for each system. Adenosine agonists can be expected to have sedative, anticonvulsant and anxiolytic actions whereas adenosine antagonists such as caffeine have stimulant and anxiogenic properties. Adenosine uptake blockers should have pharmacologic actions similar to adenosine agonists. The adenosinergic system, therefore, offers unique opportunities for developing new and potentially useful clinical agents.

Characterization of calcitonin- and adrenocorticotropin-producing human cloned cell lines.

We have developed three human cloned cell lines that produce immunoreactive human calcitonin (ihCT) and ACTH (iACTH) as well as exhibit characteristics of cultured neural cells. Clones HMS-41/I, -78/2, and -98/2 were developed from cell lines HeLa AV3, MBA 9812 (bronchogenic carcinoma), and SW 267 (pheochromocytoma), respectively. Karyological analysis of both the parent and the cloned cell lines confirmed the identity of HeLa AV3 and MBA 9812. When grown in serum-free media designed for culturing neural cells, the patterns of production for both ihCT and iACTH varied among the clones. The multiple patterns of hormone production suggest that the mechanisms involved in the biosynthesis, processing, and secretion of these hormones differ among the clones. The clones contain neuron-specific enolase and the putative neurotransmitters beta-alanine and gamma-amino butyric acid, and they respond to cAMP analogs by differentiating, as noted by the extension of neurites (except the HeLa-derived HMS-41/I). The iACTH extracted from cells and synthetic ACTH exhibited equivalent profiles upon isoelectric focusing. The forms of ihCT noted in cell extracts were similar to those observed in extracts of human tumor tissue. Our rabbit antiserum to hCT failed to detect ihCT in those cell extracts prepared for ACTH determination or in extracts of rat pituitaries, but it did detect CT in rat thyroids by both RIA and immunofluorescent procedures. We concluded that our antisera to hCT do not detect the precursor form of ACTH. The availability of these cloned cell lines provides model systems for examining the production of these peptide hormones and the concomitant expression of neural and endocrine characteristics.

The benzodiazepine--GABA--chloride ionophore receptor complex: common site of minor tranquilizer action.

The demonstration of specific recognition sites for benzodiazepines in the mammalian CNS has altered current thinking on the mechanisms of action of the benzodiazepines as well as the neurochemical events which are associated with anxiety. Recent studies suggest that the physiological regulation of the benzodiazepine receptor is far more complex than initially believed and includes a functional coupling to both a GABA receptor and an associated chloride ionophore. It now appears that a number of other psychopharmacologic agents, including minor tranquilizers other than the benzodiazepines as well as several convulsants and anticonvulsants, may exert their pharmacologic effects by affecting one or more regulatory sites on the benzodiazepine receptor complex. In addition to a number of drugs, at least one endogenous small molecular weight compound that has been isolated from the crude synaptosomal fraction of bovine cerebral cortex also appears to modulate this receptor complex.

The existence and neurobiological significance of neuronal and glial forms of the glycolytic enzyme enolase.

The isoenzymes of the glycolytic enzyme enolase have been separated and purified. The structural and functional properties of two brain enolases are described. Immunocytochemical techniques have established that one brain enolase is restricted to neuronal cells (neuron-specific enolase, NSE) while the other is localized in glial cells (nonneuronal enolase, NNE). The brain enolases, therefore, represent the first example of functional markers for neuronal and glial cell types in brain. The two enzymes are structurally distinct with the evidence establishing that they are products of separate genes. Functionally, the neuronal enolase has been demonstrated to be uniquely stable to concentrations of chloride salts that rapidly inactivate the glial enzyme. NSE may therefore represent an adaptation of this enzyme that is specifically suited to the neuronal milieu. A specific radioimmunoassay is described for NNE and NSE with the studies reported indicating that neuronal enzyme levels vary considerably when different brain areas are compared, suggesting a relationship between functional activity and levels of NSE. In addition to being a marker for neuronal cells, NSE has also been found to be present in various glands. The cells of the APUD series (amine precursor uptake and decarboxylation cells) in the pituitary, adrenal medulla, pineal, thyroid, and pancreas have been shown to contain NSE. NSE is, therefore, also a marker for these neuronlike endocrine cells since they are the only cells other than neurons that contain this protein.

