Abstract
We examined the effects of several protein kinase C (PKC) inhibitors on the murine 5-hydroxytryptamine3 (5-HT3) receptor to determine whether they acted directly on the receptor. The 5-HT-evoked currents in Xenopus laevis oocytes expressing the recombinant 5-HT3 receptor were measured with the two-electrode voltage-clamp technique. The PKC inhibitors bisindolylmaleimide I (BIM, GF109203x) and staurosporine, but not calphostin C or chelerythrine, decreased the 5-HT3receptor-mediated currents when coapplied with 5-HT. BIM blocked 0.5 μM 5-HT-elicited currents with an IC50 value of 7 nM, whereas in the presence of 5 μM staurosporine, 42% inhibition of 0.5 μM 5-HT-mediated currents was observed. Increasing concentrations of BIM resulted in a rightward shift of the 5-HT concentration-response curve, without altering efficacy. A Schild plot was generated, which had a slope of −1.01, suggesting competitive antagonism. TheKi value of BIM was determined to be 29 nM. To confirm competitive antagonism, a competitive binding assay was performed on Sf21 insect cells infected with the mouse 5-HT3 receptor cDNA in a baculovirus expression vector. BIM completely displaced binding of the selective 5-HT3receptor antagonist [3H]GR65630. BIM bound to the 5-HT3 receptor with a Ki value of 61 nM, which was slightly less potent than that of the selective 5-HT3 receptor antagonist MDL72222 (27 nM). The PKC inhibitor BIM is a potent competitive antagonist at the 5-HT3 receptor.
Footnotes
-
Send reprint requests to: Dr. Tina K. Machu, Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430.
-
↵1 This work was supported by National Institute of Alcohol Abuse and Alcoholism Grant AA10561 (to T.K.M.).
- Abbreviations:
- BIM
- bisindolylmaleimide
- 5-HT3
- 5-hydroxytryptamine3
- PKC
- protein kinase C
- PMA
- phorbol-12-myristate-13-acetate
- MBS
- modified Barth’s solution
- Received November 3, 1998.
- Accepted February 26, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|