A novel small molecule that directly sensitizes the insulin receptor in vitro and in vivo.

Insulin resistance, an important feature of type 2 diabetes, is manifested as attenuated insulin receptor (IR) signaling in response to insulin binding. A drug that promotes the initiation of IR signaling by enhancing IR autophosphorylation should, therefore, be useful for treating type 2 diabetes. This report describes the effect of a small molecule IR sensitizer, TLK16998, on IR signaling. This compound activated the tyrosine kinase domain of the IR beta-subunit at concentrations of 1 micromol/l or less but had no effect on insulin binding to the IR alpha-subunit even at much higher concentrations. TLK16998 alone had no effect on IR signaling in mouse 3T3-L1 adipocytes but, at concentrations as low as 3.2 micromol/l, enhanced the effects of insulin on the phosphorylation of the IR beta-subunit and IR substrate 1, and on the amount of phosphatidylinositol 3-kinase that coimmunoprecipitated with IRS-1. Phosphopeptide mapping revealed that the effect of TLK16998 on the IR was associated with increased tyrosine phosphorylation of the activation loop of the beta-subunit tyrosine kinase domain. TLK16998 also increased the potency of insulin in stimulating 2-deoxy-D-glucose uptake in 3T3-L1 adipocytes, with a detectable effect at 8 micromol/l and a 10-fold increase at 40 micromol/l. In contrast, only small effects were observed on IGF-1-stimulated 2-deoxy-D-glucose uptake. In diabetic mice, TLK16998, at a dose of 10 mg/kg, lowered blood glucose levels for up to 6 h. These results suggest, therefore, that small nonpeptide molecules that directly sensitize the IR may be useful for treating type 2 diabetes.

Effects of bradykinin on PC-12 cell differentiation.

PC-12 cells are used as a model for neuronal differentiation because they assume a neuronal phenotype, including the extension of neurites, when exposed to nerve growth factor (NGF). The present results show that bradykinin (BK) also causes PC-12 cells to extend neurites. In addition, BK potentiates the neurite-extending effect of nerve growth factor (NGF), an action which is attenuated by a BK antagonist. The potentiation of neurite extension produced by the combination of BK and NGF may be mediated at the receptor level, as indicated by an NGF-induced alteration of BK binding.

Identification of 3H-bradykinin binding sites in PC-12 cells and brain.

3H-Bradykinin binding sites with the characteristics of receptors were identified in homogenates of bovine hippocampus and PC-12 cells. The characteristics of the binding were similar in both types of tissue and paralleled those of the B2 bradykinin receptor. Following exposure to nerve growth factor for 72 hours, the number of BK binding sites in PC-12 cells significantly increased, but other characteristics of the binding remained unchanged.

Effects of sigma agonist compounds on local cerebral glucose utilization: relationship to psychotomimetic properties.

The effects of 3, structurally unrelated sigma agonist compounds on local cerebral glucose utilization (LCGU) were compared. The common effects of sigma agonist compounds on LCGU included a moderate, global elevation of LCGU. Superimposed upon this elevation were larger increases in LCGU occurring mainly in components of the limbic system, and the pyramidal and extrapyramidal motor systems. The only structures whose metabolic rates were significantly elevated by all 3 compounds were the frontal cortex and the anterior cingulate cortex. The altered metabolic activity in these limbic brain regions may underly the psychotomimetic effects produced by sigma agonist compounds.

Specific toxic effects of ethylcholine nitrogen mustard on cholinergic neurons of the nucleus basalis of Meynert.

The putative cholinergic neurotoxin, ethylcholine aziridinium ion (AF64A), was injected unilaterally into the nucleus basalis of Meynert (nbM) in order to determine whether it would produce specific damage to the cholinergic cell bodies of this nucleus. Injections of small amounts of AF64A (0.01 nmol in 1 microliter) or of its vehicle had little effect on the appearance of the nbM or on the levels of choline acetyltransferase (ChAT) in the cortex. Injections of larger amounts of AF64A (0.02 and 0.05 nmol in 1 microliter and 0.02 nmol in 10 microliters) produced a loss of diffuse acetylcholinesterase staining in the nbM and a loss of large positively staining neurons. Furthermore, these injections produced a significant reduction of ChAT activity in the central portion of the cortex. However, non-cholinergic neurons in the area of the nbM were not affected by these AF64A injections. In addition, cortical uptake of monoamines was not affected by these lesions. Further increases in the amount of AF64A injected (0.1 nmol in 1 microliter and 0.035 nmol in 10 microliters) caused damage at the site of the injection which was not limited to the cholinergic elements of the nbM. These results suggest that AF64A can be used to produce specific lesions of cholinergic neurons, and therefore may be useful in developing animal models of human disorders involving cholinergic hypofunction, such as senile dementia of the Alzheimer type. However, there is a narrow dose range for producing these specific effects.

