Eduardo J. Molinari

2-[125I]iodo-5-methoxycarbonylamino-N-acetyltryptamine: a selective radioligand for the characterization of melatonin ML2 binding sites.

We now describe the preparation and characterization of a novel radioligand, 2-[125I]iodo-5-methoxy-carbonylamino-N-acetyltryptamine (2-[125I]MCA-NAT), with high affinity and pharmacological selectivity for melatonin ML2 receptor sites. 2-[125I]MCA-NAT was prepared by introducing an [125I]iodine molecule on carbon 2 of 5-MCA-NAT (5-methoxycarbonylamino N-acetyltryptamine), a selective melatonin ML2 receptor ligand. The specific binding (88%) of 2-[125I]MCA-NAT (50 pM) to whole washed hamster brain membranes showed rapid kinetics of association/dissociation, and was of high affinity and saturable (Kd value = 116 +/- 14 pM; Bmax value = 15.5 +/- 1.8 fmol/mg protein, n = 3). 2-[125I]MCA-NAT showed no affinity for melatonin ML1 receptors of chicken retina. Competition curves of various melatonin analogues for 2-[125I]MCA-NAT binding to hamster brain, testes and kidney were monophasic and showed a pharmacological order of affinities (Ki values for brain, nM) identical to that of the ML2 sites [2-iodomelatonin (0.77) > 6-chloro-2-methyl-melatonin (2.56) > 6-chloromelatonin (6.8) > prazosin (21.7) > or = N-acetylserotonin (23.3 nM) > or = 5-MCA-NAT (29.5) > or = melatonin (83.9) > luzindole (1687) > serotonin (2120)]. Affinity constants for competition of melatonin analogues on [125I]MCA-NAT binding to hamster brain, testes, and kidney correlated significantly [r = 0.962, P < 0.001, n = 9; r = 0.982, P < 0.0001, n = 13; r = 0.975, P < 0.0001, n = 9, respectively) with the affinities determined on 2-[125I]iodomelatonin binding to ML2 sites (hamster brain) but not to ML1 sites (chicken retina, r = 0.33, P > 0.05, n = 16). In conclusion, 2-[125I]MCA-NAT is a specific radioligand for the identification and characterization of ML2 binding sites in brain and peripheral tissues.

Up-regulation of human α7 nicotinic receptors by chronic treatment with activator and antagonist ligands

This study examined the binding and functional properties of human alpha7 neuronal nicotinic acetylcholine receptors stably expressed in human embryonic kidney (HEK) 293 cells following chronic treatment with nicotinic receptor ligands. Treatment of cells with (-)-nicotine (100 microM) for 120 h increased the Bmax values of [125I]alpha-bungarotoxin binding 2.5-fold over untreated cells. This effect was concentration-dependent (EC50) = 970 microM) and a 6-fold upregulation was observed with the maximal concentration of (-)-nicotine tested. Also, treatment of cells with ligands of varying intrinsic activities including (+/-)-epibatidine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) also upregulated [125I]alpha-bungarotoxin binding. A concentration-dependent upregulation of binding sites was also observed following treatment with the alpha7 nicotinic receptor antagonist, methyllycaconitine (EC50 = 92 microM) with a maximal upregulation of about 7-fold. Functionally, the peak amplitude of the whole-cell currents recorded by fast application of (-)-nicotine after chronic treatment of cells with concentrations of (-)-nicotine (1000 microM) or methyllycaconitine (10 microM) that elicited similar increases in binding levels (3.5-fold) resulted in increases of 2-fold (505 +/- 21 pA) and 6-fold (1820 +/- 137 pA) respectively in whole cell current amplitude compared to untreated cells (267 +/- 24 pA). These studies clearly demonstrate that long-term exposure to both activator and antagonist ligands can increase the density of alpha7 nicotinic receptors and can differentially enhance nicotinic receptor function.

