Modification of the ANP molecule, p a rtic u la rly by carboxyl terminal deletions or disruption of its ring structure, has marked effects on the potency of the peptide to e l i c i t physiological responses (see section 1. 6. 1) . The effects of such alterations on the a b ilit y of the peptide to displace [^^^I]-ANP have been studied in a number of
tissues and can be summarised as follows: Deletion of amino acids from the amino terminal results in a gradual reduction in the a f f in it y of the peptide. ANp2_2g is equipotent or almost equipotent to ANPj^28 ' whereas ANPg_2g is 3- to 10-fold less potent. ANP7„28 the least potent of the amino-terminal deleted peptides, having an a f f in it y some 20-fold less than the 28 amino acid peptide, ANPj^_28 (S c h iffrin et a l . . 1985; De Lean et a l . . 1985; 01 ins et a l . . 1986). Deletion of T y rl^6 from the carboxyl-terminal has l i t t l e e ffe c t on binding a f f i n i t y (S c h iffrin et a l . . 1985; Carrier et a l . . 1985; De Lean
a l . . 1985). Removal of the trip ep tid e, Phe^^-Arg^^-Tyr^®, usually results in a 10- to 100-fold reduction in a f f i n i t y (S ch iffrin et a l . . 1985; 01 ins et a l . . 1986), although as much as an 8000-fold reduction in a f f i n i t y has been reported for the inhibition of [^^^I]-ANP binding to bovine adrenal cells (De Lean et a l . . 1985).
A sim ilar p ro file of a c tiv ity is observed fo r the stimulation of in tra c e llu la r cGMP production, a lb e it at concentrations some 10- to 1000-fo ld higher than those observed in the d irect binding studies; the order of potency being ANPi_2g > ANPg_2g> ANPg_25 (Leitman & Murad 1986; Nambi et a l . . 1986; Rapoport et a l . . 1986; Roubert et a l . .
1987).
There are however some analogues of ANP which show divergence between th e ir a f f in it y for ANP binding sites and th e ir a b i l it y to increase in tra c e llu la r cGMP. Scarborough et a l. (1986) showed that while ANP^_ 23 and ANP4_25 were equipotent at displacing radio-lab elled ANP, the la tt e r was more than 100-fo ld less e ffe ctive than the former at augmenting in tra c e llu la r cGMP accumulation in BASM cel l s. S im ilarly, Leitman and Murad (1986) reported a 6-fo ld difference in potency between ANPg_23 and ANP5_2g to displace of [^^^I]-ANP from cultured bovine endothelial cel l s, w hilst there was more than a 100-fol d difference in th e ir a b ilit y to increase cGMP in these cells.
A ring-deleted analogue of ANP, ANP4_23 (des-Gln^®-Gly^^)-NH2, has been shown to compete e ffe c tiv e ly with [^^^I]-ANP fo r the m ajority of binding sites in whole rat kidney and kidney cortex, with only an 8- fo ld reduction in a f f in it y re la tiv e to ANPj.28 (Maack et a l . . 1987).
Ring-deleted ANP is however without effe ct on glomerular f il t r a t i o n
isolated perfused ra t kidney (Maack et a l . , 1987). Nor does i t relax preconstricted blood vessels or in h ib it aldosterone secretion (Lewicki et a l . . 1988). In contrast, an increase in immunoreactive native ANP
is observed when ring deleted ANP is injected into anaesthetized rats, with a corresponding increase in sodium excretion (Maack et a l . .
1987). These authors concluded that the majority of ANP receptors in the kidney are "biolog ically s ile n t", and postulated that these receptors act as clearance binding sites; the increase in plasma ANP in the intact preparation being a direct consequence of these binding sites being occupied by ring-deleted ANP. These receptors have been termed C-ANP receptors, and wi l l bind many truncated analogues of ANP with high a f f i n i t y . The "biologically active" binding sites are
refered to as B-ANP receptors, and bind only ANPj^_28 with high a f f i n i t y (Maack et a l . . 1987).
At a molecular level, the 60 to 70 kDa receptor species, as id en tifie d by SDS-polyacrylamide electrophoresis under reducing conditions,
appears to be the C-ANP receptor, since truncated and ring-deleted analogues of ANP bind to this species with high a f f i n i t y (Takayanagi et a l . . 1987b; Lewicki et a l . . 1988). The 120-140 kDa receptor
species, id e n tifie d under sim ilar conditions, and which is associated with guanylate cyclase a c tiv ity , has a much lower a f f i n i t y for
truncated ANP analogues compared to ANPi_2g (Takayanagi et a l . .
1987b), and is lik e ly to be the B-ANP receptor (Lewicki et a l . . 1988). 2.6 Summarv
The evidence to date strongly suggests that most cel l s possess at least two types of ANP receptors. One receptor is intim ately
kDa under reducing conditions and appears to be responsible for the vaso-relaxant properties of ANP. The second ANP receptor has a of 60 to 70 kDa under reducing conditions, and i t has been postulated that this receptor is responsible fo r the clearance of ANP from the blood stream. No second messenger system has been id e n tifie d for this receptor.
2.7 Reasons fo r Experiments
ANP has been shown to increase in tra c e llu la r cGMP levels in intact ra t ven tricular myocytes (Aiton & Cramb, 1985). Because ANP is rapidly degraded when incubated with ventricular myocytes (see chapter 9), fu rth e r investigations into the mechanism of action of ANP were
performed in purified ra t cardiac sarcolemmal membranes isolated from ven tricular muscle. Guanylate cyclase a c tiv ity and [^^^I]-ANP binding sites were measured in this preparation and the results compared with sim ilar studies conducted on membranes isolated from bovine adrenal cortex.
[125i ]„ANP binding sites have also been id en tifie d on intact MDCK (Madin Darby canine kidney) cells (Aiton et a l . . 1987) and these were fu rth e r investigated in membrane preparations.
CHAPTER 3