Plasticity of [14C]2-deoxy-D-glucose incorporation into neostriatum and related structures in response to dopamine neuron damage and apomorphine replacement.

Unilateral injections of 6-hydroxydopamine into the lateral hypothalamus or ventral tegmental area that damage extensively the mesotelencephalic dopaminergic projection result in significant hemispheric asymmetries in autoradiographic labeling after [14C]2-deoxy-D-glucose (2DG) administration. The incorporation of this glucose analog into the ipsilateral neostriatum, nucleus accumbens septi, olfactory tubercle, and central nucleus of amygdala is decreased relative to the contralateral hemisphere. Concurrently, structures receiving projections from the neostriatum (globus pallidus, entopeduncular nucleus, and substantia nigra, pars reticulata) show enhanced labeling in the 6-hydroxydopamine-injected hemisphere, as do diencephalic structures receiving basal ganglia inputs (ventromedial nucleus and ventrolateral portion of the ventral anterolateral complex of thalamus, lateral portion of lateral habenular nucleus). Most of these 6-hydroxydopamine-induced asymmetries of [14C]2DG labelling are abolished or reversed by the injection of apomorphine i.p. (0.25 mg/kg) or directly into the denervated neostriatum (5 microgram) 7--20 days postoperatively. The metabolic effecgs of apomorphine administration are prevented by pretreatment with the dopamine receptor blocking agent, haloperidol (2mg/kg i.p., 2 h before). Thus, this autoradiographic technique can be used to demonstrate neostriatal output circuits that show altered metabolic activity in response to diminished or excessive forebrain dopamine receptor stimulation. The relevance of these findings to: (1) the behavioral abberations resulting from damage to mesotelencephalic dopaminergic systems; and (2) the 'denervation supersensitivity' to DA receptor stimulants is discussed.

Recovery of function and basal ganglia [14C]2-deoxyglucose uptake after nigrostriatal injury.

Rats with unilateral 6-hydroxydopamine injections along the mesotelencephalic dopaminergic projection showed a profound impairment in localizing somatosensory stimuli on the contralateral body surface at 3 days postoperatively. Approximately one-half of the affected animals recovered the ability to localize tactile stimuli during 6 weeks postoperatively, whereas the remainder did not. When it occurred, the recovery of sensorimotor function began between the third and fifth day postoperatively and plateaued between days 14 and 21. Unilateral damage to these dopaminergic neurons resulted in hemispheric asymmetries of [14C]2-deoxyglucose incorporation at 3 days postoperatively. For structures that normally receive a dopaminergic innervation (e.g., neostriatum, nucleus accumbens septi, olfactory tubercle) the autoradiographic density of the injured side was decreased relative to the intact hemisphere. For structures that receive striatal inputs (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), the autoradiographic density was increased on the side of the injury. This pattern of altered [14C]2-deoxyglucose incorporation was still present at 6 weeks postoperatively in animals that showed no recovery of somatosensory localization during that time. In contrast, rats that did recover showed no hemispheric asymmetries within the anterior neostriatum, globus pallidus, or substantia nigra pars reticulata at 6 weeks postoperatively, and the time course of normalization of metabolic activity in these structures was similar to that for behavioral restoration. These results directly demonstrate the importance of the neostriatum and particular structures efferent to it in the recovery of sensorimotor functions after striatal dopamine depletion. The types of neuronal plasticity within this basal ganglia circuitry responsible for the normalization of [14C]2-deoxyglucose incorporation and behavior are discussed.

Brain-derived cells contain a specific binding site for Gp120 which is not the CD4 antigen.

