Opiate peptide receptors on intact NIE-115 neuroblastoma: radioligand binding properties, intracellular response, and effects of increasing membrane cholesterol

1. Binding of [³H]methionine-enkephalin to intact NIE-115 neuroblastoma cells (competing ligand: naloxone) revealed a homogenous population of receptors with a density (B_max) of 79.0 ± 6.5 mol/mg protein (mean SEM, N=3) and an apparent K, of 5.33 ± 1.63 mM.

2. The order of displacement of [³H]met-enkephalin was met-/leu-enkephalin > naloxone > morphine, suggesting that it is of the delta receptor class.

3. Specific binding was heat-labile, stereospecific and sensitive to Na⁺.

4. Adding met-enkephalin to intact neuroblastoma caused reductions of both basal and prostaglandin E₁-stimulated levels of cyclic AMP (41.4 ± 4.0% (N=6) and 45.1 ± 2.4% (N=3) of control levels, respectively). Maximum inhibition (naloxone-reversible) was observed as low as 10⁻⁷ M met-enkephalin.

5. Preliminary results suggest that cells grown in cholesterol-supplemented medium show reduced binding of [³H]met-enkephalin.     

Cell cultures as models for studying neural functions

1. Two cell culture systems were used for studies of neural functions in vitro. A neuronal hybrid cell line (neuroblastoma x glioma hybrid cells) and primary glial-rich cultures of newborn murine brain.

2. The level of cyclic AMP in both systems is regulated by two groups of hormones, those that stimulate and those that inhibit formation of cyclic AMP.

3. Among the inhibitory hormones active on the hybrid cells are opioids. Therefore the cells are being used in the elucidation of action of opioids.

4. The list of stimulating and inhibitory hormones regulating the primary glial-rich cultures includes several peptide hormones such as the gastro- intestinal peptides secretin and vasoactive intestinal peptide, the calcaemic hormones parathyrin and calcitonin, adrenocorticotropin and melanotropins, and somatostatin.

5. Noradrenaline (via - and (β-adrenergic receptors) and adenosine (via A₁) and A₂ receptors) inhibit and stimulate cyclic AMP synthesis in the primary glial-rich cultures.

6. Bradykinin slowly hyperpolarizes the hybrid cells and elicits formation of cyclic GMP. Both responses desensitize rapidly.

7. Substance P increases the permeability of hybrid cells for Na⁺, as measured by using ¹⁴C-guanidinium as substitute for Na⁺.

8. Hybrid cells actively accumulate taurine, an amino acid that appears to fulfill important functions in the nervous system.

9. The transport of taurine across the plasma membrane is highly specific for and strictly dependent on Na⁺.

10. The pumped power station hypothesis of taurine action in the nervous system views taurine gradient plus taurine carrier as a transport system for the elimination of sodium from neurons during phases of high neuronal activity.     

Receptor expression in primary cultures of neurons or astrocytes

1. β-Adrenergic receptors are abundant on astrocytes which contain mainly the β₁ subtype. These cells also possess ₁ and ₂ receptors.

2. Neurons display fewer β-adrenergic receptor sites and those present are of the β₂ subtype.

3. Dopaminergic receptors are found on astrocytes and show a regional variation.

4. Serotonergic receptors have been demonstrated on astrocytes but are expressed to a larger extent in neurons.

5. Astrocytes display muscarinic receptors but have no receptor sites for the transmitter amino acids glutamate and GABA.

6. GABA receptors are present on cerebellar granule cells, are induced by the presence of GABA agonists in the medium and mediate an evoked release of glutamate.

7. Glutamate receptors are found on cultured cortical interneurons. Some, but not all, peptide hormones have receptors on astrocytes and/or neurons in primary cultures.     

Effect of dopaminergic agents on levels of dynorphin1–8 in rat striatum

1. In this study, the possibility that striatal dynorphin_1–8 levels would be altered by either acute or chronic treatment with dopaminergic agonists or antagonists is examined.

2. Animals were treated chronically (3 weeks) with daily injections of d-amphetamine, phencyclidine or haloperidol. For the acute studies, animals were given a single dose of either d-amphetamine or haloperidol.

