OSMOTIC AND NONOSMOTIC REGULATION OF ARGININE-VASOPRESSIN (AVP) RELEASE, MESSENGER-RNA, AND PROMOTER ACTIVITY IN SMALL-CELL LUNG-CARCINOMA (SCLC) CELLS

Arginine vasopressin (AVP) is synthesized in the magnocellular neurons of the hypothalamus and stored in the posterior pituitary. It has bee n shown that hypothalamic AVP mRNA is increased during experimental st imulation of osmotic and non-osmotic stimulation of AVP release. The m echanisms underlying the stimulation of AVP biosynthesis in these cond itions are not known. The present study was, therefore, performed to m easure AVP release, AVP mRNA level, and AVP gene promoter activity dur ing osmotic and non-osmotic stimulation of AVP secretion in the small cell lung carcinoma (SCLC) cells. AVP release was measured by radioimm unoassay, steady state levels of AVP mRNA by solution hybridization, a nd AVP gene promoter activity exhibited by a 1.5 kb 5'-flanking AVP ge ne fragment fused to a luciferase reporter after SCLC cells were subje cted to osmotic or non-osmotic conditions. High media osmolality (330 mOsm) significantly increased AVP release (control (C) 1.42 +/- 0.27 v s. High Oam 3.67 +/- 0.39 pg/2 x 10(6) cells, N = 9, P < 0.002); AVP m RNA (C 173.6 +/- 16.8 vs. High Osm 280.1 +/- 19.4 pg/2 x 10(6) cells, N = 7, P < 0.001); and AVP gene promoter activity (C 1353 +/- 99 vs. H igh Oam 2026 +/- 134 L.U./10(-4) U beta-gal, N = 8, P < 0.001). Non-os motic stimulators, 0.1 mu M endothelin 3 (ET3), 1 mu M angiotensin II (AII), and 10 mu M acetylcholine (Ach) significantly increased AVP rel ease; ET3 (C 1.78 +/- 0.20 vs. ET3 6.85 +/- 1.86 pg/2 x 10(6) cells, N = 8, P < 0.02); AII (C 1.29 +/- 0.38 vs. AII 27.80 +/- 7.09 pg/2 x 10 (6) cells, N = 5, P < 0.05) and Ach (C 1.14 +/- 0.33 vs. Ach 2.68 +/- 0.58 pg/2 x x 10(6) cells, N = 6, P < 0.05). However, only ET3 signifi cantly increased AVP mRNA (C 166.6 +/- 19.6 vs. ET3 254.4 +/- 25.6 pg/ p x 10(6) cells, N = 5, P < 0.05) and AVP promoter activity (C 1515 +/ - 163 vs. ET3 2389 +/- 342 L.U./10(-4) U beta-gal, N = 6, P < 0.05). T o localize the region of the AVP promoter that mediates the osmotic st imulation and the effect of ET3, 5' deletions of the AVP promoter frag ments terminating at - 532, - 211, and - 102, was assessed. Only the p romoter activity of the 1.5 kb construct, but not the deletion constru cts, was significantly increased by ET3 or high osmolality. These resu lts suggest that modulation of VP gene transcription is, at least in p art, responsible for increased AVP synthesis and release in response t o osmotic and non-osmotic stimulation, and that the region of 5' flank ing sequence between - 1500 and - 532 contains the elements responsibl e for the effects.