Characterization of Imidazoline Receptors on Porcine Renal Cortex Membranes

Abstract

Imidazoline receptors have been reported to play roles in kidney functions such as

natriuresis and diuresis. The processes occurred in the segment of nephron, in particular,

those that located in the cortex of kidney. The aim of this study is to characterize the subtype

of imidazoline receptors (IR) and to determine receptor density and affinity of imidazoline

receptors on porcine renal cortex membranes. From saturation binding assay, the maximum

receptor density of IR on porcine renal cortex membranes labeled by [³H]-clonidine was

390.2± 89.09 fmol/mg protein with Kd value of 9.69±3.8 nM. The maximum receptor density

of I₂ receptor on porcine renal cortex membranes labeled by [³H]-idazoxan was 655.6± 49.17

fmol/mg protein with Kd value of 8.49± 1.29 nM. The result revealed that [³H]-idazoxan

binding sites (I₂ site) were 1.7 fold higher than those of [³H]-clonidine binding whereas the

affinities were comparable. In competitive binding assay, I₁ ligands, clonidine, rilmenidine,

moxonidine, surprisingly competed with low affinities to I-site labeled by [³H]-clonidine. The

rank order of potency of competing ligands was : idazoxan ( 459± 1.33 nM) > clonidine

(730±1.31 nM) > rilmenidine (2,769±1.26 nM) > oxymetazoline (9,204±4.41 nM) >

moxonidine = efaroxan (> 10⁵ nM). The result showed that this site differed from the typical I₁

sites. On the contrary, selective I₂ receptor ligand, idazoxan, competed with very high affinity

to [³H]-idazoxan binding site whereas I₁ receptor ligands, clonidine, rilmenidine, moxonidine,

oxymetazoline and efaroxan also competed with very low affinity to I₂ site. The rank order of

potency was : idazoxan (0.579±0.06 nM) > clonidine (16,100±0.15 nM) > rilmenidine

(l8,900.±3.4 nM) > oxymetazoline (42,300±33.2 nM) > moxonidine = efaroxan (>10⁵ nM).

The results from this study suggested that the major imidazoline receptor subtype on porcine

renal cortex is I₂ site. In conclusion, I₂ receptors are the main subtype exist on porcine renal

cortex membranes which is suggested to be the functional receptors in kidney whereas I site

labeled with [³H]-clonidine is different from the typical I₁ site. This site may be a new

subtype of imidazoline receptor.