DISC U SSION

4 .2 .1 Guanvlate Cvclase A c tiv ity in Rat Cardiac Sarcolemmal Membranes These results demonstrate basal and ANP-stimulated guanylate cyclase a c t iv it y in purified sarcolemmal membranes isolated from ra t

ven tricu lar muscle. The findings are in agreement with previous reports, which have demonstrated basal guanylate cyclase a c tiv ity in p a rtic u la te fractions isolated from the heart ventricles of several species, including ra t (Kimura & Murad, 1974; Sulakhe et a l . , 1976a), ra b b it, hamster, guinea pig and mouse (Sulakhe et a l . . 1976a). The present results are also in agreement with previous studies

demonstrating ANP-stimulated increases in in tra c e llu la r cGMP levels in isolated ra t and rabbit ventricular myocytes (Aiton & Cramb, 1985; Cramb et a l . . 1987).

In ra t cardiac sarcolemmal membranes, Mn^* supported 20- to 40-fold higher levels of both basal and ANP-stimulated guanylate cyclase a c t iv it y than did Mg^*. Similar differences in the levels of basal guanylate cyclase a c tiv ity have been reported for ra t liv e r plasma membranes (Kimura & Murad, 1975a), for a particulate fraction from rabbit heart ventricles (Sulakhe et a l . . 1976a), and fo r basal and ANP-stimulated guanylate cyclase a c tiv ity in BAC membranes (Tremblay et a l . . 1986). The dose response curve fo r GTP, in the presence of excess of Mn^+, indicated positive co-operativity and/or multiple binding sites, with the addition of ANP increasing the maximum

v e lo c ity of the enzyme but having l i t t l e effe ct on the K^, or the slope of the dose response curve. Similar kinetics have been described for basal p artic u la te guanylate cyclase a c tiv ity in the ra t (Kimura & Murad, 1974) and rabbit (Sulakhe et a l . . 1976b) heart, and for basal

and ANP-stimulated a c tiv ity in bovine adrenal cortex (Tremblay et a l . . 1986). Likewise, the Hi l l coefficient calculated from the stimulatory portion of the manganese dose response curves, both in the absence and presence of ANP, suggested that this cation was exerting positive co­ operative e ffe c ts . In contrast, Mn^* concentrations in excess of 4 mM produced a marked reduction in guanylate cyclase a c tiv ity . These

results are in agreement with reports on the effects of Mn^* ion concentration on basal guanylate cyclase a c tiv ity in ra t (Kimura & Murad 1974) and rabbit (Sulakhe et a l . . 1976a) heart membranes.

Differences in ANP-stimulated guanylate cyclase a c tiv ity were observed between fra c tio n A and fraction B. Both these fractions are enriched in membrane enzyme markers, but electron micrographs indicate that the membranes in fraction A have a tendency to 'stick together', forming

liposom al-like structures (Cramb & Dow 1983). I t is possible that w hilst these structures do not affect basal guanylate cyclase

a c tiv ity , they do lim it the access of ANP to its receptor, and hence reduce ANP-stimulated a c tiv ity .

Half-maximal stimulation of guanylate cyclase a c tiv ity was produced by 1 nM ANP. Sim ilar concentrations of ANP have been reported to produce half-maximal stimulation of guanylate cyclase a c tiv ity in BAC

membranes (Tremblay et a l . . 1986), and to produce half-maximal

elevation of in tra c e llu la r cGMP levels in cortical collecting tubules (Narav-FeTes-Toth et a l . . 1988) and LLC-PKj cells (Inui et a l . , 1985). The dose response curve fo r ANP stimulation of guanylate cyclase

a c t iv it y was shallow in nature, with a H i l l co e ffic ien t of less than 1, indicating negative co-operativity.

truncated analogues of ANP to relax preconstricted smooth muscle, and in a ll cases ANPg_28 less potent that ANP (Thibault et a l . . 1984b;

Garcia et a l . . 1985; 01 ins et a l . . 1986). Physiological studies have

shown that ANPg_28 effective at producing natriuresis and diuresis in ra t but not in dog, indicating species variation in the response to these two peptides (Wakitani et a l . . 1985). This is supported by the observation that while ANPg_2g is 10-fold less potent than ANP at producing a half-maximal increase in in tra c e llu la r cGMP in ra t and human c e ll lines, i t is 1000-fold less potent in bovine and canine cells (Leitman & Murad, 1986). In the present experiments, the concentrations of ANP and ANPg_2g required to produce half-maximal stimulation of guanylate cyclase a c tiv ity in ra t cardiac sarcolemmal membranes were not sig n ifican tly d iffe re n t. There was however a

sig n ifican t difference between the maximum responses e lic ite d by these two peptides, with ANPg_2g producing less than 50% of the response produced by ANP.

In agreement with a previous report on the e ffe c t of

ANP5„25 guanylate cyclase a c tiv ity , this peptide was more than 1000-fold weaker than ANP in stimulating guanylate cyclase a c tiv ity in ra t cardiac sarcolemmal membranes (Leitman & Murad 1986).

4. 2. 2 Degradation of ANP

Endopeptidase 24.11 has been implicated in the breakdown of ANP by pig