DITHIOTHREITOL, SODIUM-CHLORIDE, AND ETHYLENEDIAMINETETRAACETIC ACID INCREASE THE BINDING-AFFINITY OF [I-125] ANGIOTENSIN-IV TO AT(4)-RECEPTORS IN BOVINE ADRENAL-CORTEX

The present studies demonstrate that the sulkydryl reducing agent, dithiothreitol (DTT), increases the specific binding of [I-125]angiotnesin IV ([I-125]AIV) to AT(4) receptors in bovine adrenal cortical membranes. Both the degree of stimulation and the pharmacological selectivity of [I-125]AIV binding in the presence of DTT were quantitatively different depending on the contents of the assay buffer. Similar effects were also observed using a different sulfhydryl reducing agent, 2-mercaptoethanol (2-MCE). These sulkydryl reducing agents (100 mM) produced a 200% increase in specific [I-125]AIV binding in an assay buffer that has been used to characterize the novel AT(4) receptor subtype. A much larger stimulation (700%) of specific [I-125]AIV binding was found when the assay was conducted in a buffer that has been used to characterize ligand binding to the AT(1) receptor. Ligand association studies indicated that 0.3 nM [I-125]AIV displayed similar equilibrium kinetics and stability in both the AT(4) and AT(1) buffers. Ligand saturation studies indicated that [I-125]AIV bound with high affinity (K-d = 6 mM) in the AT(4) buffer system, but bound with lower affinity (K-d = 32 nM) in the AT(1) buffer system. Removal of NaCl and EDTA from the AT(4) buffer also resulted in low-affinity [I-125]AIV binding (K-d = 33 nM. The subsequent inclusion of NaCl, EDTA, or DTT resulted in higher-affinity [I-125]AIV binding (KdS = 3-14 nM). No significant effects on the apparent density (B-max) of AT(4) receptors were observed. These results show that DTT specifically increases the specific binding of [I-125]AIV under conditions of both high and low affinity for the AT(4) receptor, which can be regulated by NaCl and/or EDTA. Further, the effects of DTT shown here on the AT(4) receptor are similar to its reported actions on the AT(2) receptor and indicate that putative receptor disulfide bridges may be important in the regulation of receptor affinity.