Guinea Pig Striatal Membranes Research Articles

Benzo- and cyclohexanomazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of mazindol (1), homomazindol (2), and bishomomazindol (3) derivatives with a benzo or cyclohexano ring fused at various sites were prepared as part of an SAR study to determine the effect of increased aliphatic and aromatic lipophilicity on selected in vitro assays used to identify potential cocaine-like and cocaine antagonism activity. Very good (IC(50) = 2-3 nM) inhibition of [(3)H] WIN 35,428 and [(125)I] RTI-55 binding on rat or guinea pig striatal membranes and HEK cells expressing cDNA for the human dopamine transporter (HEK-hDAT) was shown by the 8,9-benzomazindol 25 and 9,10-benzohomomazindol 28. All new compounds were weaker inhibitors of [(3)H] DA uptake in HEK-hDAT cells than 1 and 2. No improvement in the binding selectivity ratio (SERT/DAT and NET/DAT) was found when compared to 2. Compounds 25and 28 showed a considerable increase versus 1 in uptake/binding discrimination ratios at the DAT (311.0 and 182.1 vs 0.9), SERT (33.6 and 127.3 vs 1.9), and NET (7.3 and 10.0 vs 0.3).

Mazindol Analogues as Potential Inhibitors of the Cocaine Binding Site at the Dopamine Transporter

A series of mazindol (2) and homomazindol (3) analogues with a variety of electron-donating and electron-withdrawing groups in the pendant aryl group and the benzo ring C, as well as H, methoxy, and alkyl groups replacing the hydroxyl group were synthesized, and their binding affinities at the dopamine transporter (DAT) on rat or guinea pig striatal membranes were determined. Several active analogues were also evaluated for their ability to block uptake of DA, 5-HT, and NE and inhibit binding of [(125)I] RTI-55 at HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Mazindane (26) was found to be a pro-drug, oxidizing (5-H --> 5-OH) to mazindol on rat striatal membranes and HEK-hDAT cells. The 4',7,8-trichloro analogue (38) of mazindol was the most potent and selective ligand for HEK-hDAT cells (DAT K(i) = 1.1 nM; SERT/DAT = 1283 and NET/DAT = 38). Experimental results strongly favor the cyclic or ol tautomers of 2 and 3 to bind more tightly at the DAT than the corresponding keto tautomers.

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Serotonin 5-HT 1B/1D agonist-stimulated [ 35S]GTPγS binding in rat and guinea pig striatal membranes

Serotonin (5-hydroxytryptamine; 5-HT) receptor ligands were used to assess agonist-stimulated [ 35S]GTPγS binding in rat and guinea pig striatal membranes. The assay contained 45–60 μg protein, 300 μM GDP and 0.1 nM [ 35S]GTPγS, incubated at 37°C for 20 min. The non-selective agonists 5-HT, 5-CT (5-carboxyamidotryptamine), and L-694,247, and the selective 5-HT 1B receptor agonist CP 93,129 produced concentration-dependent increases in [ 35S]GTPγS binding in rat striatum, whereas the selective 5-HT 1A receptor agonist R(+)-8-OH-DPAT [R(+)-8-hydroxy-2-(di- n-propylamino)tetralin] was inactive. Cyanopindolol, a 5-HT 1A/1B receptor antagonist, completely blocked the effect of 5-HT. Methiothepin, yohimbine and cyanopindolol also blocked 5-CT-stimulated [ 35S]GTPγS binding with the following rank order of potency: cyanopindolol ≥ methiothepin >>> yohimbine, consistent with rat 5-HT 1B receptor pharmacology. Neither cyanopindolol nor methiothepin altered basal [ 35S]GTPγS binding by themselves while yohimbine had weak partial agonist activity. Furthermore, cyanopindolol shifted the 5-CT concentration-response curve rightward, increasing the EC 50 and decreasing the maximal response, but did not affect L-694,247-stimulated [ 35S]GTPγS binding. The ability of cyanopindolol or spiperone (a 5-HT 1A/1D receptor antagonist) to alter CP 93,129-stimulated [ 35S]GTPγS binding was determined. Cyanopindolol produced a rightward shift in the CP 93,129 concentration-response curve, while spiperone had no affect. Finally, in guinea pig striatum and hippocampus, L-694,247 produced a concentration dependent increase in [ 35S]GTPγS binding. In conclusion, these studies indicate that 5-HT 1B receptor function can be assessed using agonist-stimulated [ 35S]GTPγS binding in rat striatal membranes using CP 93,129, 5-HT or 5-CT, but not L-694,247.

