Modulation of alpha2-adrenoceptor functions by heterotrimeric Galphai protein isoforms

Abstract

Subtype diversity of heterotrimeric G proteins and G protein-coupled receptors enables a wide spectrum of signal transduction. However, the significance of isoforms within receptor or G protein subfamilies has not been fully elucidated. In the present study, we have tested whether alpha(2)-adrenoceptors require specific Galpha isoforms for their function in vivo. In particular, we analyzed the role of the highly homologous Galpha(i) isoforms, Galpha(i1), Galpha(i2), and Galpha(i3), in typical alpha(2)-adrenoceptor-controlled functions. Mice with targeted deletions in the genes encoding Galpha(i1), Galpha(i2), or Galpha(i3) were used to test the effects of alpha(2)-adrenoceptor stimulation by the agonist medetomidine. The alpha(2)-adrenoceptor agonist medetomidine inhibited [(3)H]norepinephrine release from isolated prefrontal brain cortex or cardiac atria tissue specimens with similar potency and efficacy in tissues from wild-type or Galpha(i)-deficient mice. In vivo, bradycardia, hypotension, induction of sleep, antinociception, and hypothermia induced by alpha(2)-adrenoceptor activation did not differ between wild-type and Galpha(i)-knockout mice. However, the effects of the alpha(2)-agonists medetomidine or 5-bromo-6-(2-imidazolin-2-ylamino)quin-oxaline tartrate (UK14,304) on spontaneous locomotor activity or anesthetic sparing were reduced or absent, respectively, in mice lacking Galpha(i2). In microdissected locus coeruleus neurons or postganglionic sympathetic neurons from stellate ganglia, all three Galpha(i) subunits were expressed as determined by quantitative reverse transcription-polymerase chain reaction, with Galpha(i1) and Galpha(i2) dominating over Galpha(i3). Functional redundancy of the highly homologous Galpha(i) isoforms may predominate over specificity to regulate distinct intracellular pathways downstream of alpha(2)-adrenoceptors in vivo. In contrast, inhibition of locomotor activity and anesthetic sparing may be elicited by a specific coupling of alpha(2A)-adrenoceptors via the Galpha(i2) isoform to intracellular pathways.

Fig. 1.

Effect of the α₂-adrenoceptor agonist medetomidine on [³H]norepinephrine release from brain cortex slices. a, tritium efflux- versus time-curves from brain cortex slices of Gα_i1-KO mice. Cortex slices were stimulated with four rectangular electrical pulses at 100 Hz (2-ms pulse width; 80 mA) applied at 8-min intervals. [³H]Norepinephrine release was inhibited by addition of medetomidine. b to d, concentration-response curves for the inhibition of [³H]norepinephrine release by medetomidine. For ease of comparison, results from wild-type specimens (open squares) were reproduced in b to d. Data are means ± S.E.M. from n = 8 to 12 mice.

Fig. 2.

Inhibition of locomotor activity and loss of righting reflex by medetomidine (a–c) or pentobarbital (d) in mice lacking Gα_i isoforms. a, spontaneous locomotor activity as determined in an infrared photobeam cage system did not differ between genotype groups. b, medetomidine (50 μg/kg) reduced activity by >90% in WT, Gα_i1-KO, or Gα_i3-KO mice but to 37.2 ± 8.1% in Gα_i2-KO mice (n = 6–8/genotype; ∗, p < 0.01 versus WT). c, higher doses of medetomidine induced a strong sedative effect, i.e., loss of the righting reflex, in all genotype groups (percentage of mice that lost the righting reflex; p > 0.05 versus WT). d, pentobarbital-induced loss of righting reflex. Time to present loss of righting after administration of 50 mg/kg i.p. pentobarbital (mean ± S.E.M. represented by left margin of the horizontal bars) or time to recovery from loss of righting (mean ± S.E.M. represented by right margin of horizontal bars) did not significantly differ between genotypes. Data are means ± S.E.M. from n = 6 mice/group (p > 0.05 versus WT).

Fig. 3.

Anesthetic sparing effect of α₂-adrenoceptor activation in mice deficient in individual Gα_i protein subunits. a to d, medetomidine was administered at doses that did not cause loss of the righting reflex (50 or 100 μg/kg i.p.) 30 min before exposure to increasing concentrations of isoflurane (0–1.2 vol% in O₂). Medetomidine caused a leftward shift of the isoflurane-mediated loss of righting reflex curves in WT (50 or 100 μg/kg) mice and in animals lacking Gα_i1 (100 μg/kg) and Gα_i3 (100 μg/kg) but not in Gα_i2-deficient mice (n = 12 mice/genotype). e, concentration of isoflurane required to induce loss of righting reflex in 50% of wild-type or Gα_i-deficient mice in the absence (0, control) or presence of medetomidine (50 and 100 μg/kg i.p.; ∗, p < 0.05 versus control).

Fig. 4.

Inhibition of locomotor activity and anesthetic sparing effect of the α₂-adrenoceptor agonist UK14,304. a, spontaneous locomotor activity as determined in an infrared photobeam cage system did not differ between genotype groups. b, UK14,304 (100 μg/kg)-mediated reduction in activity was significantly blunted in Gα_i2-KO mice (n = 8–12/measurements per genotype; ∗∗, p < 0.01 versus WT). c to f, UK14,304 was administered at 300 μg/kg i.p. 30 min before exposure to increasing concentrations of isoflurane (0–1.2 vol% in O₂). UK14,304 caused a leftward shift of the isoflurane-mediated loss of righting reflex curves in WT mice and in animals lacking Gα_i1 or Gα_i3 but not in Gα_i2-deficient mice (n = 6 mice/genotype, log rank test).

Fig. 5.

Antinociceptive and hypothermic effects of α₂-adrenoceptor stimulation in mice lacking Gα_i protein isoforms. a, pain threshold was assessed by determining the tail-flick latency. Administration of medetomidine (100 or 200 μg/kg i.p.) prolonged the tail-flick latency in all groups, although this effect did not differ significantly between genotypes (∗, p < 0.001 versus respective saline group; n = 8/genotype). b, body core temperature was determined during isoflurane anesthesia by a rectal probe. Administration of medetomidine (100 or 200 μg/kg i.p.) caused a reduction in body temperature in all genotype groups (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 versus basal values in the absence of medetomidine; n = 12/genotype).

Fig. 6.

Hemodynamic effects of α₂-adrenoceptor stimulation in mice deficient in Gα_i protein isoforms. Heart rate (a) and aortic systolic (b) and diastolic (c) pressures were determined during isoflurane anesthesia by Millar microtip catheterization. Medetomidine elicited similar bradycardic (a) and hypotensive (b and c) effects in mice from all genotype groups (n = 6–8 mice/group; p > 0.05 versus WT).

Fig. 7.

Expression of Gα_i isoform mRNA in locus coeruleus and stellate ganglia from wild-type mice. a and b, identification of the locus coeruleus in cryostat sections through wild-type mouse brain (a) or sympathetic ganglion stellata (b) by anti-tyrosine hydroxylase immunostaining (green fluorescence). Nuclei were identified by blue 4′,6-diamidino-2-phenylindole (Dapi) fluorescence. Right, overlay of tyrosine hydroxylase and Dapi signals. Scale bars, 200 μm (a and b). c, quantification of Gα_i isoform mRNA in microdissected locus coeruleus or total stellate ganglia specimens by quantitative reverse transcription-PCR (∗, p < 0.05 versus Gα_i1).