Characterization of µ-opioid receptors in BT474 cells.

<p>(<b>A</b>) Detection of µ-opioid receptors by RT-PCR. Reactions contained 10 ng of cDNA from BT474 cells or from the corresponding cloned receptors (Cn). Only a 293 bp fragment of the µ-opioid receptor is present (arrow). Control PCRs with primers for GAPDH or water instead of cDNA demonstrated the integrity of cDNA and the specificity of the RT-PCR reaction. Ladder: 100 bp. (<b>B</b>) Determination of G proteins in membranes from BT474 cells by Western blot using subtype-specific antibodies. BT474 cells contain inhibitory G_iα2 and G_iα3 as well as stimulatory G_sα subunits. They also contain G_q/11α, G₁₂α, G₁₃α, G₁₄α and Gβ subunits. Membranes of MCF-7 cells served as control. (<b>C</b>) Identification of adenylyl cyclase (AC) isoforms in BT474 cells by RT-PCR. Reactions contained 10 ng of cDNA and primer pairs for all mammalian AC types. The following fragments were amplified (arrows): AC type 1 (143 bp), 2 (323 bp), 3 (259 bp), 4 (368 bp), 5 (312 bp), and 9 (325 bp). The quality of cDNA was verified by amplification of GAPDH, the specificity by using water instead of cDNA. Ladder: 100 bp. (<b>D</b>) Regulation of intracellular cAMP production by opioids. Control and chronically Morphine treated BT474 cells were stimulated for 15 min with Forskolin (10 µM). µ-Opioid receptor-mediated inhibition of cAMP production was assessed in the presence of Morphine (10 µM). Receptor specificity was determined by co-incubation with Naloxone (100 µM). The data shown are from 3 experiments. ***; significantly different at p<.001. n.s.; not quite significant.