Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function

Type 1 and type 2 diabetes result from a deficit in insulin production and beta-cell mass. Methods to expand beta-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [(3)H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates beta-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating beta-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [(3)H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human beta-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [(3)H]thymidine incorporation. CCK is a beta-cell proliferative factor that is effective in both mouse and human islets. CCK triggers beta-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.     

CCK/dopamine interactions in Fawn-Hooded and Wistar-Kyoto rat brain

The aim of this study was to compare the actions of CCK neuropeptides within the nucleus accumbens (N.Acc) of alcohol preferring (Fawn-Hooded, FH) and alcohol nonpreferring (Wistar-Kyoto, WKY) rats. CCK-8S (30-300 nM) facilitated the K(+) stimulated release of [(3)H]dopamine (DA) from N.Acc prisms in both rat strains, whereas CCK-4 (30 nM-1 microM) caused a significant decrease of evoked [(3)H]DA in the FH rat only. A scattered distribution of CCK-A and -B receptor immunopositive varicose fibers were visualized throughout the N.Acc of both rat strains along with a topographic distribution of CCK receptor positive cells throughout the ventral mesencephalon.     

High and low affinity receptors mediate growth effects of gastrin and gastrin-Gly on DLD-1 human colonic carcinoma cells

Gastrin (G17) and N-carboxymethylgastrin (G17-Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these effects is controversial. A recent study demonstrated the presence of a low affinity binding site for G17 and G17-Gly on the DLD-1 human colon cancer cell line. The goal of the current study was to further investigate the role of this receptor in mediating the growth-promoting effects of gastrin peptides. Binding of [Leu(15)]G17 and [Leu(15)]G17-Gly to DLD-1 cell membranes in competition with [(3)H]G17-Gly was examined. Binding of [(3)H]cholecystokinin-8 (CCK8) to DLD-1 cell membranes was also assessed. Whole cell binding experiments were carried out using [(125)I-Tyr(12),Leu(15)]G17-Gly. In addition, the ability of [Leu(15)]G17 and [Leu(15)]G17-Gly to stimulate cell growth, as determined by cell counting, was tested. [Leu(15)]G17 and [Leu(15)]G17-Gly competed with [(3)H]G17-Gly at both a high and a low affinity site on DLD-1 membranes. The IC(50) values for [Leu(15)]G17 were 6.0 x 10(-8) M and 6.9 x 10(-6) M while those for [Leu(15)]G17-Gly were 3.2 x 10(-9) M and 4.9 x 10(-6) M. [(3)H]CCK8 did not bind to either site. [Leu(15)]G17-Gly also competed with [(125)I-Tyr(12),Leu(15)]G17-Gly at both a high and a low affinity site on DLD-1 cells with similar affinities as observed with membranes. [Leu(15)]G17 and [Leu(15)]G17-Gly significantly stimulated the growth of DLD-1 cells in a dose-dependent and biphasic manner. The binding profiles of the peptides tested suggest that these sites are different from previously identified wild-type and mutant CCK(1) or CCK(2) receptors.     

Modification by Cholecystokinin Octapeptide of the Binding ofμ-, δ-, and K-Opioid Receptors

Previous study has shown that cholecystokinin (CCK) octapeptide (CCK-8) suppressed the binding of opioid receptors to the universal opioid agonist [3H]etorphine. In the present study, highly selective tritium-labeled agonists for the mu-[(tryrosyl-3,5-3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAGO], delta- ([tyrosyl-3,5-3H][D-Pen2,5]enkephalin ([3H]DPDPE], and kappa- ([3H]U69,593) opioid receptors were used to clarify which type(s) of opioid receptor in rat brain homogenates is suppressed by CCK-8. In the competition experiments, CCK-8 suppressed the binding of [3H]DAGO and [3H]U69,593 but not that of [3H]DPDPE to the respective opioid receptor. This effect was blocked by the CCK antagonist proglumide at 1 mumol/L. In the saturation experiments, CCK-8 at concentrations of 0.1 nmol/L to 1 mumol/L decreased the Bmax of [3H]DAGO binding sites without affecting the KD; on the other hand, CCK-8 increased the KD of [3H]U69,593 binding without changing the Bmax. The results suggest that CCK-8 inhibits the binding of mu- and kappa-opioid receptors via the activation of CCK receptors.     

