NCI-H295R, a human adrenal cortex-derived cell line, expresses purinergic receptors linked to Ca²⁺-mobilization/influx and cortisol secretion.
Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A(2A) and A(2B)), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10-1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1-1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10-100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.
Pharmacology, The Jikei University School of Medicine, Tokyo, Japan. harrywestjpjp@yahoo.co.jp
Haruhisa
H
Nishi
Hirokazu
H
Arai
Toshihiko
T
Momiyama
D000255:Adenosine Triphosphate / Q000378:metabolism; D000302:Adrenal Cortex / Q000166:cytology*; D000804:Angiotensin II / Q000378:metabolism; D003994:Bucladesine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D015870:Gene Expression; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism*; D058913:Purinergic Agonists / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D011983:Receptors, Purinergic / Q000235:genetics / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1 / Q000235:genetics* / Q000378:metabolism
D005938:Glucocorticoids; D058913:Purinergic Agonists; D012333:RNA, Messenger; D011983:Receptors, Purinergic; D058472:Receptors, Purinergic P2Y1; D000804:Angiotensin II; D003994:Bucladesine; D000255:Adenosine Triphosphate; D002118:Calcium; D006854:Hydrocortisone