Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR.
Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.
Miguel
João C
JC
School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, Northern Ireland BT52 1SA, UK. jcmiguel@uem.br
Patterson
Steven
S
Abdel-Wahab
Yasser H A
YH
Mathias
Paulo C F
PC
Flatt
Peter R
PR
D000109:Acetylcholine / Q000494:pharmacology; D000409:Alanine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D015590:Fluorophotometry / Q000379:methods*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007424:Intracellular Fluid / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
D005456:Fluorescent Dyes; D007328:Insulin; D011189:Potassium Chloride; D010738:Type C Phospholipases; D005947:Glucose; D000109:Acetylcholine; D000409:Alanine; D002118:Calcium