Characterisation of the vascular angiotensin receptor

McQueen, James (1986) Characterisation of the vascular angiotensin receptor. PhD thesis, University of Glasgow.

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Binding sites of high affinity and low capacity for 125 I-angiotensin II have been characterised in a cell membrane fraction derived from mesenteric arterial arcades of the rat, resistance-type vessels which are representative of the vascular target organs for angiotensin II. Degradation of tracer angiotensin II and heterogeneity of binding sites were accounted for by use of nonlinear regression methods for the analysis of radioligand binding data. Binding constants for 125 I-angiotensin II obtained by different experimental approaches were in good agreement and gave a dissociation equilibrium constant of 0.013-0.098nM (95% confidence interval). Affinities for a number of angiotensin-related peptides derived from competitive binding curves paralleled the pressor activities of these peptides with the order 125 I-angiotensin II = angiotensin II > angiotensin III > C-terminal hexapeptide > C-terminal pentapeptide >> angiotensin I = bradykinin. The iodinated peptide was found to retain full pressor activity in bioassay experiments. These binding sites therefore exhibit properties of a physiological receptor for angiotensin II. The mechanism by which alteration of sodium balance brings about changes in pressor sensitivity to angiotensin II has been investigated by radioligand receptor assay. Sodium depletion (low salt diet + diuretic) caused a rapid increase in plasma angiotensin II concentration and a corresponding fall in receptor density from 102 +/- 4 fmol/mg membrane protein (control) to 44 +/- 5 fmol/mg protein at 12 days. Sodium loading and inhibition of angiotensin converting enzyme with captopril were equally effective in suppressing plasma angiotensin II concentration, but only in the captopril treated animals was an increase in receptor density observed (118 +/- 6 fmol/mg protein at 12 days). Sodium loading caused a slow fall in receptor density, not significant at 5 days, to 77 +/- 4 fmol/mg protein at 12 days. This indicates that plasma angiotensin II concentration is not the sole determinant of vascular receptor status. The results described above were obtained under standard assay conditions with a Tris buffer containing 4. 8mM Ca ++. Using tissue from normal animals, the apparent receptor density was found to vary with the assay incubation medium calcium concentration over the range 0-10mM. Receptor densities of 50 +/- 4, 102 +/- 4 and 156 +/- 5 fmol/mg membrane protein were obtained in low- (0mM), normal- (4.8mM) and high- (25mM) [Ca++] assay respectively. After 2 days of sodium loading, sodium depletion and converting enzyme blockade, the receptor densities determined in high- and low-[Ca++] assays did not differ significantly from those of control animals. Thus the altered receptor densities seen in normal-[Ca++] assay were due to shifts in the [Ca++]-receptor density relationship without apparent change in the total number of receptors. Similar results were obtained for the 12 day sodium loading and converting enzyme blockade experiments. In contrast, after 12 days of sodium depletion there was no difference in receptor density between normal- and high-[Ca++ ] assays (49 +/- 7 fmol/mg) and the value obtained in low-[Ca++] assay was significantly below that for all the other groups. These findings indicate two stages in the regulation of the vascular angiotensin receptor; an initial masking or inactivation of binding sites through a mechanism which is reversed by calcium, and (with elevation of plasma angiotensin II concentration) a subsequent loss of receptors. The factor responsible for the differing effects of converting enzyme blockade and sodium loading on receptor status appears to modulate this receptor masking/inactivating process. A mechanism by which this process operates is proposed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Medicine, Biochemistry
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-76595
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:05
Last Modified: 19 Nov 2019 14:05

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