Purification and characterization of the rat pancreatic cholecystokinin receptor

Cholecystokinin (CCK) is a peptide hormone that has a variety of physiologically important functions in the gastrointestinal tract, in which distinct high affinity receptors have been identified. We describe here the purification of the digitonin-solubilized rat pancreatic receptor as an initial step in the determination of its primary structure. Solubilization of total pancreatic membranes using 1% digitonin resulted in a single class of binding sites with a specific content of 4 pmol/mg as measured in a soluble binding assay using the nonpeptidyl CCK antagonist [3H]3S[-]-N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl]-1H-indole-2-carboxamide [( 3H]364,718). The solubilized receptor was purified using the following chromatographic steps: 1) cation exchange; 2) Ulex europaeus agglutinin-I-agarose; and 3) Sephacryl S-300. The final preparation of the purified receptor had a specific content of 8,055 pmol/mg, which represented a 9,051-fold purification from intact membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified receptor preparation under reducing conditions resulted in a predominant polypeptide with an Mr = 85,000-95,000 and minor polypeptides of Mr = 57,000 and 26,000 as determined by radiolabeling and silver staining. Solubilized pancreatic membranes were affinity labeled with the peptidyl CCK agonist 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Phe33)CCK-26-33] and chromatographed under conditions similar to those described for untreated membranes. Elution of radioactive peaks from each chromatographic column was coincident with [3H]364,718 binding activity and resulted in a labeled polypeptide having the same electrophoretic mobility as receptor derived from freshly labeled membranes and purified from untreated membranes. High performance liquid-gel exclusion chromatography of the crude digitonin-solubilized membrane preparation revealed an estimated molecular size for the [3H]364,718-binding activity of 370,000, which was consistent with the size determined by nondenaturing gel electrophoresis of the purified receptor complexed with the labeled nonpeptidyl antagonist. Binding of [3H]364,718 to the purified receptor preparation was comparable to that observed with the crude solubilized pancreatic membrane preparation; and both the homologous ligand 364,718 (Ki = 0.5 nm) and CCK-8 (Ki = 1.4 microM) competed for binding to both preparations in a similar manner.     

Stimulation of δ-Opioid Receptors Reduces the In Vivo Binding of the Cholecystokinin (CCK)-B-Selective Agonist [3H]pBC 264: Evidence for a Physiological Regulation of CCKergic Systems by Endogenous Enkephalins

Cholecystokinin (CCK) and enkephalins appear to be colocalized in several brain structures, and a physiological interaction between these peptides has been suggested by a large number of pharmacological studies. In this work we have shown, by in vivo binding experiments, that the endogenous enkephalins, protected from degrading enzymes by mixed inhibitors such as kelatorphan and N-[(R,S)-2-benzyl-3-[(S)-2-amino-4-methylthiobutyldithio]-1-oxo pro pyl]- L-phenylalanine benzyl ester (RB 101), a systemically active prodrug, modulate CCK release in mouse brain, leading to an overall increase in the extracellular levels of CCK. This was quantified by measuring the effects of both inhibitors on the in vivo binding of [3H]propionyl-Tyr(SO3H)-gNle-mGly-Trp-(N-Me)Nle-Asp-Phe-NH2 ([3H]pBC 264), a selective and highly potent CCK-B agonist. Thus, intracerebroventricular injection of kelatorphan produced a dose-dependent inhibition of the in vivo binding of [3H]pBC 264 with a maximal effect (40%) at 50 nmol. A similar response was observed after intravenous injection of RB 101 (40 mg/kg). The specific binding of [3H]pBC 264 was also inhibited (25%) by intravenous injection of the selective delta-opioid agonist H-Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu)-OH (BUBUC; 2 mg/kg) but not by the mu-agonist H-Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (5 mg/kg), suggesting a preferential involvement of delta-opioid receptors in the modulation of CCK release. This was confirmed by using the selective delta-opioid antagonist naltrindole, which prevented the inhibitory effects of BUBUC and of enkephalin-degrading enzyme inhibitors on [3H]pBC 264 binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of an estrogen receptor form devoid of estrogen binding ability in MCF-7 cells