Binding profile of the novel 5-HT 1B/1D receptor antagonist, [ 3H]GR 125,743, in guinea-pig brain: a comparison with [ 3H]5-carboxamidotryptamine

Native brain 5-HT 1B/1D receptors were studied using the novel antagonist, [ 3H]GR 125,743 ( N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide). In guinea-pig striatal membranes, [ 3H]GR 125,743 displayed rapid association ( t 1/2=4.5 min), high (90%) specific binding and high affinity ( K d=0.29 nM), although B max values (fmol/mg protein) varied according to brain region-striatum: 199; frontal cortex: 89; hippocampus: 79; cerebellum: 26. In frontal cortex, the B max determined with [ 3H]5-CT ([ 3H]carboxamidotryptamine) was significantly higher (178; P<0.05), suggesting that it also labels other binding sites. In striatal membranes, guanylylimidodiphosphate (GppNHp) inhibited [ 3H]5-CT but not [ 3H]GR 125,743 binding, suggesting that the latter has antagonist properties. Nevertheless, in competition binding experiments, the p K i values obtained with [ 3H]GR 125,743 and [ 3H]5-CT for 20 serotonergic ligands, including L 694,247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1 H-indole-3-yl]ethylamine), GR 46,611 (3-[3-(2-dimethylamino-ethyl)-1 H-indol-6-yl]- N-(4-methoxybenzyl)acrylamide), sumatriptan and alniditan, were highly correlated ( r=0.99). Ketanserin and ritanserin showed low affinity for [ 3H]GR 125,743 binding to guinea-pig striatal sites ( K i=12600 and 369 nM), suggesting that 5-HT 1B (rather than 5-HT 1D) receptors are predominantly labelled in this tissue. The present data indicate that [ 3H]GR 125,743 is a useful tool for studying native 5-HT 1B/1D receptors.

3H]RS 57639, a High Affinity, Selective 5-HT4 Receptor Partial Agonist, Specifically Labels Guinea-pig Striatal and Rat Cloned (5-HT4S and 5-HT4L) Receptors

RS 57639‡‡4-Amino-5-chloro-2-methoxy benzoic acid 1-(3-[2,3-dihydrobenzo[1,4]dioxin-6-yl)-propyl]-piperidin-4-yl methyl ester., by being a partial agonist in rat esophagus but a competitive antagonist in guinea-pig ileum, is one of several ligands which operationally discriminate among 5-HT4 receptors in different tissues. The discovery of splice variants of the 5-HT4 receptor, 5-HT4S and 5-HT4L, raises the possibility that this functional heterogeneity among 5-HT4 receptors may be due to differences in the interaction of ligands with different isoforms of the receptor. To test this idea, the functional and binding interactions of RS 57639 with rat 5-HT4S and 5-HT4L receptors were characterized, RS 57639 stimulated adenylate cyclase in cells expressing 5-HT4S or 5-HT4L receptors with similar potency (pec50 = 7.9 ± 0.1 and 7.6 ± 0.1) and efficacy (71 ± 3 and 59 ± 4% of 5-HT). [3H]RS 57639 also bound to 5-HT4S and 5-HT4L receptors with similar affinity (Kd = 0.09 ± 0.01 and 0.11 ± 0.01 nM) and specificity (SB204070 > GR113808 > SDZ 205557 > cisapride > renzapride > α me-5-HT > 5-CT). Therefore, the operational differences among 5-HT4 receptors, detected with RS 57639, are not explained by differences in the interaction of the ligand with 5-HT4S and 5-HT4L receptors. [3H]RS 57639 binding to guinea-pig striatal membranes was also characterized. [3H]RS 57639 bound with high affinity (Kd = 0.25 ± 0.07 nM) and a specificity similar to that of the 5-HT4 receptor antagonist, [3H]GR113808. Therefore, while the mechanism by which RS 57639 operationally distinguishes among 5-HT4 receptors was not determined, [3H]RS 57639 was shown to specifically label native and cloned 5-HT4 receptors. As the first selective agonist radioligand to be described for this receptor, [3H]RS 57639 may prove useful in further studies of receptor coupling and ligand interactions. © 1997 Elsevier Science Ltd.

3H]LY303870, a novel nonpeptide radioligand for the NK-1 receptor.

We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. (R)-N-[2-[Acetyl[3H3][(2-methoxyphenyl)-methyl]amino]- 1-(1H-indol-3-ylmethyl) ethyl][1,4'-bipiperidine]- 1'-acetamide ([3H]LY303870) binds to a single class of sites with an equilibrium KD of 0.22 nM and a Bmax of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC50 of 0.56 nM, whereas the less active (S)-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (+/-)-CP96,345 and (+/-)-RP 67580 had IC50 values of 0.74 and 49 nM, respectively. Substance P (SP) was also a potent inhibitor with with an IC50 of 3.1 nM. The inhibition by SP could be separated into two components: a high-affinity component with a Ki of 0.53 nM and a lower-affinity component with a Ki of 155 nM. Addition of 100 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [3H]-LY303870 was not changed by the presence of 100 microM Gpp(NH)p. The distribution of [3H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [3H]SP. Therefore, [3H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.

RS 39604: a potent, selective and orally active 5-HT4 receptor antagonist.

1. Selective antagonism of 5-HT4 receptors may provide therapeutic benefit in certain disorders of the myocardium, alimentary and lower urinary tract. We now report on RS 39604, a novel and selective 5-HT4 receptor antagonist and compare its pharmacological properties with those of SB 204070. 2. In guinea-pig striatal membranes, both RS 39604 and SB 204070 inhibited specific binding of [3H]-GR 113808 in a concentration-dependent manner yielding pKi estimates of 9.1 and 10.9, respectively. RS 39604 displayed a low affinity (pKi < 6.5) for 5-HT1A, 5-HT2C, 5-HT3, alpha 1c, D1, D2, M1, M2, AT1, B1 and opioid mu receptors and moderate affinity for sigma 1, (pKi = 6.8) and sigma 2 (pKi = 7.8) sites. 3. In the rat isolated oesophagus, precontracted with carbachol, RS 39604 (30-300 nM) behaved as a competitive antagonist towards 5-HT-induced relaxation (pA2 = 9.3; Schild slope = 1.0). We and others have shown previously that SB 204070 behaves as an unsurmountable antagonist in this preparation (pA2 approximately 10.5). In the guinea-pig isolated ileal mucosa, RS 39604 (30 nM) antagonized 5-MeOT-induced increase in short-circuit current (pA2 = 9.1). 4. In anaesthetized vagotomized micropigs, RS 39604, administered by the i.v. or intraduodenal (i.duod.) route, produced dose-dependent inhibition of 5-HT-induced tachycardia (ID50 = 4.7 micrograms kg-1, i.v. and 254.5 micrograms kg-1, i.duod). At maximal doses of 30 micrograms kg-1, i.v. and 6 mg kg-1, i.duod., the inhibitory effects of RS 39604 lasted for more than 6 h. In this preparation, SB 204070 was as potent as RS 39604by the i.v. route but was inactive by the intraduodenal route at doses up to 3 mg kg-1.5. In conscious mice, RS 39604, administered by the i.p. or p.o. route, produced dose-depend entinhibition of 5-hydroxytryptophan (5-HTP)-induced diarrhoea (ID50= 81.3 microg kg-1, i.p. and 1.1 mg kg-1,p.o.). In this assay, SB 204070 was inactive by the oral route at doses up to 30 mg kg-1.6. In anaesthetized guinea-pigs, RS 39604 antagonized the contractile effect of 5-HT in the proximal colon by producing parallel, dextral displacement of the dose-response curve to 5-HT. The mean dose ratios to 5-HT at 0.1 mg kg-1, i.v., 1 mg kg-1, i.v. and 10 mg kg-1, i.duod. were 4.6, 30.7 and 10.8,respectively. SB 204070 behaved as an unsurmountable antagonist in this assay.7. In a model of visceral pain in conscious rats, RS 39604 (0.01-1 mg kg-1, i.v.) did not affect colorectal distension-induced increases in arterial pressure whereas morphine (1 mg kg-1, i.v.) produced significant inhibition of the response, implying that 5-HT4 receptors are not involved in nociception in this model.8. The data suggest that RS 39604 is a high affinity and selective 5-HT4 receptor antagonist that is orally active and long-lasting in vivo. It is concluded that RS 39604 may be the preferable probe to use for investigating the physiological and pathophysiological role of 5-HT4 receptors in vivo.

