Glucose-mediated Ca(2+) signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation.
Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](i). The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells.
Salgado
A P
AP
Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
Santos
R M
RM
Fernandes
A P
AP
Tomé
A R
AR
Flatt
P R
PR
Rosário
L M
LM
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D016257:Fura-2 / Q000737:chemistry; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D007651:Keto Acids / Q000378:metabolism / Q000494:pharmacology; D008356:Mannoheptulose / Q000378:metabolism / Q000494:pharmacology; D051381:Rats
D007328:Insulin; D007651:Keto Acids; D008356:Mannoheptulose; C013082:alpha-ketoisocaproic acid; D005947:Glucose; D016257:Fura-2