Characterization of a novel 3H-5-hydroxytryptamine binding site subtype in bovine brain membranes

Abstract

3H-5-Hydroxytryptamine (5-HT) binding sites were analyzed in bovine brain membranes. The addition of either the 5-HT1A-selective drug 8-OH-DPAT (100 nM) or the 5-HT1C-selective drug mesulergine (100 nM) to the assay resulted in a 5-10% decrease in specific 3H-5-HT binding. Scatchard analysis revealed that the simultaneous addition of both drugs decreased the Bmax of 3H-5-HT binding by 10-15% without affecting the KD value (1.8 +/- 0.3 nM). Competition studies using a series of pharmacologic agents revealed that the sites labeled by 3H-5-HT in bovine caudate in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine appear to be homogeneous. 5-HT1A selective agents such as 8-OH-DPAT, ipsapirone, and buspirone display micromolar affinities for these sites. RU 24969 and (-)pindolol are approximately 2 orders of magnitude less potent at these sites than at 5-HT1B sites which have been identified in rat brain. Agents displaying nanomolar potencies for 5-HT1C sites such as mianserin and mesulergine are 2-3 orders of magnitude less potent at the 3H-5-HT binding sites in bovine caudate. In addition, both 5-HT2- and 5-HT3-selective agents are essentially inactive at these binding sites. These 3H-5-HT sites display nanomolar affinity for 5-carboxyamidotryptamine, 5-methoxytryptamine, metergoline, and 5-HT. Apparent Ki values of 10-100 nM are obtained for d-LSD, RU 24969, methiothepin, tryptamine, methysergide, and yohimbine, whereas I-LSD and corynanthine are significantly less potent. In addition, these 3H-5-HT labeled sites are regulated by guanine nucleotides and calcium. Regional studies indicate that this class of sites is most dense in the basal ganglia but exists in all regions of bovine brain. These data therefore demonstrate the presence of a homogeneous class of 5-HT1 binding sites in bovine caudate that is pharmacologically distinct from previously defined 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 receptor subtypes. We therefore suggest that this class of sites be designated the 5-HT1D subtype of binding sites labeled by 3H-5-HT.

Further characterization of the 5‐HT receptor mediating vascular relaxation and elevation of cyclic AMP in porcine isolated vena cava

“…Both stimulant (Barbaccia et al, 1983;Shenker et al, 1983;1987;Markstein et al, 1986) and inhibitory (DeVivo & Maayani, 1986;Weiss et al, 1986;Hoyer & Schoeffter, 1988) effects have been described and assigned to various types of 5-HT1 binding site (for review see Roth & Chuang, 1987 (Fozard, 1987), were similarly devoid of appreciable activity in our study. The possibility that 5-HT1c or 5-HT1D sites could mediate the effects of 5-HT, as might be suggested by the antagonist potencies of mesulergine and metergoline (Table 2), is excluded on the basis of the high affinity of spiperone for the porcine vena cava receptor as compared to its low affinity at 5-HT1c and 5-HTlD binding sites (Heuring & Peroutka, 1987).…”

[3H]‐5‐carboxamidotryptamine labels 5‐HT1D binding sites in bovine substantia nigra

“…(Pedigo et al, 1981;Heuring & Peroutka, 1987 Hoyer et al, 1985;Heuring & Peroutka, 1987;Leonhardt et al, 1989), which increases the complexity of binding curves and data analysis.…”

“…The ability to mask selectively 5-HTlA and 5-HTIc receptors with 8-OH-DPAT and mesulergine, respectively (Heuring & Peroutka, 1987), has decreased the complexity of radioligand binding analysis. The hypothesis that 5-HTIB receptors represent the species homologue of the 5-HTID receptor (Adham et al, 1992) (Leonhardt et al, 1989;Weisberg & Teitler, 1992;Beer et al, 1992 (Hamblin & Metcalf, 1991) and a 5-HTIE receptor (McAllister et al, 1992).…”

“…The 5-HTID receptor has been defined as the [3H]-5-HT binding remaining in the presence of 100 nM (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene (8-OH-DPAT) and 100 nM mesulergine, which selectively masks the 5-HT,A and 5-HT,c receptors, respectively (Heuring & Peroutka, 1987). However, [3H]-5-HT in the presence of the above masking ligands has been shown to recognize a heterogeneous population of binding sites in porcine caudate (Sumner & Humphrey, 1989) and human cortex (Leonhardt et al, 1989).…”

“…Radioligand binding assays ([3H]-5-HT, [3H]-5-CT) were carried out in duplicate in a final volume of 1.0 ml consisting of 100 tlI radioligand, 50 lal each of 8-OH-DPAT and mesulergine (100 nM final concentration), to inhibit binding to 5-HT,A or 5-HT,c receptors, respectively (Heuring & Peroutka, 1987) Bound radioactivity was determined after rapid filtration through Whatman GF/B filter strips, using a Brandel cell harvester (Gaithersburg, MD, U.S.A.), and washing with three washes (5 ml each) of ice-cold wash buffer. Filters were counted by liquid scintillation spectrometry in Atomlight (New England Nuclear) at approximately 47% efficiency.…”