The high affinity melatonin binding site probed with conformationally restricted ligands- I. Pharmacophore and minireceptor models

The affinities of enantiomers of conformationally restricted melatonin analogues for the ML-1 and ML-2 putative melatonin receptor subtypes are reported. Most ligands exhibited reversed stereoselectivity when competing with 125I 2-iodomelatonin binding to chicken retinal (ML-1) and hamster brain (ML-2) membranes, further supporting the biochemical and pharmacological differences reported for these two sites. Based on the data for the ML-1 site and thorough conformational analyses of several ligands, two pharmacophore models were derived using the program APOLLO. The pharmacophoric elements included were putative receptor points from the amide NH, the amide CO, and the methoxy-O, together with the normal through the phenyl ring. The large drop in ML-1 affinity observed for 4-methoxy-2-acetamido-indan (6a) could not be explained from either of these models. Minireceptors were subsequently built around the two pharmacophores using Yak. Analysis of the resulting ligand-minireceptor interactions offered an explanation for the low affinity of 6a and allowed one of the pharmacophore models to be selected for use in future drug design.

Pharmacological and molecular characterization of ATP-sensitive K+ channels in the TE671 human medulloblastoma cell line.

ATP-sensitive K+ (K(ATP)) channels in the human medulloblastoma TE671 cell line were characterized by membrane potential assays utilizing a potentiometric fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)), and by mRNA analysis. Membrane potential assays showed concentration-dependent and glyburide-sensitive changes in fluorescence upon addition of (-)-cromakalim, pinacidil, diazoxide and P1075. The rank order of potency for these openers was P1075 > (-)-cromakalim approximately = pinacidil > diazoxide. Additionally, glyburide and glipizide inhibited P1075-evoked responses in TE671 cells with half-maximal inhibitory concentrations of 0.22 and 14 microM, respectively. The rank order potencies of both openers and inhibitors were similar to those observed in the rat smooth muscle A-10 cell line. In contrast, in the rat pancreatic insulinoma RIN-m5F cell line, only diazoxide was effective as an opener. Reverse transcription-polymerase chain reaction (RT-PCR) studies detected sulfonylurea receptors SUR2B and SUR1 mRNA in TE671 cells whereas only SUR2B and SUR1 mRNA were, respectively, detected in A-10 and RIN-m5F cells. The inward rectifier Kir6.2 mRNA was detected in all three cell types whereas Kir6.1 was detected only in A-10 cells. Collectively, the molecular and pharmacologic studies suggest that K(ATP) channels endogenously expressed in TE671 medulloblastoma resemble those present in the smooth muscle.

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells.

The pharmacological properties of KATP channels generated by stable co‐expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K+ channel Kir6.2 were characterized in HEK‐293 cells. [3H]‐Glyburide (glibenclamide) bound to transfected cells with a Bmax value of 18.5 pmol mg−1 protein and with a KD value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. Functional activity of KATP channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 μM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC50=0.92 nM). Metabolic inhibition by 2‐deoxyglucose and oligomycin treatment triggered 86Rb+ efflux from transfected cells that was sensitive to inhibition by glyburide (IC50=3.6 nM). Diazoxide, but not levcromakalim, evoked concentration‐dependent decreases in DiBAC4(3) fluorescence responses with an EC50 value of 14.1 μM which were attenuated by the addition of glyburide. Diazoxide‐evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co‐expressed in HEK‐293 cells resemble those typical of native KATP channels described in pancreatic and neuronal tissues.

Characterization of a novel ATP-sensitive K+ channel opener, A-251179, on urinary bladder relaxation and cystometric parameters

ATP‐sensitive K+ channels (KATP) play a pivotal role in contractility of urinary bladder smooth muscle. This study reports the characterization of 4‐methyl‐N‐(2,2,2‐trichloro‐1‐(3‐pyridin‐3‐ylthioureido)ethyl)benzamide (A‐251179) as a KATP channel opener.

Pharmacological characterization of a 1,4‐dihydropyridine analogue, 9‐(3,4‐dichlorophenyl)‐3,3,6,6‐tetramethyl‐3,4,6,7,9,10‐hexahydro‐1,8(2H,5H)‐acridinedione (A‐184209) as a novel KATP channel inhibitor