Brain-specific benzodiazepine receptors and putative endogenous benzodiazepine-like compounds.

The recent demonstration of high-affinity binding sites for the benzodiazepines in the mammalian CNS has provided new information on the mechanism of action of this important class of drugs. The presence of these binding sites has prompted studies on their pharmacological and physiological significance, including attempts at isolating an endogenous ligand. The results presented here support the pharmacological importance of these binding sites as receptors, since there is a highly significant correlation between the occupation of these sites by various benzodiazepines and their clinical effects. Our results further support the physiological significance of the benzodiazepine receptor since brain-specific receptors can be demonstrated in the intact animal under physiological conditions. The isolation of a number of substances from bovine brain that competitively inhibit 3H-diazepam binding to synaptosomal membranes suggests the presence of an endogenous ligand. Two of these substances have been identified as the purines inosine and hypoxanthine. Pharmacological studies of these purines suggest that they may have diazepam-like effects in vivo. These results support the existence in brain of endogenous benzodiazepine-like compounds that could normally be involved in ameliorating anxiety and (or) seizure activity.

Cerebrospinal fluid neuron-specific enolase is reduced in Alzheimer's disease.

Neuron-specific enolase (NSE), a glycolytic enzyme enolase found in brain, was examined in the cerebrospinal fluid and serum of 30 patients with presumptive Alzheimer's disease (AD) and of 13 healthy controls and evaluated as a measure of neuronal functional activity associated with AD. The cerebrospinal fluid NSE levels of patients with AD were significantly reduced and serum NSE levels were significantly increased from controls. Cerebrospinal fluid NSE levels may be representative of central nervous system cell loss or a decrease in neuronal functional activity associated with AD.

Evidence for neural origin and PAS-positive variants of the malignant small cell tumor of thoracopulmonary region ("Askin tumor").

The differential diagnoses of childhood and adolescent tumors composed of small round cells include a distinctive clinicopathological entity called malignant small cell tumor (MSCT) of the thoracopulmonary region in childhood. In the present study, 15 such tumors that fulfilled the criteria by Askin et al. were examined for features of possible neural differentiation by light and electron microscopy (EM). With hematoxylin-eosin stain (H&E) the tumors were made up of small undifferentiated cells; rosette formation was noticed in four cases. By immunohistochemistry all 15 tumors were positive for neuron/specific enolase (NSE), which is a specific marker for neural elements and their tumors including neuroblastomas. Ten of 15 MSCT had positive PAS staining. Ultrastructurally dense core (neurosecretory) granules and cell processes indicative of neuronal differentiation could be recognized in 10 of 14 tumors. The dense core granules were often atypical. Filamentous cytoskeleton, never observed in Ewing's sarcoma, was often present. Based on the current results, MSCT of the thoracopulmonary region can be considered a peripheral neuroectodermal tumor with the possible origin in intercostal nerves. MSCTs are generally misdiagnosed as Ewing's sarcoma due to their primitive appearance in H&E sections and their periodic acid-Schiff positivity. NSE immunostaining, preferably augmented by electron microscopy, is necessary for their correct diagnosis.

Chronic benzodiazepine treatment increases [3H]muscimol binding in mouse brain.

High affinity [3H]muscimol receptors were analyzed in the forebrain and cerebellum of mice that had been treated on a chronic regimen with chlordiazepoxide and clonazepam. In the forebrain, only clonazepam induced an increase in the number of muscimol binding sites 2 hr after drug treatment. In the cerebellum, both the chlordiazepoxide and clonazepam-treated animals showed an increased number of muscimol binding sites 2 hr after drug treatment. At 26 hr after drug treatment, only the clonazepam-treated animals still revealed an increased number of cerebellar muscimol binding sites. Chronic benzodiazepine administration, therefore, induced an increase in the apparent number of high affinity muscimol binding sites in both the forebrain and cerebellum.

Adenosine antagonists. Lack of effect on the inhibition of kindled seizures in rats by carbamazepine.

