Osmotic and non-osmotic regulation of arginine vasopressin (AVP) release, mRNA, and promoter activity in small cell lung carcinoma (SCLC) cells

Abstract

Arginine vasopressin (AVP) is synthesized in the magnocellular neurons of the hypothalamus and stored in the posterior pituitary. It has been shown that hypothalamic AVP mRNA is increased during experimental stimulation of osmotic and non-osmotic stimulation of AVP release. The mechanisms underlying the stimulation of AVP biosynthesis in these conditions are not known. The present study was, therefore, performed to measure AVP release, AVP mRNA level, and AVP gene promoter activity during osmotic and non-osmotic stimulation of AVP secretion in the small cell lung carcinoma (SCLC) cells. AVP release was measured by radioimmunoassay, steady state levels of AVP mRNA by solution hybridization, and AVP gene promoter activity exhibited by a 1.5 kb 5′-flanking AVP gene fragment fused to a luciferase reporter after SCLC cells were subjected to osmotic or non-osmotic conditions. High media osmolality (330 mOsm) significantly increased AVP release (control (C) 1.42 ± 0.27 vs. High Osm 3.67 ± 0.39 pg/2 × 10 6 cells, N = 9, P < 0.002); AVP mRNA (C 173.6 ± 16.8 vs. High Osm 280.1 ± 19.4 pg/2 × 10 6 cells, N = 7, P < 0.001); and AVP gene promoter activity (C 1353 ± 99 vs. High Osm 2026 ± 134 L.U./10 −4 U β-gal, N = 8, P < 0.001). Non-osmotic stimulators, 0.1 μM endothelin 3 (ET3), 1 μM angiotensin II (AII), and 10 μM acetylcholine (Ach) significantly increased AVP release; ET3 (C 1.78 ± 0.20 vs. ET3 6.85 ± 1.86 pg/2 × 10 6 cells, N = 8, P < 0.02); AII (C 1.29 ± 0.38 vs. AII 27.80 ± 7.09 pg/2 × 10 6 cells, N = 5, P < 0.05) and Ach (C 1.14 ± 0.33 vs. Ach 2.68 ± 0.58 pg/2 × x10 6 cells, N = 6, P < 0.05). However, only ET3 significantly increased AVP mRNA (C 166.6 ± 19.6 vs. ET3 254.4 ± 25.6 pg/p × 10 6 cells, N = 5, P < 0.05) and AVP promoter activity (C 1515 ± 163 vs. ET3 2389 ± 342 L.U./10 −4 U β-gal, n = 6, P < 0.05). To localize the region of the AVP promoter that mediates the osmotic stimulation and the effect of ET3, 5′ deletions of the AVP promoter fragments terminating at −532, −211, and −102, was assessed. Only the promoter activity of the 1.5 kb construct, but not the delection constructs, was significantly increased by ET3 or high osmolality. These results suggest that modulation of AVP gene transcription is, at least in part, responsible for increased AVP synthesis and release in response to osmotic and non-osmotic stimulation, and that the region of 5′ flanking sequence between −1500 and −532 contains the elements responsible for the effects.