The hormonal regulation of a Na(+)-K(+)-Cl- cotransport was investigated in a renal tubule cell line (RC.SV2 cells) transformed by the simian virus 40. This cell line has the main characteristics of cells from the thick ascending limb of Henle, including the presence of Tamm-Horsfall protein and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production by calcitonin (CT). Kinetic studies with 22Na+, 36Cl-, and 86Rb+ indicated the existence of a Na(+)-K(+)-Cl- cotransport with a stoichiometry of 1Na+:1K+: 2Cl-. All compounds stimulating cAMP production enhanced the ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx mediated by Na(+)-K(+)-Cl- cotransport. CT (100 ng/ml) increased the Or-Bs influx twofold by enhancing maximum velocity without changing the apparent Michaelis constant. The K(+)-channel blocker barium blunted the CT-stimulated Or-Bs influx by 64-74%, whereas the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate reduced the CT-stimulated influx by 28-40%. These results suggest that CT stimulates the Na(+)-K(+)-Cl- cotransport by a cAMP-dependent mechanism and that K+ recycling through K+ membrane channels is an important modulator of cotransporter-mediated ion fluxes.

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Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed‐batch cell culture batches yielded consistent glycoprofiles of a Fc‐fusion antibody comprizing three different N‐glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N‐glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site‐specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell‐based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N‐glycan structures, a higher degree of antennarity, and a site‐specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc‐site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N‐glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.