This study reports on the identification and characterization of a 1,4‐dihydropyridine analogue, 9‐(3,4‐dichlorophenyl)‐3,3,6,6‐tetramethyl‐3,4,6,7,9,10‐hexahydro‐1,8(2H,5H)‐acridinedione (A‐184209) as a novel inhibitor of ATP‐sensitive K+ channels. A‐184209 inhibited membrane potential changes evoked by the prototypical cyanoguanidine ATP‐sensitive K+ channel opener (KCO) P1075 in both vascular (A10) and urinary bladder smooth muscle cells with IC50 values of 1.44 and 2.24 μM respectively. P1075‐evoked relaxation of 25 mM K+ stimulated aortic strips was inhibited by A‐184209 in an apparently competitive fashion with a pA2 value of 6.34. The potencies of A‐184209 to inhibit P1075‐evoked decreases in membrane potential responses in cardiac myocytes (IC50=0.53 μM) and to inhibit 2‐deoxyglucose‐evoked cation efflux pancreatic RINm5F cells (IC50=0.52 μM) were comparable to the values for inhibition of smooth muscle KATP channels. On the other hand, a structural analogue of A‐184209 that lacked the gem‐dimethyl substituent, 9‐(3,4‐dichlorophenyl)‐3,4,6,7,9,10‐hexahydro‐1,8(2H,5H)‐acridinedione (A‐184208), was found to be a KATP channel opener, evoking membrane potential responses in A10 smooth muscle cells (EC50=385 nM) and relaxing aortic smooth muscle strips (IC50=101 nM) in a glyburide‐sensitive manner. Radioligand binding studies demonstrated that A‐184209 displaced SUR1 binding defined by [3H]glyburide binding to RINm5F cell membranes with a Ki value of 0.11 μM whereas A‐184208 was ineffective. On the other hand, both A‐184209 (Ki=1.34 μM) and A‐184208 (Ki=1.14 μM) displaced binding of the KCO radioligand, [125I]A‐312110 in guinea‐pig bladder membranes with similar affinities. These studies demonstrate that A‐184209 is a novel and structurally distinct compound that inhibits KATP channels in smooth muscle with potencies comparable to glyburide. The structural overlap between DHP openers and blockers, together with their differential interaction with ligand binding sites, support the notion that both openers and blockers bind to similar or very closely coupled sites on the sulfonylurea receptor and that subtle changes in the pharmacophore itself could switch functional properties from KATP channel activation to inhibition.

Characterization and modulation of [125I]iberiotoxin-D19Y/Y36F binding in the guinea-pig urinary bladder.

The radioligand binding characteristics of the Ca(2+)-activated K(+) channel ligand [125I]iberiotoxin-D19Y/Y36F were examined in guinea-pig urinary bladder membranes. Saturation analysis revealed a single class of high affinity binding sites in the bladder with a K(D) value of 45.6 pM and a B(max) value of 112 fmol/mg protein. Specific binding was displaced by unlabeled iberiotoxin and penitrem A, but not by blockers of other classes of K(+) channels including alpha-dendrotoxin, margatoxin and apamin. The indole alkaloids, paxilline and verruculogen, significantly increased binding by 4.5- and 4.3-fold, respectively. Tetraacetic acid derivatives such as ethylenediamine tetraacetic acid and ethyleneglycoltetraacetic acid enhanced specific [125I]iberiotoxin-D19Y/Y36F binding about 2.5-fold, which was not attributable to calcium chelation. This increase was due to a significant change in ligand binding affinity (K(D)=6.3 pM), but not due to a change in the B(max), indicating that these compounds may enhance toxin binding via allosteric interactions. Collectively, these results demonstrate that the binding sites for [125I]iberiotoxin-D19Y/Y36F present in the urinary bladder shows a pharmacological profile typical of maxi-K(+) channels and can be modulated, not only by previously known indole alkaloids, but also by tetraacetic acid analogs.

Expression of Cloned Receptors in Mammalian Cell Lines

The expression of cloned receptor and ion-channel proteins is a very useful tool for studying the physiological and pharmacological properties of receptors. The process of introducing a gene of interest into eukaryotic cells by biochemical or physical methods is termed transfection. After transfection, some of the DNA is transported into the nucleus, where it is transcribed. The resulting mRNA is transferred into the cytoplasm and translated into protein. The transfection method described in this unit is a versatile, plasmid-based, liposome-mediated gene-transfer technique known as lipofection. This technique has gained wide acceptance because of its simplicity, suitability for a wide range of cell types, reasonable transfection efficiencies, and reproducibility. This unit also includes a procedure for assessing transfection efficiency via cotransfection with a reporter gene (lacZ) and assay for coexpression.