Ca/Calmodulin Inhibition and Phospholipase C-Linked Ca Signaling in Clonal b-Cells*

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca 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 Ca 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 mM) and W-13 (30–100 mM), but not W-5 (100 mM) and W-12 (300 mM), reversibly inhibited the AVPand bombesin-induced Ca transients. As the generation of PLC-linked Ca transients requires mobilization of internal Ca and Ca influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K (45 mM)induced Ca signals (IC50, ;25 mM), and W-13 (100 mM) reduced the K (45 mM)-induced [Ca]i rise by about 40–60%, whereas W-5 (100 mM) and W-12 (300 mM) had no effect. In addition, W-7 (100 mM) inhibited whole cell Ca currents in mouse b-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 mM), but not W-5 (30 mM), inhibited agonist-induced internal Ca mobilization by about 75% in Ca-free medium. Neither W-7 (30 mM) nor W-5 (30 mM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca influx through VICC activated by thapsigargin (2 mM) in the presence of verapamil (50 mM) was inhibited by W-7 (30 mM) but not by W-5 (30 mM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca via activation of CaM-dependent processes could provide positive feedback on the generation of PLClinked Ca transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca and Ca influx through VSCC and VICC. (Endocrinology 140: 5516– 5523, 1999) I RELEASE from pancreatic b-cells is stimulated by a variety of nutrients and nonnutritional factors (1–3). All insulin secretagogues elevate cytosolic free Ca ([Ca]i), and numerous studies have shown that Ca 21 plays a key role in triggering insulin secretion (2). Neurotransmitters and hormones, such as acetylcholine, arginine vasopressin (AVP) or bombesin, that activate the Ca-phosphoinositide (PI) pathway cause a rise in [Ca]i and stimulate insulin secretion from normal and transformed b-cells in the presence of glucose (2–8). The generation of Ca signals by PLC-linked hormones requires inositol 1,4,5-trisphosphate (IP3)-mediated mobilization of intracellular Ca 21 and influx of Ca from the outside through voltage-sensitive (VSCC) and voltage-insensitive (VICC) Ca channels in hamster insulinoma tumor (HIT-T15) cells (9–11). In HIT-T15 cells and in primary b-cells, PLC-linked agonists at low, nearphysiological concentrations cause repetitive Ca transients whose frequency is determined by the extracellular agonist concentration while the amplitude remains constant (9–14). This indicates that the cytosolic Ca signal evoked by PLClinked agonists might be primarily frequency encoded. As transient rises in [Ca]i increase the rate of exocytosis from single HIT-T15 and primary b-cells, a functional role of PLClinked Ca transients can be assumed (15, 16). Although the basic features of PLC-linked Ca transients have been elucidated in recent years, the mechanisms underlying the PLClinked Ca transients are still unknown. Various positive and negative feedback mechanisms, usually assumed to involve Ca itself, have been described and used to model transient Ca oscillations in a number of cell systems (17). Ca-dependent regulatory mechanisms are often mediated by the activation of the ubiquitous cellular protein calmodulin (CaM), which, in turn, activates a range of cellular proteins, such as CaM-dependent protein kinase II (CaM kinase II) (18, 19). Several lines of evidence suggest that CaM and/or CaM kinase II could be involved in the regulation of PLC-linked Ca signals (20–25). To investigate the role of CaM activity in PLClinked Ca signals in b-cells, the effects of naphthalenesulfonamide anticalmodulin agents, the W compounds, on AVPand bombesin-induced Ca signals were studied in single fura-2loaded, insulin-secreting HIT-T15 cells. Materials and Methods HIT-T15 cell culture HIT-T15 cells were provided by Dr. Knepel (Göttingen, Germany). The cells were grown in RPMI 1640 medium containing 10 mm glucose supplemented with 10% FCS (vol/vol), 100 U penicillin/ml, and 100 mg streptomycin/ml at 37 C in 5% CO2 and 95% air (vol/vol). All experiments were performed with cells from passage 65–86. Received April 16, 1999. Address all correspondence and requests for reprints to: Dr. Christof Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany. * This work was supported by Deutsche Forschungsgemeinschaft Grant Scho 466/1–3. 0013-7227/99/$03.00/0 Vol. 140, No. 12 Endocrinology Printed in U.S.A. Copyright © 1999 by The Endocrine Society