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, dit hiothreitol (DTT), increases the specific binding of [I-125]angiotnesi n IV ([I-125]AIV) to AT(4) receptors in bovine adrenal cortical membra nes. Both the degree of stimulation and the pharmacological selectivit y of [I-125]AIV binding in the presence of DTT were quantitatively dif ferent depending on the contents of the assay buffer. Similar effects were also observed using a different sulfhydryl reducing agent, 2-merc aptoethanol (2-MCE). These sulkydryl reducing agents (100 mM) produced a 200% increase in specific [I-125]AIV binding in an assay buffer tha t has been used to characterize the novel AT(4) receptor subtype. A mu ch larger stimulation (700%) of specific [I-125]AIV binding was found when the assay was conducted in a buffer that has been used to charact erize ligand binding to the AT(1) receptor. Ligand association studies indicated that 0.3 nM [I-125]AIV displayed similar equilibrium kineti cs and stability in both the AT(4) and AT(1) buffers. Ligand saturatio n 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 = 3 2 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 ef fects of DTT shown here on the AT(4) receptor are similar to its repor ted actions on the AT(2) receptor and indicate that putative receptor disulfide bridges may be important in the regulation of receptor affin ity.