Comparison of amperometric methods for detection of exocytosis from single pancreatic beta-cells of different species.
Two methods for amperometric detection of exocytosis at single pancreatic beta-cells were compared. In the first, direct detection of insulin was accomplished using an insulin-sensitive chemically modified electrode. In the second, 5-hydroxytryptamine (5-HT) that had been allowed to accumulate within the beta-cell secretory vesicles was detected with a bare carbon electrode. The goal of the comparison was to determine whether 5-HT secretion was a valid marker of insulin secretion in single beta-cells. To aid in this comparison, some experiments involved simultaneous measurement of insulin and 5-HT at cells previously allowed to accumulate 5-HT. Upon application of common insulin secretagogues, current spikes resulting from detection of 5-HT, insulin, or both compounds were obtained indicative of secretion via exocytosis. The mean area of current spikes obtained from simultaneous measurements equaled the sum of the mean area of insulin and 5-HT measured independently. Additionally, analyses of the number of spikes obtained for detection of insulin, 5-HT, or both compounds were similar for several common secretagogues. These data support the hypothesis that accumulated 5-HT is released from insulin containing secretory vesicles, exclusively. In addition, measurement of insulin and 5-HT from beta-cells of different species was compared to determine whether a species dependence exists for the two methods compared here. Detection of 5-HT results in a similar number of spikes that are equivalent to insulin in frequency and amplitude in human, porcine, and canine beta-cells; however, in mouse and INS-1 beta-cells, 5-HT is more readily detected than insulin.
Aspinwall
C A
CA
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Huang
L
L
Lakey
J R
JR
Kennedy
R T
RT
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004563:Electrochemistry / Q000379:methods*; D005089:Exocytosis; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D012701:Serotonin / Q000032:analysis / Q000378:metabolism; D013045:Species Specificity; D013552:Swine; D014407:Tumor Cells, Cultured
D007328:Insulin; D012701:Serotonin