3. Dynorphin_1–8 levels were estimated using a radioimmunoassay. Only chronic treatment with d-amphetamine has any effect on dynorphin levels. Following 3 weeks of chronic d-amphetamine, the level of dynorphin_1–8 increased in 2 separate experiments.

4. Acute d-amphetamine or haloperidol treatment or chronic haloperidol or phencyclidine treatment had no effect on striatal dynorphin_1–8 levels. However, chronic (but not acute) d-amphetamine administration does produce a small increase in striatal dynorphin_1–8 levels.     

The effects of conditioning with amphetamine on the thermic effects of amphetamine and pentobarbital

1. Rats were injected with amphetamine (1.5 mg/kg) in the presence of a distinctive set of environmental stimuli (CS₁) and saline in the presence of a different set of environmental stimuli (CS₂) on different days for a total of 10 amphetamine and 20 saline injections.

2. The hyperthermic effect of amphetamine first increased but then declined to levels seen during the very first drug administration.

3. Following the conditioning phase, half the rats were injected with amphetamine in CS₁ and half in CS₂.Although there was little thermic effect of amphetamine injected in CS₁, there was pronounced hyperthermia following amphetamine in CS₂.

4. Next, pentobarbital (30 mg/kg) was administered to half the rats in CS₁ and half in CS₂. The hypothermic effect of pentobarbital was attenuated in CS₂.     

The role of cyclic AMP in chemoreception in the rabbit caroid body

The present study identified physiological factors which influence the generation (and degradation) of cyclic AMP (cAMP) in the arterial chemoreceptor tissue of the mammalian carotid body. Experiments established a 3-way correlation between cAMP generation, neurotransmitter release from chemoreceptor cells, and carotid sinus nerve (CNS) activity. Incubation of carotid bodies in vitro for 10 min in media equilibrated with different low O₂ (‘hypoxic’) gas mixtures (5% O₂ or 10% O₂, balance N₂) elevated basal cAMP levels (100% O₂ media) in proportion to the stimulus intesity. Similar experiments using nodose sensory ganglia showed that low O₂ stimulation did not alter cAMP levels in this non-chemosensory tissue. However, the adenylate cyclase (AC) activator, forskolin (10 μM), evoked large increases in the cyclic nucleotide content in both carotid bodies and nodose ganglia. After chronic (10 days) CSN denervation or synpathectomy, the basal levels of cAMP in the carotid body were elevated; the cAMP response to low O₂ media (stimulus minus control) was increased after CSN denervation but remained unaltered after sympathectomy. The effects of zero Ca²⁺ media on cAMP generation was examined in order to assess whether feedback from released neurotransmitters acting on known (presynaptic) type I cell receptors could have contributed to the observed changes in cAMP. Basal levels of cAMP were increased 2.8-fold, and the response to hypoxic stimulation was elevated 5-fold, in the absence of extracellular Ca²⁺. Forskolin (10 μM) did not alter basal release of [³H]-catecholamines ([³H]CA: synthesized from [³H]tyrosine, or resting CSN discharge; however, stimulus-evoked [³H]CA release and CSN discharge were potentiated in the presence of forskolin. This increased release was primarily due to enhanced efflux of dopamine (DA). At increasing stimulus strengths, however, the relative effect of forskolin on [³H]CA release was diminished. The data suggest that the chemoreceptor type I cells in the carotid body generate cAMP in their transductive response to hypoxia, but that the net levels of cAMP in the tissue are also regulated by both feedback actions of released neurotransmitters and by the sympathetic and sensory innervation to the organ. The effects of forskolin on [³H]CA release and CSN activity, combined with the finding that hypoxia increases the cAMP content of the carotid body, suggest the immediate invlovement of this classical second messenger in chemotransduction and chemotransmission of natural carotid body stimuli.     

