Pindolol-insensitive [3H]-5-hydroxytryptamine binding in the rat hypothalamus; identity with 5-hydroxytryptamine7 receptors.
Pindolol-insensitive [3H]-5-hydroxytryptamine ([3H]-5-HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5-HT7 receptors. Consistent with a previous report, 3 microM and not 100 nM pindolol was required to occupy fully 5-HT1A and 5-HT1B receptors. Remaining [3H]-5-HT binding was saturable (KD, 1.59+/-0.21 nM; Bmax, 53.8+/-3.1 fmol x mg protein(-1)). Displacement of [3H]-5-HT with metergoline and 5-CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pKi values and the rank order of affinity were significantly correlated (r = 0.81 and 0.93, respectively) with published [3H]-5-HT binding to rat recombinant 5-HT7 receptors. In the presence of pindolol, 5-HT-enhanced accumulation of [32P]-cyclic AMP was unaffected by the 5-HT4 antagonist RS39604 (0.1 microM) or the 5-ht6 antagonist Ro 04-6790 (1 microM) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5-HT7 receptor. Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine, 5,7-DHT, elevated the [3H]-5-HT Bmax 2 fold, indicating that the hypothalamic 5-HT7 receptor is post-synaptic to 5-HT nerve terminals and regulated by synaptic 5-HT levels. These results suggest that, in the presence of 3 microM pindolol, [3H]-5-HT selectively labels hypothalamic binding sites consistent with functional 5-HT7 receptors.
Clemett
D A
DA
School of Biomedical Sciences, Queen's Medical Centre, Nottingham University.
Kendall
D A
DA
Cockett
M I
MI
Marsden
C A
CA
Fone
K C
KC
D015116:5,7-Dihydroxytryptamine / Q000378:metabolism / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D007031:Hypothalamus / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D010869:Pindolol / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D044302:Receptor, Serotonin, 5-HT1B; D017661:Receptors, Presynaptic / Q000187:drug effects / Q000378:metabolism; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D012701:Serotonin / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism / Q000494:pharmacology*
D044302:Receptor, Serotonin, 5-HT1B; D017661:Receptors, Presynaptic; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D012702:Serotonin Antagonists; C104425:serotonin 7 receptor; D015116:5,7-Dihydroxytryptamine; D012701:Serotonin; D010869:Pindolol; D000262:Adenylyl Cyclases