In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [(3)H]estradiol (E(2)). We show here that this property is not restricted to this antiestrogen. [(3)H]E(2) binding assays (whole cell assays, DCC assays on cell extracts) and enzyme immunoassays (Abbott) performed in parallel, establish the permanent presence of such unusual ERs in the absence of any exposure of the cells to a ligand. E(2) and the pure antiestrogen RU 58 668, which down-regulate ER, also decrease [(3)H]E(2) binding. In control cells, these ERs represent about the half of the whole receptor population; they also display a tendency to stabilize within the cell nucleus. Loss of E(2) binding ability appears irreversible, since we failed to label receptor accumulated under OH-Tam with [(3)H]E(2) or [(3)H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E(2)-induced down regulation of ER, failed to stabilize [(3)H]E(2) binding (whole cell assay) after an [(3)H]E(2) pulse (1 h), confirming that regulation of E(2) binding and peptide level are related to different regulatory mechanisms. Loss of binding ability could not be ascribed to any ER cleavage as demonstrated by Western blotting with a panel of ER antibodies raised against its various domains (67 kDa ER solely detected). We propose that loss of E(2) binding ability is related to the aging process of the receptor, i.e. it is progressively converted to a form devoted to degradation after it has accomplished its physiological role. Ligands may favor (E(2), RU 58 668) or impede (OH-Tam) this elimination process.     

Multiple neurotransmitter receptors in the brain: amines, adenosine, and cholecystokinin

Radioligand binding studies of neurotransmitter receptors have provided discrimination at the molecular level, permitting the differentiation of multiple receptor subtypes for several biogenic amines. Using this paradigm we have labeled two distinct receptors each for cholecystokinin (CCK) and for adenosine. Adenosine receptors were labeled in brain with [3H]N6-cyclohexyladenosine (3H-CHA) and [3H]1,3-diethyl-8-phenylxanthine (3H-DP). The adenosine receptor labeled by 3H-CHA appears to be an A1 site, associated with reduction of adenylate cyclase activity, while 3H-DP sites resemble A2 receptors linked to adenylate cyclase enhancement. Cholecystokinin-33 labeled by the Bolton-Hunter procedure with 125I(125I-BH-CCK) labels different receptors in brain and pancreas. The pancreatic receptor does not react with CCK derivatives of fewer than eight amino acids, while the brain receptor does recognize pentagastrin, the carboxyl-terminal five amino acids of CCK. The "brain type" CCK receptor may normally interact with CCK-4, the carboxyl-terminal tetrapeptide of CCK, recently identified as a unique neuropeptide highly concentrated in the brain. CCK-8, the other major molecular form of CCK, may be the endogenous ligand for the "pancreatic type" receptor.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.     

Stimulation of cannabinoid receptor agonist 2-arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons

In an earlier study, we reported that chronic ethanol (EtOH) stimulates the formation of anandamide in human SK-N-SH cells. In the present study, we investigated the effect of chronic EtOH on the formation of yet another cannabinoid receptor (CB1) agonist, 2-arachidonylglycerol (2-AG), in cerebellar granule neurons (CGNs). The formation of 2-[(3)H]AG without any stimulation was more pronounced in the older cultures than in younger cultures. Exposure of CGNs to EtOH led to a significant increase in the level of 2-[(3)H]AG (P<0.05). Incubation with the anandamidehydrolase inhibitor phenylmethylsulfonyl fluoride and EtOH did result in an additive increase in 2-[(3)H]AG, but did not with E-6-(bromomethylene)tetrahydro-3-(1-naphthelenyl)-2H-pyran-2-one. The formation of 2-[(3)H]AG was enhanced by ionomycin in both the control and EtOH-exposed CGNs, and the ionomycin-stimulated 2-[(3)H]AG synthesis was inhibited by the intracellular chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Further, glutamate increased the formation of 2-[(3)H]AG only in control CGNs. MK-801 inhibited the EtOH-induced 2-[(3)H]AG synthesis, suggesting the participation of intracellular Ca(2+) in EtOH-induced 2-[(3)H]AG synthesis. The dopamine receptor (D2) agonist did not modify the 2-AG synthesis in either the control or EtOH-exposed CGNs. However, the D2 receptor antagonist inhibited the EtOH-induced formation of 2-[(3)H]AG. The EtOH-induced 2-[(3)H]AG formation was inhibited by SR141716A and pertussis toxin, suggesting the CB1 receptor- and Gi/o-protein-mediated regulation of 2-AG. The observed increase in 2-AG level in CGNs is possibly a mechanism for neuronal adaptation to the continuous presence of EtOH. These findings indicate that some of the pharmacological actions of EtOH may involve alterations in the endocannabinoid signaling system.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [(3)H]8-OH-DPAT and other serotonergic ligands

[(3)H]8-OH-DPAT is a selective ligand for labeling 5-HT(1A) receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [(3)H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [(125)I]RTI-55 and [(3)H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [(3)H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [(3)H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [(125)I]cyanopindolol, [(3)H]ketanserin/[(3)H]mesulergine, [(3)H]GR-65630, [(3)H]GR-113808 and [(3)H]LSD) that specifically labeled 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4) and 5-HT(5-7) receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors

Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.     

Ca(2+)-activated but not G protein-mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4, 5-trisphosphate generation

Inositol phosphate (InsP) responses to receptor activation are assumed to involve phospholipase C cleavage of phosphatidylinositol 4,5-bisphosphate to generate Ins(1,4,5)P(3). However, in [(3)H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [(3)H]InsP responses to alpha(1)-adrenergic receptor stimulation with norepinephrine (100 microM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 microM unlabeled Ins(1,4, 5)P(3) into guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-stimulated, permeabilized [(3)H]inositol-labeled NCM increased [(3)H]Ins(1,4,5)P(3) slightly but did not significantly reduce levels of its metabolites [(3)H]Ins(1,4)P(2) and [(3)H]Ins(4)P, suggesting that these [(3)H]InsPs are not formed principally from [(3)H]Ins(1,4,5)P(3). In contrast, the calcium ionophore A23187 (10 microM) provoked [(3)H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [(3)H]InsP responses characterized by marked increases in [(3)H]Ins(1,4,5)P(3) (2.9 +/- 0.2% of total [(3)H]InsPs after 20 min of high Ca(2+) treatment in comparison to 0. 21 +/- 0.05% of total [(3)H]InsPs accumulated after 20 min of GTPgammaS stimulation). These data provide evidence that Ins(1,4, 5)P(3) generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P(3) generation occurs under conditions of Ca(2+) overload. Thus in NCM, Ca(2+)-induced Ins(1,4,5)P(3) generation has the potential to worsen Ca(2+) overload and thereby aggravate Ca(2+)-induced electrophysiological perturbations.     

Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.     

The differential loss of [3H]pirenzepine vs [3H] (-) quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor

[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

Pharmacological heterogeneity of release-regulating presynaptic AMPA/kainate receptors in the rat brain: study with receptor antagonists

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [(3)H]noradrenaline or [(3)H]serotonin release, striatal [(3)H]dopamine release and cortical [(3)H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [(3)H]noradrenaline, [(3)H]dopamine or [(3)H]serotonin, while it weakly affected the AMPA-mediated release of [(3)H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [(3)H]dopamine and [(3)H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [(3)H]noradrenaline, while the AMPA-mediated [(3)H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]serotonin, but not that of [(3)H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [(3)H]noradrenaline, [(3)H]dopamine, [(3)H]acetylcholine or [(3)H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [(3)H]noradrenaline and [(3)H]acetylcholine, but it potently blocked the AMPA-induced release of [(3)H]serotonin and, less so, of [(3)H]dopamine. AMOA inhibited the AMPA-induced release of [(3)H]serotonin or [(3)H]acetylcholine, but not that of [(3)H]noradrenaline or [(3)H]dopamine. GAMS prevented the AMPA-mediated release of [(3)H]acetylcholine and, more weakly, that of [(3)H]dopamine, but it failed to inhibit the release of [(3)H]noradrenaline or [(3)H]serotonin elicited by AMPA. gamma-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes.     

Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha-latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [(3)H]GABA release evoked by alpha-latrotoxin.     

Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.     

Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands for the cannabinoid receptors.

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of 10 N-palmitoylethanolamide homologues and analogues, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds to cannabinoid receptors in the rat spleen, RBL-2H3 cells and CHO-CB(1) and CHO-CB(2) receptor-transfected cells. In rat spleen slices, CB(2) receptors were the predominant form of the cannabinoid receptors. No binding of [(3)H]SR141716A was observed. [(3)H]CP-55,940 binding was displaced by WIN 55,212-2 and anandamide. No displacement of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 by palmitoylethanolamide derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 could be observed and conversely, no inhibitory activity of N-palmitoylethanolamide derivatives and analogues was measurable. These compounds do not recognize the human CB(1) and CB(2) receptors expressed in CHO cells. In conclusion, N-palmitoylethanolamide was, in our preparations, a weak ligand while its synthesized homologues or analogues were essentially inactive. Therefore, it seems unlikely that N-palmitoylethanolamide is an endogenous agonist of the CB(2) receptors but it may be a compound with potential therapeutic applications since it may act via other mechanisms than cannabinoid CB(1)-CB(2) receptor interactions.