Urea transport in freshly isolated and cultured cells from rat inner medullary collecting duct

Abstract

Regulation of urea transport by vasopressin in inner medullary collecting duct (IMCD) cells is thought to be important for the urinary concentrating mechanism. Isolated tubule perfusion studies suggest the existence of a saturable urea carrier. We have measured¹⁴C-urea efflux in IMCD cells which were freshly isolated and grown in primary culture. Cells were isolated from rat papilla by collagenase digestion and hypotonic shock. In suspended cells,¹⁴C-urea efflux (J _urea from loaded cells was exponential with time constant 59±3 sec (sem,n=6, 23°C).J _urea had an activation energy of 4.1 kcal/mole and was inhibited 42±7% by 0.25mm phloretin and 30–40% by the high affinity urea analogues dimethylurea and phenylurea.J _urea was increased 40–60% by addition of vasopressin (10⁻⁸ m) or 8-bromo-cAMP (1mm); stimulatedJ _urea was inhibited 55±8% by the kinase A inhibitor H-8. Phorbol esters and epidermal growth factor did not alterJ _urea. IMCD cells grown in primary culture were homogeneous in appearance with>fivefold stimulation of cAMP by vasopressin. The exponential time constant for urea efflux was 610±20 sec (n=3).J _urea was not altered by vasopressin, cAMP or phloretin. Another function of in vivo IMCD cells, vasopressin-dependent formation of endosomes containing water channels, was absent in the cultured cells. These results demonstrate presence of a urea transporter on suspended IMCD cells which is activated by cAMP and inhibited by phloretin and urea analogues. The urea transporter and its regulation by cAMP, and cAMP-dependent apical membrane endocytosis, are lost after growth in primary culture.