Infection with the human immunodeficiency virus (HIV-1) often produces a set of neuropsychiatric dysfunctions which have been termed the AIDS dementia complex. This complex appears due to the infection of brain cells by HIV-1. If so, brain cells might be expected to contain a binding site for the same viral envelope glycoprotein that enables HIV-1 to bind to other cells (e.g. CD4+ T-cells), gp120. The present study shows that the cells of the brain-derived U-138MG, U-373MG, SK-N-MC and SK-N-SH cell lines bind gp120 in an inhibitable fashion. Binding of gp120 to these cells is inhibited by the dyes Aurintricarboxylic acid (ATA) and Evans blue (EB), which are known to inhibit specific gp120 and HIV-1 binding, and block HIV-1 infection, in CD4-expressing cells. Binding is not inhibited by Aurin, a dye related to ATA but lacking its anti-HIV effects. As expected, anti-CD4 antibodies are ineffective in blocking gp120 binding to brain-derived cells. These results suggest that human brain-derived cells possess a specific binding site for gp120 that is not the CD4 antigen.

Acetylcholinesterase associated with dopaminergic innervation of the neostriatum: histochemical observations of a heterogeneous distribution.

Unilateral injections of 6-hydroxydopamine that damaged the mesotelencephalic dopaminergic projection resulted in decreased acetylcholinesterase (AChE) staining in the ipsilateral neostriatum. This reduction, which was most apparent in the posterior and medial anterior neostriatum, occurred in rats killed 3 days or more after the 6-hydroxydopamine injection. No hemispheric asymmetries of AChE staining were found in the medial nucleus accumbens septi, olfactory tubercle or amygdala. Thus, some of the AChE in the neostriatum appears to be localized to its dopaminergic innervation; however, the enzyme may be heterogeneously distributed throughout this structure's dopaminergic afferent fibers. AChE seems not to be associated with dopaminergic axons innervating the limbic forebrain.

Altered succinate dehydrogenase activity of basal ganglia following to mesotelencephalic dopaminergic projection.

Unilateral damage to the mesotelencephalic dopamine-containing projection of rats by intracerebral 6-hydroxydopamine injection, alters the staining of the tricarboxylic acid cycle enzyme succinate dehydrogenase in the basal ganglia. Relative to the intact hemisphere, the neostriatum ipsilateral to the damage shows diminished enzyme staining, while the ipsilateral globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata all show enhanced deposition of reaction product. These asymmetries are detectable in some animals by 3 days after surgery and increase in magnitude at longer postoperative survival times. The alterations in succinate dehydrogenase staining appear to be related to the severity of the sensorimotor impairments resulting from the injury.

Plasticity of neostriatal dopamine receptors after nigrostriatal injury: relationship to recovery of sensorimotor functions and behavioral supersensitivity.

Rats given unilateral injections of 6-OHDA along the course of the mesotelencephalic dopaminergic projection show impairments in contralateral sensorimotor functions from which they often recover. Such rats also display an enhanced sensitivity to DA receptor stimulants, e.g. apomorphine, as revealed by contralateral turning, and an increased binding of neuroleptic compounds (e.g. [3H]spiroperidol) to the denervated striatum. This research examines the relationship of these receptor changes to both behavioral supersensitivity and recovery of sensorimotor functions by quantifying the time course of each phenomenon after injury. The supersensitivity to apomorphine and the behavioral recovery developed with a similar time course after injury, being evident within 1.5-3 days and reaching nearly maximal levels by 2 weeks postoperatively. A significant increase in in vivo [3H]spiroperidol binding to the denervated striatum occurred by 4 days postoperatively, and the magnitude of this change increased linearly during the first postoperative month. In contrast, the in vitro binding of this ligand to membranes of the denervated striatum was not increased until 3 weeks after the lesion. The results suggest that a proliferation of DA receptors may contribute to the pharmacological supersensitivity and the recovery of function, and that these early receptor changes may be revealed with greater sensitivity using in vivo binding techniques.

Chronic lithium administration alters behavioral recovery from nigrostriatal injury: effects on neostriatal [3H]spiroperidol binding sites.