Heat shock protein 72 restores cyclic AMP accumulation after heat shock in N18TG2 cells

Although there are several reports on the alteration of intracellular signal transduction during heat shock in somatic cells, the long term effects of heat shock on neuronal cells remain unknown. In this report, we investigated cyclic AMP (cAMP) accumulation and the expression of heat shock proteins following heat shock in mouse neuroblastoma N18TG2 cells. Basal cAMP accumulation, or that stimulated by serotonin (10 μM), cholera toxin (1 μg/ml), and forskolin (1 μM) was suppressed at 0, 3, and 6 h following heat shock (45°C for 30 min). The cAMP levels were restored at 15 and 24 h after heat shock, corresponding with the expression of stress-induced heat shock protein 72 (HSP72). Quercetin, an inhibitor of HSP expression, decreased the expression of HSP72 and inhibited the recovery of cAMP levels 24 h after heat shock. Quercetin also decreased the basal expression of the constitutive heat shock cognate protein 70 (HSC70) and suppressed cAMP accumulation in non-heat shocked cells. These results suggest that stress-induced HSP72 restores cAMP accumulation to control levels following heat shock and that constitutive HSC70 is related to cAMP levels in non-stress conditions.     

Cyclic Nucleotide-gated Channels in Identified Human Olfactory Receptor Neurons

Patch-clamp recordings revealed the presence of a non-desensitizing cyclic nucleotide-gated channel on human olfactory receptor neurons and a fast-desensitizing non-specific cation channel activated by nucleotides on human supporting cells. Cyclic nucleotide-gated channels on olfactory receptor neurons showed selective channel activation by cAMP (K_1/2= 5 μM) and cGMP (K_1/2= 2 μM), a unitary conductance of ∼20 pS, a reversal potential of single-channel currents close to 0 mV, a linear current-voltage relationship over the range of −80 to 80 mV and a strong extracellular but a weaker intracellular blocking effect of Ca²⁺. The channel activity outlasted the cyclic nucleotide pulses for hundreds of milliseconds when higher agonist concentrations (>50 μM cAMP) were applied. The duration of the response was longer than in cyclic nucleotide-gated channels from other species studied so far. The plateau duration and the decay remained constant for pulses with a length of 50−150 ms, whereas pulses shorter than 50 ms successively reduced the time required by shortening the plateau phase. A larger difference for the K_1/2 values of cAMP (K_1/2= 22 μM) and cGMP (K_1/2= 2.5 μM) were found for a small group (n = 3) of cyclic nucleotide-gated channels, pointing to the selective expression of the a-subunit in a small subgroup of olfactory receptor neurons.     

Noradrenergic actions of purkinje and locus coeruleus neurons in culture

1. Explant cultures prepared from neonatal mice were used to study the actions of ionto-phoretically applied noradrenaline (NA) on Purkinje and locus coeruleus neurons, with extracellular and intracellular recording respectively.

2. NA depressed spontaneous activity of Purkinje neurons and enhanced excitatory responses to glutamate in 14/16 cells.

3. In cultures older than 26 days, NA hyperpolarized LC neurons but had no effect on or depressed depolarizing responses to glutamate. The hyperpolarizations were blocked by the selective ₂ antagonist yohimbine.

4. Enhancement of glutamate responses is not a ubiquitous characteristic of NA action and appears not to be associated with ₂ adrenergic receptors.     

Platelet H imipramine binding: A possible predictor of response to antidepressant treatment

1. No significant difference in the density (Bmax) of platelet ³H imipramine recognition sites were found between the group of 20 unmedicated depressed patients and 10 healthy volunteers. The mean K_D value was significantly higher in the population of depressives than in controls.

2. Non-responders (after 2 weeks of treatment with antidepressants) had significantly lower initial Bmax values than responders or control subjects. Density of platelet ³H imipramine site may thus be a predictor of early response to antidepressant therapy.

3. No significant sex differences were found in K_D or Bmax values in the depressed group or in control subjects. There was, however, a seasonal variation in Bmax but not in K_D values of platelet ³H imipramine binding.     

Functional and biochemical basis for multiple muscarinic acetylcholine receptors

1. The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity.

2. [³H]PZ has been used extensively to identify and characterize the putative M₁ (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia.

3. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists.

4. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M₁ and M₂ subtypes.

5. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.     

H-Clonidine binding to membranes of platelets prepared under physiological conditions

(1) A reproducible binding assay has been established to measure ₂-adrenoreceptors on membranes of human platelets prepared under physiological conditions.

(2) Washed platelet suspensions were obtained from fresh blood by a modified method (Mustard et al., 1972) and membranes were prepared by mechanical homogenization.

