Characterization of the postjunctional alpha 2C-adrenoceptor mediating vasoconstriction to UK14304 in porcine pulmonary veins

Abstract

Background and purpose: In terms of postjunctional alpha(2)-adrenoceptors in the pulmonary circulation, no evidence is available with regard to the receptor subtypes mediating vasoconstriction. Therefore, we characterized the alpha(2)-adrenoceptor subtypes mediating contraction in isolated porcine pulmonary veins.

Experimental approach: alpha-adrenoceptor-mediated vasoconstriction was studied using a tissue bath protocol. mRNA profile and relative quantification of alpha(2)-adrenoceptor subtypes were determined in porcine pulmonary veins using reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time PCR.

Key results: In porcine pulmonary veins, noradrenaline, phenylephrine (alpha(1)-adrenoceptor agonist), UK14304 and clonidine (alpha(2)-adrenoceptor agonists) caused concentration-dependent contractions. The rank order of agonist potency was: NA approximately UK14304 approximately clonidine > phenylephrine. UK14304 responses were antagonised by MK912 (noncompetitive antagonist parameter pD'(2): 10.1), rauwolscine (pK(B): 9.5), yohimbine (pK(B): 9.1), WB4101 (pK(B): 8.7), ARC239 (pK(B): 7.5), prazosin (pK(B): 7.1) and BRL44408 (pK(B): 7.0). Antagonist potencies fitted best with radioligand binding data (pK(i)) at the human recombinant alpha(2C)-adrenoceptor (r(2)=0.96, P=0.0001). Correlation with alpha(2B)-adrenoceptors was lower (r(2)=0.74, P>0.01) and no correlation was obtained with alpha(2A)-adrenoceptors. Moreover, RT-PCR studies in porcine pulmonary veins showed mRNA signals for alpha(2A)- and alpha(2C)-adrenoceptors, but not for alpha(2B)-adrenoceptors, whilst real-time PCR studies indicated a prominent expression of alpha(2C)-adrenoceptor mRNA.

Conclusions and implications: Postjunctional alpha(2C)-adrenoceptors mediated contraction in porcine pulmonary veins. alpha(1)-Adrenoceptors also seem to be present in this tissue. Since alpha(2)-adrenoceptor responsiveness is increased when pulmonary vascular tone is elevated, alpha(2C)-adrenoceptor antagonists may be beneficial in diseases such as pulmonary hypertension or congestive heart failure.

Figure 1

Cumulative log CRCs to α-adrenoceptor agonists in rings of porcine pulmonary veins. Values are mean±s.e.m. (vertical bars) from n animals as indicated in parentheses.

Figure 2

Cumulative log CRCs to UK14304 in the absence or presence of ARC239 (a) BRL44408 (b) prazosin (c) WB4101 (d) and yohimbine (e) in rings of porcine pulmonary veins. The insets represent the Schild regression analysis of the respective CRCs. Values are means±s.e.m. (vertical bars) from n animals as indicated in parentheses.

Figure 3

Cumulative log CRCs to UK14304 in the absence or presence of rauwolscine (a) and MK912 (b) in rings of porcine pulmonary veins. Values are means±s.e.m. (vertical bars) from n animals as indicated in parentheses.

Figure 4

Correlation of antagonist affinity estimates (pK_B; ^†pD′₂ in the case of MK912) against UK14304 at the contractile α₂-adrenoceptor in porcine pulmonary veins and binding affinity (pK_i) at human recombinant α_2A-, α_2B- and α_2C-adrenoceptors. pK_i values were taken from Uhlén et al. (1994). The dashed line represents the line of identity and the solid line, the regression line of the plotted points. The numbering of the drugs is as follows: MK912 (1), rauwolscine (2), yohimbine (3), WB4101 (4), ARC239 (5), prazosin (6) and BRL44408 (7).

Figure 5

Agarose gel electrophoresis of RT-PCR products showing the presence of mRNA for α_2A-, α_2B- and α_2C-adrenoceptors in pig cerebral cortex (upper panel) and the presence of mRNA for α_2A- and α_2C-adrenoceptors in porcine pulmonary veins (lower panel). The marked lanes denote: 100 bp DNA ladder (M), positive control showing RT-PCR product of 383 bp using GAPDH primers (1), RT-PCR product of 455 bp obtained using forward and reverse primers of α_2A-adrenoceptor (2), RT-PCR product of 179 bp obtained using forward and reverse primers of α_2B-adrenoceptor (3), RT-PCR product of 220 bp obtained using forward and reverse primers of α_2C-adrenoceptor (4), negative control, that is, a sample without reverse transcriptase to monitor genomic contamination (5) and negative control, that is, a PCR sample without template to monitor contamination of the reaction mixture (6). The size (bp) of three marker bands is indicated in the right margin.

Figure 6

Real-time PCR results showing the relative expression of mRNA for α_2A- and α_2C-adrenoceptors (AR) compared to GAPDH mRNA. Note that mRNA for the α_2B-adrenoceptor was not detectable (n.d.), confirming the RT-PCR results. Values are means±s.e.m. (vertical bars) from three independent experiments.