Unilateral damage to the mesotelencephalic dopaminergic projection of the rat produces impairments in sensorimotor functions, including an inability to localize contralateral somatosensory stimuli. Many rats with 6 hydroxydopamine injections along this pathway show a gradual improvement in somatosensory localization during the first post-operative month. One mechanism that may contribute to this behavioral recovery is the proliferation of dopamine receptor sites in the affected neostriatum. The role of these binding site changes in the recovery was tested by chronic administration of lithium to rats. Rats given lithium in their drinking water for 4 weeks after the 6-hydroxydopamine injection showed a greatly attenuated recovery of sensorimotor functions, compared to brain-damaged rats drinking unadulterated water. When rats that were given lithium for the first four postoperative weeks were subsequently given unadulterated water to drink, they recovered normally. Lithium treatment did not prevent the augmentation of [3H]spiroperidol binding in the neostriatum ipsilateral to the lesion, relative to the contralateral control neostriatum, at 4 or 8 weeks post-operatively. Thus, lithium treatment dissociates behavioral recovery after nigrostriatal injury from the lesion-induced relative proliferation of neostriatal [3H]spiroperidol binding sites. However, the lithium treatment decreased the absolute amount of specific [3H]spiroperidol binding to both the ipsilateral and contralateral neostriata. The significance of these findings for the neural mechanism underlying this recovery sequence is discussed.

Quantitative morphometric analysis of the neurotoxic effects of the excitotoxin, ibotenic acid, on the basal forebrain.

The effects of injections of the excitotoxin, ibotenic acid, into the nucleus basalis magnocellularis (NBM) were quantitatively studied. Besides destroying NBM neurons, ibotenic acid also produced comparable cell destruction within the adjacent medial amygdaloid nucleus and globus pallidus. Since the globus pallidus is spared in victims of senile dementia of the Alzheimer type (SDAT), although the adjacent NBM and amygdala are damaged, these data are not consistent with the theory that the damage to the basal forebrain seen in SDAT victims is produced by elevated levels of endogenous excitotoxin. These data also question the validity of using ibotenic acid-induced NBM lesions as a model of the cholinergic deficit in SDAT.

Inhibition of the binding and the behavioral effects of thyrotropin-releasing hormone (TRH) by the triazolobenzodiazepines.

The abilities of 4 triazolobenzodiazepines, adinazolam, alprazolam, estazolam and triazolam, to inhibit thyrotropin-releasing hormone (TRH) receptor binding and to antagonize the narcoleptic effects of TRH were examined. The IC50 values for inhibition of 3H-3-methyl-His-2-TRH (MeTRH) binding ranged from 19 microM to 477 microM, and the Hill coefficient from 0.53 to 0.98. Similar ranges of values were obtained from benzodiazepines of other structural classes. Thus, the inhibition of TRH receptor binding by the triazolobenzodiazepines is similar to that produced by other types of benzodiazepines. Furthermore, the triazolobenzodiazepine, alprazolam, antagonized the narcoleptic effect of TRH. However, this action is not necessarily linked to its inhibition of TRH receptor binding since alprazolam also inhibited the narcoleptic effect of amphetamine.

Rotation induced by intranigral injections of GABA agonists and antagonists: zone-specific effects.

Recent investigations of the function of the strionigral pathway have utilized the intranigral injection of gamma-aminobutyric acid (GABA) agonist and antagonist drugs. While the unilateral application of these substances typically produces rotational behavior, the direction of this turning (ipsilateral or contralateral to the injected hemisphere) differs in several reports. The present study determines whether the direction of this drug-induced turning depends upon the locus of migral stimulation. Picrotoxin and bicuculline methiodide were injected into either the pars compacta or the pars reticulata of the substantia nigra at several anterior-posterior levels. Injection of these drugs into the pars compacta resulted in ipsilateral turning while injection into the pars reticulata produced contralateral rotation. Both of these effects were dose-dependent and were elicited by similar threshold doses of picrotoxin. Prior 6-hydroxydopamine treatment abolished the ipsilateral but not the contralateral rotation. In contrast, muscimol injections produced contralateral turning independent of whether they were made into the pars compacta or pars reticulata. However, 6-hydroxydopamine treatment only attenuated the contralateral turning produced by pars compacta injections. These findings provide a histological basis for understanding the different types of turning behavior elicited by the intranigral injection of GABA agonists and antagonists. In addition, they suggest that GABA receptors mediate at least two independent actions in substantia nigra.

Discriminative stimulus properties of phencyclidine (PCP)-related compounds: correlations with 3H-PCP binding potency measured autoradiographically.

