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Ca²⁺/Calmodulin Inhibition and Phospholipase C-Linked Ca²⁺ Signaling in Clonal ß-Cells¹

Abteilung für Klinische Endokrinologie, Medizinische Hochschule Hannover (C.S., T.M., M.W., K.P., A.v.z.M., G.B.), 30623 Hannover; and Pharmazeutisches Institut, Eberhard-Karls-Universität Tubingen (C.K., P.K.-D., G.D.), 72076 Tubingen, Germany

Address all correspondence and requests for reprints to: Dr. Christof Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany.

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca²⁺ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca²⁺ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10–30 µM) and W-13 (30–100 µM), but not W-5 (100 µM) and W-12 (300 µM), reversibly inhibited the AVP- and bombesin-induced Ca²⁺ transients. As the generation of PLC-linked Ca²⁺ transients requires mobilization of internal Ca²⁺ and Ca²⁺ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca²⁺ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K⁺ (45 mM)-induced Ca²⁺ signals (IC₅₀, 25 µM), and W-13 (100 µM) reduced the K⁺ (45 mM)-induced [Ca²⁺]_i rise by about 40–60%, whereas W-5 (100 µM) and W-12 (300 µM) had no effect. In addition, W-7 (100 µM) inhibited whole cell Ca²⁺ currents in mouse ß-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 µM), but not W-5 (30 µM), inhibited agonist-induced internal Ca²⁺ mobilization by about 75% in Ca²⁺-free medium. Neither W-7 (30 µM) nor W-5 (30 µM) affected AVP (100 nM)-stimulated formation of IP₃. Third, capacitative Ca²⁺ influx through VICC activated by thapsigargin (2 µM) in the presence of verapamil (50 µM) was inhibited by W-7 (30 µM) but not by W-5 (30 µM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca²⁺ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca²⁺ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca²⁺ and Ca²⁺ influx through VSCC and VICC.

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