(3) ³H-clonidine, a selective ₂-adrenoreceptor agonist was selected as the ligand in a concentration range of 2–64 nM. Specific binding of ³H-clonidine was defined as the difference between total bound radioactivity and that not displaced by excess cold clonidine (6.4 × 10⁻⁵M).

(4) Platelets from 14 healthy young male volunteers were analysed using this technique, and one high affinity binding site, with K_D and B_max values in the reported range was found (K_D: 10.14 ± 1.95 nM; B_max: 428 ± 33 fmol/mg protein (Mean) ± S.E.M.).

(5) This technique will be used in future studies that will examine ₂-adrenoreceptor changes in specific subgroups of depressed patients.     

Platelet H-imipramine binding: A state-dependent marker in depression

1. Reduced density of ³H-imipramine binding sites (B_max) to platelets has been reported in depressed patients during an episode of illness. In the present study we assessed the usefullness of decreased B_max of platelet ³H-imipramine binding as an indicator of the depressed state. We also investigated the effect of long-term treatment with imipramine on platelet ³H-imipramine binding.

2. A comparison of platelet ³H-imipramine binding in 10 drug-free depressed patients and 8 normal volunteers revealed significantly lower mean Bmax values in depressed patients, whereas the affinity (K_d) of ³H-imipramine binding was identical in both groups.

3. A longitudinal study of platelet ³H-imipramine binding in 10 depressed patients during and after imipramine treatment (125–200 mg/day) revealed consistently low B_max values despite clinically meaningful improvement. However, B_max values increased significantly following complete remission and remained elevated even after imipramine had been discontinued for 4 weeks.

4. These findings suggest that decrease in the sensity of platelet ³H-imipramine binding sites in depressed patients is not likely to be a direct drug effect and that normalization of this variable may follow clinical remission.     

Immunoreactive thymopoietin in the mouse central nervous system

A thymopoietin-immunoreactive substance (TP-IRS) has been detected in homogenates of mouse spinal cord and brain using a radioimmunoassay; levels were maximal at birth. TP-IRS was also detected in supernatants of mouse neuroblastoma (NIE-115) and primary spinal cord cultures but not human astrocytic and meningeal tumors or mouse primary astrocyte cultures. With affinity purified rabbit anti-TP globulin, immunofluorescent staining was seen in mouse spinal cord cultures in association with nuclear membranes of neurons and, to a lesser degree, flat background cells. From supernatants of NIE-115 cells grown in tritiated leucine and lysine, proteins of approximately 8000 and 4500 Da were isolated by TP affinity chromatography (compared with 5562 Da for thymic thymopoietin). When injected into mice, these neural proteins partially blocked neuromuscular transmission in a manner similar to thymic thymopoietin.     

Bilateral ablation of the corticostriatal projection: Behavioral, biochemical and electrophysiological correlates

1. Striatal neuronal responses to dexamphetamine, 2.5 mg/kg i.p., were examined in normal and bilateral cortically ablated freely moving rats using multiunit recording.

2. Striatal glutamate levels, D₁ and D₂ receptor binding, and haloperidal catalepsy were examined in both sham-operated and ablated animals.

3. Bilateral ablation of the cortex, while not affecting dexamphetamine-induced behavioral activation, changed the Striatal neuronal response from predominantly excitation to inhibition. Striatal glutamate levels were reduced 22% in ablated animals; and dopamine D-2 receptor binding sites were similarly decreased by 20%. In bilaterally ablated animals, haloperidol-induced catalepsy was greatly reduced.     

Effects of prostaglandin E2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus

