Ca2+/calmodulin inhibition and phospholipase C-linked Ca2+ Signaling in clonal beta-cells.
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 microM) and W-13 (30-100 microM), but not W-5 (100 microM) and W-12 (300 microM), 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, approximately 25 microM), and W-13 (100 microM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40-60%, whereas W-5 (100 microM) and W-12 (300 microM) had no effect. In addition, W-7 (100 microM) inhibited whole cell Ca2+ currents in mouse beta-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 microM), but not W-5 (30 microM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 microM) nor W-5 (30 microM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 microM) in the presence of verapamil (50 microM) was inhibited by W-7 (30 microM) but not by W-5 (30 microM). 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.
Schöfl
C
C
Abteilung für Klinische Endokrinologie, Medizinische Hochschule Hannover, Germany.
Mader
T
T
Krämer
C
C
Waring
M
M
Krippeit-Drews
P
P
Prank
K
K
von zur Mühlen
A
A
Drews
G
G
Brabant
G
G
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001839:Bombesin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004553:Electric Conductivity; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D010190:Pancreatic Neoplasms; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D002121:Calcium Channel Blockers; D002147:Calmodulin; D007328:Insulin; D013449:Sulfonamides; D001127:Arginine Vasopressin; C017967:W 7; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D010738:Type C Phospholipases; D005947:Glucose; D001839:Bombesin; D002118:Calcium