Several PCP analogs, the putative PCP agonist MDP, and the sigma receptor agonists SKF-10,047 and dexoxadrol were tested for their ability to substitute for PCP in animals trained to discriminate PCP from saline. The potencies of these compounds in substituting for PCP in the behavioral task correlated with their abilities to inhibit the specific binding of 3H-PCP to rat hippocampal sections measured autoradiographically, which occurred at a single class of sites with an affinity of 85 nM and a capacity of 2646 fmol/mg protein. In addition to this specific binding, an additional nonspecific but displaceable fraction of total 3H-PCP binding was present. These results suggest that the specific 3H-PCP binding site measured in the hippocampus may be the type of binding site which mediates the behavioral effects of PCP and related compounds. Therefore, measurement of the inhibition of 3H-PCP binding at this site might aid in the search for PCP antagonists.

Comparison of the binding and functional actions of angiotensin agonists in clone 9 cells: additional evidence for angiotensin II receptor heterogeneity.

The effects of the angiotensin-II (AII) agonists and antagonists on both 125I-SARILE binding and phosphoinositol (PI) accumulation in clone 9 cells were examined. Clone 9 cells, which are derived from rat liver, have been shown to respond to AII agonists with an increase in PI accumulation which is inhibitable by Sar1,Ile8-AII (SARILE) and DUP-753 but not PD-123319, suggesting that they possess the AT1 subtype of AII receptor. The present results confirmed these properties. The order of potency of AII agonists was AII > AIII > AI. Clone 9 cells also possessed binding sites for 125I-SARILE. The majority of these were AT1 type receptors, although a small number of AT2 receptors may also have been present. The order of potency of AII agonists in inhibiting 125I-SARILE binding was AII >> AIII = AI. The difference in rank order of potency between the functional and binding assay was due to AIII being much less potent in the binding assay than the functional assay. Since the potency of AIII relative to AII was lower than that at either AT1 or AT2 subtypes of AII receptor, these data suggest that an additional subtype, with selectively low affinity for AIII, exists.

Dopamine D2 receptor stimulation of Na+/H+ exchange assessed by quantification of extracellular acidification.

A microphysiometer was used to quantify the rate of extracellular acidification by C6 glioma cells and L fibroblasts expressing recombinant dopamine D2 receptors. The dopamine D2 receptor agonist, quinpirole, accelerated the rate of acidification of the medium by C6 cells expressing either the short or long form of D2 receptors, D2(415) and D2(444), but not by wild-type cells that were not transfected with a D2 receptor cDNA. The rate of acidification increased with increasing concentrations of quinpirole up to 100 nM. Inhibition of the response by the dopamine D2 antagonist, spiperone, provided additional evidence that the enhanced extracellular acidification resulted from stimulation of D2 receptors. To test the hypothesis that D2 receptor-stimulated extracellular acidification was due to transport of protons by a Na+/H+ antiporter and reflected intracellular alkalinization, the effect of two inhibitors of Na+/H+ exchange, amiloride and methyl-isobutyl-amiloride, was determined. Both compounds inhibited quinpirole-induced extracellular acidification at concentrations that did not alter D2 receptor-mediated inhibition of adenylylcyclase or radioligand binding to D2 receptors. In addition, quinpirole-induced extracellular acidification was greatly inhibited by removal of sodium from the extracellular medium, confirming the participation of Na+/H+ exchange in the extrusion of acid. Quinpirole (100 nM) also increased the rate of extracellular acidification by L cells expressing D2(415), LZR1 cells. Treatment with pertussis toxin (100 ng/ml for 18 h) had no effect on the quinpirole-induced acid extrusion by C6D2(415) and LZR1 cells, although the same pertussis toxin treatment regimen completely prevented inhibition of adenylylcyclase. We conclude that recombinant D2 receptors accelerate Na+/H+ exchange in C6 cells and L fibroblasts by a pathway that does not involve inhibition of adenylylcyclase or pertussis toxin-sensitive G proteins.

Antimuscarinic effects of (R)- and (S)- oxyphencyclimine hydrochloride.

The (R)-(+)- and (S)-(-)-enantiomers of the anticholinergic compound, oxyphencyclimine, were synthesized from (R)-(-)- and (S)-(+)-2-cyclohexyl-2-hydroxy-2-phenylethanoic acid, respectively. The potencies of the enantiomers were compared using a cholinergic receptor binding assay. The (R)-(+)-enantiomer inhibited binding 29 times more potently than the (S)-(-)-enantiomer.