This study investigates the ability of adenosine antagonists to reverse the anticonvulsant effects of carbamazepine on amygdala-kindled seizures in order to elucidate the possible physiological relevance of the potent effects of carbamazepine on adenosine receptors. At large but subconvulsant doses, neither caffeine nor theophylline altered the anticonvulsant potency of carbamazepine, even though caffeine by itself significantly increased the duration of the kindled afterdischarge. The adenosine agonist cyclohexyladenosine (CHA), administered intraperitoneally at a dose that produced sedation, had no effect on the kindled seizures. Although carbamazepine potently displaces the binding of several adenosine ligands in vitro, the present data do not suggest that the anticonvulsant effects of carbamazepine on amygdalakindled seizures are mediated by an adenosine agonist-like action.

Chronic clonazepam administration induces benzodiazepine receptor subsensitivity.

Clonazepam and chlordiazepoxide were administered chronically in increasing doses for three weeks in two different strains of mice. Forebrain [3H]diazepam binding was assayed in groups of mice sacrificed at 2, 26, 50 hr and 10 days following the last dose. Scatchard and single point analyses revealed a significant decrease in the number of [3H]diazepam binding sites [Bmax] which persisted for at least two days following chronic clonazepam treatment. The Bmax changes observed following chlordiazepoxide treatment were less pronounced than those elicited by clonazepam. No significant changes in receptor binding affinity (Kd) were detected with either drug. In the clonazepam-treated animals, Bmax values returned to normal by day 10 after drug treatment. Chronic benzodiazepine administration therefore induced a decrease in the apparent number of benzodiazepine binding sites in the mouse forebrain. The magnitude and duration of the observed subsensitivity appears to depend on the potency of the administered benzodiazepine.

Interactions between calcium channel compounds and adenosine systems in brain of rat.

A number of organic ligands of calcium channels were investigated for possible actions on several aspects of adenosine systems in the cerebral cortex of rat. The principle findings of the present study were that a number of antagonists of calcium channels and the agonist compound Bay K 8644 inhibited binding to adenosine receptors, binding to nucleoside transporters, and the accumulation of [3H]adenosine with a low microM potency. 2-Nitrophenyl dihydropyridine derivatives were more potent than 3-nitrophenyl dihydropyridine or non-dihydropyridine ligands of calcium channels at inhibiting binding to adenosine receptors. Dihydropyridine ligands of calcium channels were more potent than non-dihydropyridine ligands of calcium channel in inhibiting the binding of [3H]nitrobenzylthioinosine to cortical membranes or inhibiting the accumulation of [3H]adenosine into synaptoneurosomes. However, unlike the case of adenosine receptors, no distinction between 2-nitrophenyl and 3-nitrophenyl dihydropyridine derivatives was observed. In addition, the non-dihydropyridine ligand of calcium channels, diltiazem was a weak inhibitor of the accumulation of [3H]adenosine. These results demonstrate that organic ligands of calcium channels, particularly dihydropyridine compounds, can interact with several aspects of adenosine systems.

The development of immunoreactivity for neuron-specific enolase of preoptic and septal neurons in dissociated cultures.

The immunocytochemical visualization of neuron-specific enolase, which is a marker protein for differentiated neurons, was applied to follow the differentiation of preoptic and septal neurons in dissociated cultures. From 4 to 24 days in vitro, the relative numbers of stained neurons were counted and the staining intensity of individual neurons determined by absorbency measurements using a television-based densitometer. Whereas few stained cells could be observed at 4 DIV, 80% of the neurons were neuron-specific enolase-positive at 13 days in vitro. This value remained constant up to 24 days in vitro. The density of the immunoreaction product increased dramatically from 13 to 17 days in vitro and was still higher at 24 days in vitro. The glial and ependymal cells of the carpet, as well as neuroblasts, remained unstained. Comparison with morphological observations and immunocytochemical demonstration of neuronal peptides made earlier shows that expression of neuron-specific enolase closely parallels neuronal differentiation. These observations indicate that cultures derived from preoptic and septal neurons represent a viable model system for the study of neuronal maturation in vitro.