The present study attempts to elucidate the possible role of adenosine 3′,5′-monophosphate (cAMP) and prostaglandin E₂ (PGE₂) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH-RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P₂ membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 μg/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step-wise increment in several doses of PGE₂ (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE₂ induced significant (P<0.01) increments in LH-RH release at doses of 5.68 × 10⁻⁷, 5.68 × 10⁻⁶, and 5.68 × 10⁻⁵ M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH-RH release was also obtained; forskolin and cholera toxin significantly (P<0.01) stimulated LH-RH release at doses of 1 × 10⁻⁴ and 5.4 × 10⁻¹⁰ M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium. An intermittent infusion of dibutyryl cAMP (dbcAMP: 1 × 10⁻⁷ M, 10-min on, 20-min off) resulted in rhythmic LH-RH release from median eminences superfused in vitro. In separate experiments, to examine adenylate cyclase activity, P₂ membrane fractions from the mediobasal hypothalamus were preincubated with appropriate test agents. Adenylate cyclase reaction was initiated by adding adenosine triphosphate. After a 15-min incubation, the reaction was terminated by boiling, the supernatant recovered and subjected to cAMP determination by RIA. The following results were obtained: (1) in vivo E priming of ovariectomized animals significantly increased basal adenylate cyclase activity of P₂ membrane preparations as compared to those from unprimed rats; (2) known adenylate cyclase activators, such as forskolin and cholera toxin clearly produced a dose-related increase in cAMP production; and (3) PGE₂ at the concentration of 5.68 × 10⁻⁶ M stimulated cAMP production. It appears that cAMP and PGE₂ may be involved in the activation of the LH-RH neural apparatus. It is tempting to postulate that PGE₂ may stimulate adenylate cyclase to increase intracellular cAMP levels which, in turn, trigger the release of LH-RH from the median eminence nerve terminals.     

Distribution of D2 dopamine receptor mRNA in the primate brain.

1. The distribution of the messenger RNA (mRNA) encoding the D₂dopamine receptor has been mapped in the monkey brain by hybridization.

2. Using [35s]-labelled riboprobes corresponding to the region of the D2 dopamine receptor spanning the third cytosolic loop and the sixth and seventh transmembrane domains, specific hybridization was observed in a number of neural structures.

3. High levels of mRNA expression were observed in the caudate, putamen, and claustrum. Significant amounts were also identified in the hippocampus, lateral geniculate nucleus, much of the cortex, amygdala, pons, and thalamus. High levels of this mRNA were also visualized in the substantia nigra, likely reflecting autoreceptor synthesis.

4. While the distribution of D₂ dopamine receptor mRNA was similar between the monkey and previously published maps in the rat, several differences were noted.

5. These results demonstrate the feasibility of visualizing this mRNA in the primate brain, and suggest that a similar analysis of human postmortem brain material may be possible.     

Factors affecting solubilization of the D1 receptor from bovine caudate nucleus

1. The D₁ dopanine receptor was identified using the dopanine agonist ³-ADTN.

2. The ability of various detergents to solubilize this receptor from bovine caudate nucleus was examined.

3. In order to maintain high affinity binding, receptors were solubilized in the presence of dopanine and manganese. GPP(NH)P reduced the yield of soluble receptors.

4. Of the detergents tested, n-octyl-B-D-glucopyranoside gave the best yield of receptors capable of binding H-ADTN and related compounds with high affinity.     

Differing in vitro survival dependency of mouse and rat NG2+ oligodendroglial progenitor cells

NG2 chondroitin sulfate proteoglycan is a surface marker of oligodendroglial progenitor cells (OPCs) in various species. In contrast to well‐studied rat OPCs, however, we found that purified mouse NG2 surface positive cells (NG2⁺ cells) require additional activation of cyclic AMP (cAMP) signaling for survival in a medium containing 30% B104 neuroblastoma conditioned medium supplemented with fibroblast growth factor‐2 (B104CM+FGF2), whereas B104CM+FGF2 alone is sufficient for survival and selective proliferation of rat OPCs. After induction of in vitro differentiation, more than 90% of mouse NG2⁺ cells became O4‐positive, and a majority expressed myelin basic protein by 5 day of differentiation, which confirmed the identity of isolated mouse NG2⁺ cells as OPCs. In comparison to rat OPCs, mouse OPCs in B104CM+FGF2 were less motile, and demonstrated lower basal phosphorylation levels of ERK1/2 and cAMP response element‐binding protein (CREB) and a higher incidence of apoptosis mediated by the intrinsic pathway. Transient up‐regulation of cAMP‐CREB signaling partially inhibited apoptosis of mouse OPCs independently of the ERK pathway. This study demonstrates a difference in trophic requirements between mouse and rat OPCs, with an essential role for cAMP signaling to preserve viability of mouse OPCs. © 2009 Wiley‐Liss, Inc.