Dual coupling of cloned human 5-hydroxytryptamine1D alpha and 5-hydroxytryptamine1D beta receptors stably expressed in murine fibroblasts: inhibition of adenylate cyclase and elevation of intracellular calcium concentrations via pertussis toxin-sensitive G protein(s).
The second messenger coupling of cloned human 5-hydroxytryptamine (5-HT)1D alpha and 5-HT1D beta receptors stably expressed in murine fibroblasts (LM (tk-)) was investigated. Clonal cell lines expressing similar receptor densities (Bmax = 750-950 fmol/mg) were used in this study. 5-HT (EC50 = 1.5-2.0 nM) and sumatriptan (EC50 = 6-14 nM), a selective 5-HT1D agonist, produced dose-dependent inhibition of forskolin-stimulated cAMP accumulation in intact cells transfected with the 5-HT1D alpha or 5-HT1D beta receptor gene. The maximal inhibitory responses elicited by these agonists were slightly greater with the 5-HT1D alpha receptor (approximately 90%) than the 5-HT1D beta receptor (approximately 80%). 5-HT (EC50 = 1.7-2.4 nM) and sumatriptan (EC50 = 8-18 nM) also evoked dose-dependent elevations in intracellular calcium concentrations ([Ca2+]i), with EC50 values that were indistinguishable from those for inhibition of forskolin-stimulated cAMP accumulation. Cells expressing 5-HT1D beta receptors displayed significantly larger 5-HT-induced increases in [Ca2+]i than did cells expressing 5-HT1D alpha receptors (206 nM versus 114 nm increase; p < 0.01). Dose-dependent elevations in inositol phosphates (IP) were also observed after application of 5-HT (EC50 = 29-54 nM) or sumatriptan (EC50 = 73-481 nM); the maximal increases in IP accumulation were modest (51-69%) for both 5-HT1D subtypes. In contrast to the cAMP and calcium responses, the concentration-response curves for IP accumulation were shifted to the right at least 10-fold. Methiothepin, a nonselective 5-HT1 antagonist, competitively antagonized the cAMP response, yielding an apparent dissociation constant (Kb) of 3-4 nM for the 5-HT1D receptors. Methiothepin (10 microM) significantly reduced the elevations in [Ca2+]i (> 90%) and IP (> 75%) evoked by saturating concentrations (1 microM) of agonists. All three functional responses were significantly attenuated (> 90%) by pretreatment with 100 ng/ml pertussis toxin. The sumatriptan-induced elevation of [Ca2+]i via activation of the 5-HT1D subtypes may provide a molecular mechanism of action by which sumatriptan could directly constrict cerebral blood vessels and alleviate migraine symptoms.
Synaptic Pharmaceutical Corporation, Paramus, New Jersey 07652.
J M
JM
Zgombick
L A
LA
Borden
T L
TL
Cochran
S A
SA
Kucharewicz
R L
RL
Weinshank
T A
TA
Branchek
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D051379:Mice; D037342:Pertussis Toxin; D011985:Receptors, Serotonin / Q000145:classification / Q000502:physiology*; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium