| pA2 online © Copyright 2004 The British Pharmacological Society |
069P
University of Bath Summer Meeting July 2004 |
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NM Broxton, BA Evans & RJ Summers. Dept of Pharmacology, Monash University, Clayton, VIC 3800 Australia |
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1A- adrenoceptor isoforms display different signalling profiles following agonist stimulation
Print abstract The 
1-adrenoceptor (1-AR) family consists of the 1A , 1B and 1D subtypes. The 1A-AR has been reported to have twelve isoforms, four of which are functional, the others being truncated and non-functional (Chang et al 1998). The functional isoforms denoted 1A-1,2,3&4 have similar pharmacological properties and all couple to Gq and signal through the IP3/DAG pathway (Chang et al1998). Studies with various tissues and recombinant receptors suggest that 1A-AR couple to multiple second messenger pathways including those modulated by PLA2, PLD, adenylate cyclase, MAP kinase and protein kinase C (Graham et al 1996).The current study examined the ability of the four 1A-AR isoforms to activate signalling pathways studied by cytosensor microphysiometry, cAMP accumulation and [3H]arachidonic acid release.
Agonist stimulation of 1A-AR in the cytosensor microphysiometer increased cellular activity, a summation of all the signalling pathways activated by agonist stimulation. All isoforms produced an increase in the extracellular acidification rate (ECAR) to agonists in a concentration-dependent manner. However responses of the 1A-2 to A61603 were significantly greater (p<0.05) than all other isoforms (Emax value m µsec-1 at 80.8 ± 8.7(4) for 1A-2 , 27.9 ± 1.9 (6) for 1A-1, 59.7 ± 5.0 (5) for 1A-3 and 33.0 ± 3.0 (4) for 1A-4 .) The maximal responses ( m µsec-1) of the 1A-AR isoforms to noradrenaline ( 1A-1 -AR: 28.5 ± 2.4 vs 40.5 ± 2.7, (6) P<0.0001), methoxamine (19.4 ± 1 vs 34.5 ± 2.6, (6) P<0.0001) and A61603 (27.4 ± 1.8 vs 36.7 ± 2.7, (4) P<0.0001) were increased following pertussis toxin (PTX, 100ng/ml, 16 hr) treatment. There was no significant effect on the response to the oxymetazoline (P=0.15). Stimulation of cAMP accumulation occurred for the 1A-1,3&4 isoforms in a ligand and concentration dependent manner (Emax % forskolin 10-4M, pEC50(n): A61603; 1A-1 51.4 ± 3.1, 8.0 ± 0.3(4); 1A-3 41.4 ± 3.7, 8.8 ± 5.3 (3); 1A-4 38.6 ± 2.1, 7.5 ± 0.1(4)). Oxymetazoline had no significant effect on cAMP accumulation for any of the isoforms (p<0.05). The 1A-2 isoform did not produce cAMP accumulation at levels of expression comparable with the other isoforms. cAMP production was PTX sensitive for only the 1A-3-AR with an increase in the maximal response to agonist in the presence of PTX (Emax % forskolin 10-4M: noradrenaline 31.2 ± 4.3 vs 65.09 ± 8.4 (4) (P<0.0001), methoxamine 23.64 ± 3.7 vs 43.3 ± 6.07 (4) (P<0.0001)). Stimulation of arachidonic acid release was also shown to occur for the 1A-1,3&4 isoforms in a concentration-dependent manner with rank orders of agonist potency of cirazoline>noradrenaline>phenylephrine>oxymetazoline for 1A and 1A-4 and oxymetazoline 
noradrenaline>cirazoline>phenylephrine for 1A-2 -AR. The 1A-2 -AR was unable to stimulate this signalling pathway at levels of expression comparable with the other isoforms (Bmax fmol mg-1 protein: 1A-1 :509 ± 93(5), 1A-2 : 237 ± 66(5), 1A-3 : 432 ± 74(3), 1A-4 : 433 ± 77(5)).
These results confirm that 1A-AR isoforms couple to a number of different signalling pathways, but suggest that these responses are ligand and isoform dependent.
Chang DJ et al., (1998) FEBS Lett. 422: 279-283
Graham RM et al., (1996) Circ Res. 78: 737-49