Gi-Protein alpha-subunit mRNA antisense oligonucleotide inhibition of Gi-coupled receptor contractile activity in the epididymis of the guinea-pig

Abstract

We have used a reversible permeabilization method to facilitate the entry of Gialpha1, 2 and 3 G-protein subunit mRNA antisense or mismatch oligonucleotides into intact tissue, to investigate the G-protein alpha-subunit coupling of alpha2-adrenoceptors, neuropeptide Y (NPY) Y1, and A1 adenosine receptors in preparations of the epididymis of the guinea-pig. The alpha2-adrenoceptor agonist, xylazine, elicited concentration dependent contractions from preparations of phenylephrine (3 microM)-stimulated epididymis (pEC50 value 6.52+/-0.39, maximum response 236+/-41 mg force). Compared to respective mismatch controls the incubation of preparations with Gialpha2, but not with Gialpha1 or Gialpha3 mRNA antisense oligonucleotides (30 microM) reduced the maximal xylazine-potentiation of phenylephrine (3 microM)-stimulated contractility (to 51+/-12% of Gialpha2 mismatch control). The oligonucleotide incubations had no effect upon the pEC50 values of xylazine. The A1 adenosine receptor agonist, cyclopentyladenosine (CPA) elicited concentration dependent contractions from preparations of phenylephrine (3 microM)-stimulated epididymis (pEC50 value 7.66+/-0.57, maximum response 208+/-54 mg force). Incubation of preparations of epididymis with Gialpha1, but neither Gialpha2 nor Gialpha3 antisense oligonucleotides reduced the maximal CPA-potentiation of phenylephrine (3 microM)-stimulated contractions (to 55+/-17% of Gialpha1 mismatch control), pEC50 values were not affected. The incubation of preparations with Gialpha2 antisense mRNA oligonucleotides reduced the maximal NPY-potentiation of phenylephrine (3 microM)-stimulated contractions (to 62+/-15% of Gialpha mismatch control). Compared with Gialpha2 mismatch controls, the incubation of preparations with Gialpha1 and Gialpha3 oligonucleotides also reduced the NPY-potentiation of phenylephrine (3 microM)-stimulated contractions. These studies indicate that, in the guinea-pig epididymis, alpha2-adrenoceptors and A1 adenosine receptors preferentially couple to effectors through Gialpha2 and Gialpha1 subunits respectively. In contrast NPY receptors may elicit effects through either Gialpha1, 2 or 3 subunits.

Figure 1

Effects of reversible permeabilization (RP) and RP with incubation (48 h) upon xylazine concentration-response curves. Preparations were permeabilized or were both permeabilized and incubated (48 h) prior to the construction of xylazine concentration-response curves upon phenylephrine (3 μM)-stimulated preparations of epididymis. Bars represent s.e.mean (some omitted for clarity) of six experiments.

Figure 2

Effects of Giα2 mRNA antisense or mismatch oligonucleotides upon response to xylazine or CPA in threshold (phenylephrine, 3 μM)-stimulated preparations of epididymis. (a) shows the effects of Giα2 antisense (AS) or mismatch (MM) oligonucleotides (10 μM) upon responses to xylazine. (b) shows the effects of Giα2 antisense (AS) or mismatch (MM) oligonucleotides (10 μM) upon responses to CPA. Bars represent s.e.mean (some omitted for clarity) of 9–12 experiments.

Figure 3

Effects of antisense mRNA or mismatch oligonucleotides upon responses to xylazine in threshold (phenylephrine, 3 μM)-stimulated preparations of epididymis. (a), (b) and (c) show the respective effects of Giα1, 2 or 3 antisense (AS) or mismatch (MM) oligonucleotides (30 μM) upon responses to xylazine. Bars represent s.e.mean (some omitted for clarity) of 6–12 experiments.

Figure 4

Effects of antisense mRNA or mismatch oligonucleotides upon responses to CPA in threshold (phenylephrine, 3 μM)-stimulated preparations of epididymis. (a), (b) and (c) show the respective effects of Giα1, 2 or 3 antisense (AS) or mismatch (MM) oligonucleotides (30 μM) upon responses to CPA. Bars represent s.e.mean (some omitted for clarity) of six experiments.

Figure 5

Effects of Giα1, 2 or 3 mRNA antisense (AS) or of Giα2 mismatch (MM) oligonucleotides upon responses to NPY in threshold (phenylephrine, 3 μM)-stimulated preparations of epididymis. Bars represent s.e.mean (some omitted for clarity) of six experiments.

Figure 6

G-protein subunits in membrane fractions of the epididymis of the guinea-pig. Lanes a–c show the relative densities of Giα1, Giα1/2 and Giα3 subunit primary antibody bands in membrane preparations of the cauda epididymis. Each lane shows membrane bands of pooled tissues from six animals. →Indicates molecular weight standard markers.