Guanylin and its lysine-containing analogue in the isolated perfused rat kidney: Interaction with chymotrypsin inhibitor

Abstract

Guanylin and uroguanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GME Their natriuretic and kaliuretic activities are well documented. Since guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of guanylin. Using the isolated perfused rat kidney, guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-guanylin (0.1-2.5 μg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 μg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 μg/ml), guanylin (0.3 μg/ml) was placed into solution. A maximal decrease in fractional Na+ reabsorption (%TNa+) was achieved at 1.0 μg/ml of lys-guanylin (from 73.2±2.29 to 54.9±0.10, P<0.05). Lys-guanylin (1.0 μg/ml) also decreased fractional K+ reabsorption (%TK+) from 59.26±3.93 to 30.75±0.78 (P<0.05). Chymostatin had no detectable effects in electrolyte reabsorption in this assay. When introduced after chymostatin, guanylin lowered %TNa+ (from 81.2±1.86 to 72.6±2.45, P<0.05) and %TK+ (from 69.4±4.12 to 65.8±2 .81, P<0.05). At this subthreshold concentration, guanylin alone lacks effects in %TNa+ or %TK+ Furthermore, the ability of both peptides to promote increases in intestinal fluid secretion was evaluated in the in vivo suckling mouse model. When administered per os, guanylin failed to stimulate intestinal secretion. When chymostatin was present in the test solution, guanylin induced intestinal secretion in this assay. In marked contrast, lys-guanylin alone induced diarrhoea in the suckling mouse. The present paper concludes that guanylin undergoes metabolism in target tissues such as the intestine and kidney and its lysine-containing analogue retains full biological activity.