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Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways

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Abstract

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)1 (serotonin1) receptor class that inhibits adenylyl cyclase, namely the human 5-HT1F receptor (Adham et al. 1993 a). In the present study we have examined in greater detail the functional coupling of the 5-HT1F receptor in two different cell lines, NIH-3T3 and LM(tk) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk) cells, whereas the EC50 values were comparable.

To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88±2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (Kb = 438 nM).

We have also investigated activation of additional signal transduction pathways by the 5-HT1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca2+]i was observed even at very high agonist concentrations (100 μM). In contrast, in LM(tk) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca2+]i. The 5-HT1F receptor failed to alter arachidonic acid release in either cell line.

The maximal increase in IP accumulation in LM(tk) cells was modest, averaging about 100% above basal. The increases of IP and [Ca2+]i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC50 = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 μM methiothepin. All three responses (cAMP, IP, and [Ca2+]i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of Gi/Go protein(s) in these signal transduction pathways. [Ca2+]i was also elevated by sumatriptan, which may provide a mechanism by which this drug causes constriction of the vasculature. In conclusion, these data indicate that the human 5-HT1F receptor can couple to multiple effectors, and that this coupling is cell-type dependent.

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Abbreviations

FSCA:

forskolin-stimulated cAMP accumulation

[Ca2+]:

intracellular free calcium concentration

AA:

arachidonic acid

EEDQ:

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

CHO:

chinese hamster ovary cell

LM(tk):

mouse fibroblast cell

Bmax :

maximal binding site density

Ki :

apparent dissociation constant obtained from competition binding studies

G protein:

guanine nucleotide-binding protein

HBS:

HEPES-buffered saline

IP:

inositol phosphates

IP3 :

inositol 1,4,5 trisphosphate

PLC:

phospholipase C

Kb :

antagonist dissociation constant

Kd :

equilibrium dissociation constant

N-1F-6:

stable NIH-3T3 cells expressing the cloned 5-HT1F receptor

L-1F-3:

stable LM(tk) cells expressing the cloned 5-HT1F receptor

PTX:

pertussis toxin

BSA:

bovine serum albumin

METH:

methiothepin

SUMA:

sumatriptan

5-MeO-DMT:

5-methoxy-N,N-dimethyltryptamine

1-NP:

1-(1-napthyl)piperazine

5-CT:

5-carboxyamidotryptamine

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Correspondence to: N. Adham at the above address

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Adham, N., Borden, L.A., Schechter, L.E. et al. Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways. Naunyn-Schmiedeberg's Arch Pharmacol 348, 566–575 (1993). https://doi.org/10.1007/BF00167231

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  • DOI: https://doi.org/10.1007/BF00167231

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