Cisapride, a gastroprokinetic agent, binds to 5-HT4 receptors

The affinity of cisapride for 5-HT4 receptors was investigated in comparison with those of other 5-HT4 receptor agonists and antagonists, such as 5-HT, 5-MeOT, mosapride, zacopride, metoclopramide, BIMU8, GR113808, SDZ 205-557 and ICS 205-930. Cisapride and the other compounds dose-dependently inhibited specific 3H-GR113808 binding to 5-HT4 receptors in guinea pig striatal membranes, and complete inhibition of specific 3H-GR113808 binding was achieved at the high concentrations of these compounds. Cisapride was 1.9-, 7.3-, 4.3-, 11- and 26-fold more potent than 5-HT, 5-MeOT, mosapride, zacopride and metoclopramide, respectively, in competing for 5-HT4 receptors. To determine the manner of interaction between cisapride and 5-HT4 receptors, Scatchard analysis of 3H-GR113808 specific binding to striatal membranes was performed. Cisapride increased the Kd value of 3H-GR113808 in striatal membranes in a dose-dependent manner without any influence on the binding density (Bmax) of 3H-GR113808. These findings indicate that cisapride binds to 5-HT4 receptors competing with 3H-GR113808 in guinea pig striatal membranes.

Homologous and heterologous desensitization of δ-opioid receptor in Xenopus oocytes, COS-cells and guinea pig striatal membranes

Homologous and heterologous desensitization of δ-opioid receptor in <i>Xenopus</i> oocytes, COS-cells and guinea pig striatal membranes

Species and age-dependent differences of functional coupling between opioid δ-receptor and G-proteins and possible involvement of protein kinase C in striatal membranes

[ d-Ser 2,Leu 5]-enkephalin-Thr 6 (DSLET) and [ d-Pen 2,5]-enkephalin (DPDPE), both δ-agonists, stimulated the high affinity GTPase in the rat striatal membranes in a naltrindole-reversible manner. Similar stimulation was also observed in the striatal membrane preparations of 16-week-old guinea pigs, while not in those preparations of 4-week-old ones. When calphostin C, a protein kinase C inhibitor, was added to the reaction mixture, DSLET showed a marked stimulation in this activity in 4-week guinea pig striatal membranes. There was no effect of KT5720, a cyclic AMP-dependent protein kinase inhibitor, on such δ-opioid-mediated responses. These findings suggest that protein kinase C is locally involved in the functional uncoupling of δ-receptors to G-proteins in the striatum of young guinea pigs.

Functional Reconstitution of Purified Gi and Go with μ‐Opioid Receptors in Guinea Pig Striatal Membranes Pretreated with Micromolar Concentrations of N‐Ethylmaleimide

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.