Expression of mRNA and functional alpha(1)-adrenoceptors that suppress the GIRK conductance in adult rat locus coeruleus neurons

Abstract

1. Locus coeruleus neurons in adult rats express binding sites and mRNA for alpha(1)-adrenoceptors even though the depolarizing effect of alpha(1)-adrenoceptor agonists on neonatal neurons disappears during development. 2. In this study intracellular microelectrodes were used to record from locus coeruleus neurons in brain slices of adult rats and reverse transcription-polymerase chain reaction (RT - PCR) was used to investigate the mRNA expression of alpha(1)- and alpha(2)-adrenoceptors in juvenile and adult rats. 3. The alpha(1)-adrenoceptor agonist phenylephrine had no effect on the membrane conductance of locus coeruleus neurons (V(hold) -60 mV) but decreased the G protein coupled, inward rectifier potassium (GIRK) conductance induced by alpha(2)-adrenoceptor or mu-opioid agonists. The GIRK conductance induced by noradrenaline was increased in amplitude when alpha(1)-adrenoceptors were blocked with prazosin. 4. RT - PCR of total cellular RNA isolated from microdissected locus coeruleus tissue demonstrated strong mRNA expression of alpha(1a)-, alpha(1b)- and alpha(1d)-adrenoceptors in both juvenile and adult rats. However, only mRNA transcripts for the alpha(1b)-adrenoceptors were consistently detected in cytoplasmic samples taken from single locus coeruleus neurons of juvenile rats, suggesting that this subtype may be responsible for the physiological effects seen in juvenile rats. 5. Juvenile and adult locus coeruleus tissue expressed mRNA for the alpha(2a)- and alpha(2c)-adrenoceptors while the alpha(2b)-adrenoceptor was only weakly expressed in juveniles and was not detected in adults. 6. The results of this study show that alpha(1)-adrenoceptors expressed in adult locus coeruleus neurons function to suppress the GIRK conductance that is activated by mu-opioid and alpha(2)-adrenoceptors.

Figure 1

Alpha₁-adrenoceptors suppress outward currents induced by α₂-adrenoceptors. Intracellular microelectrode recordings obtained from rat locus coeruleus neurons voltage clamped at −60 mV. (a) After the outward current produced by 30 μM noradrenaline (NA) had reached steady-state, a further increase in amplitude was produced when α₁-adrenoceptors were blocked with 300 nM prazosin. (b) The steady-state outward current produced by the α₂-adrenoceptor agonist UK14304 was reduced in amplitude when the α₁-adrenoceptor agonist phenylephrine (PE) was co-applied. The α₂-agonist, idazoxan, blocked the effect of UK14304.

Figure 2

Effects of phenylephrine on currents activated by α₂-adrenoceptor and μ-opioid receptor agonists. (a) Concentration-effect curves for the inhibition by phenylephrine of α₂-adrenoceptor currents activated by UK14304 (1 μM) and opioid currents activated by [Met⁵]enkephalin (10 μM). The curves are the fits of a logistic function (equation 1) to the data, which are means and s.e.means. The effect of a single concentration of phenylephrine (100 μM) on the maximal current activated by DAMGO (1 μM) is also shown. (b) The α₁-adrenoceptor agonist phenylephrine (PE) caused concentration-dependent inhibition of outward currents activated by the μ-opioid agonist [Met⁵]enkephalin. (c) The concentration-effect curve for the selective μ-opioid agonist DAMGO was shifted to the right and had a reduced maximal response in response to 100 μM phenylephrine. The curves were derived from using average estimates of the parameters obtained by fitting a logistic function to data obtained in single neurons. Data are the means and s.e.means.

Figure 3

α₁- and α₂-adrenoceptor mRNA expression in the adult locus coeruleus. (a) Agarose gel electrophoresis of 8 μl of PCR product of α_1b- and α_1d-adrenoceptor subtypes. A band of 405 bp (α_1b+RT) represents α_1b. Likewise, mRNA expression for α_1a- and α_1d-adrenoceptors are confirmed by the presence of bands of 251 bp (α_1a,+RT) and 517 bp (α_1d,+RT; top band). (b) PCR products defined by the α₂ subtype specific primers were amplified from the same cDNA pool as the three α₁-receptors. PCR product of 312 bp (α_2a,+RT) represents α_2a and 425 bp (α_2c,+RT) represents α_2c, while the predicted 456 bp product defined by the α_2b subtype specific primers was not amplified after 30 cycles (α_2b,+RT). No PCR products were seen in the control experiments that contained no reverse transcriptase in the RT reaction (−RT), nor in the second control that did not contain RNA (H₂O). DNA size markers, ΦX174/HaeIII (MW).

Figure 4

α₁-adrenoceptor mRNA expression in single cells of juvenile rat locus coeruleus. Agarose gel electrophoresis of 8 μl of PCR product of adrenergic α_1b-, α_1a- and α_1d-receptor subtypes. The cytoplasmic contents of five individual locus coeruleus neurons were examined for α_1b expression and a representative band of 405 bp is shown (α_1b,+RT). No PCR product was amplified from six locus coeruleus neurons after two rounds of PCR with α_1a specific primers (α_1a,+RT). Three out of five cells examined for α_1d expression demonstrated the presence of α_1d (517 bp, α_1d,+RT). No PCR products were amplified from the mRNA harvested from 14 locus coeruleus neurons with any of the three α₁-adrenoceptor primers when the RT enzyme was omitted from the RT reaction (−RT). DNA size markers, ΦX174/HaeIII (MW).

Figure 5

Summary of α₁-adrenoceptor modulation of GIRK currents induced by different agonists. Potassium currents induced by noradrenaline were potentiated by prazosin (300 nM), while potassium currents induced by UK14304 (1 μM), [Met⁵]enkephalin (10 μM) and DAMGO (1 μM) were inhibited by phenylephrine (100 μM). Changes are expressed as percentage of the currents induced by agonist alone. Data are means and s.e.means.