Uroguanylin was isolated initially from opossum urine as 13 to 15 amino acid peptides that activate the transmembrane receptor-guanylate cyclases expressed in cultured T84 intestinal and opossum kidney (OK) cells. Cloning of an intestinal cDNA encoding the 109 residue preprouroguanylin provided the means to detect the expression of uroguanylin mRNA in the intestinal mucosa and myocardium, but not in kidney (Fan et al, these Proc.). We postulated that urinary uroguanylin was derived from the circulation by glomerular filtration. Opossum plasma (0.8 L) was extracted using C18 cartridges, fractionated by gel filtration and preparative isoelectric focusing and bioassayed by measuring cGMP accumulation in T84 cells. Uroguanylin bioactivity eluted in the internal volume of Sephadex G-25 and this peptide fraction migrated with a pi of 3.9 on isoelectric focusing. Because prouroguanylin is inactive, a method was developed using chymotrypsin to release the bioactive C-terminal uroguanylin peptide from the prohormone. Using the same purification procedure beginning with 1.3 L of plasma, we isolated the active uroguanylin (pi 3.9) peptide. A second peak of prouroguanylin-fike activity (pi 5) that was dependent on chymotrypsin pretreatment for detection in the bioassay was also isolated. The prouroguanylin peak was combined and subjected to additional purification by reverse phase-HPLC. A single peak of prouroguanylin that was dependent on chymotrypsin pretreatment for bioactivity eluted at a position identical to that of authentic prouroguanylin24-109. We conclude that both uroguanylin and prouroguanylin circulate in the plasma of opossums under normal physiological conditions, thus plasma may be the source of urinary uroguanylin. The nathuretic actions of uroguanylin, its production by the intestine and presence in plasma are key components of the hypothesis that uroguanylin serves as an intestinal natriuretic hormone.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology