Craig A Aspinwall

PMID: 15609009;Abstract:

Many common metabolic and neurological disorders are related to defective regulation of exocytosis at the level of single cells. In exocytosis, vesicles containing the secretory product of a given cell type fuse with the plasma membrane allowing release of the vesicular contents into the extracellular environment where the physiological action can be exerted. The typical secretory vesicle contains between 0.15 and 10 attomoles of material that is released on a millisecond timescale. Hence, detection of this process presents several chemical and analytical challenges. In this work, we utilize the native ATP, stored at high concentrations within the secretory vesicles of most neuroendocrine cells and co-released during exocytosis and during cell lysis, as a universal tracer of cellular secretion events. Organisms studied include pancreatic islets, mast cells, and Escherischia coli. Cellular processes investigated include exocytotic release, stimulated cell lysis, and programmed cell lysis. © Springer-Verlag 2004.

PMID: 10489519;Abstract:

An oxygen microsensor with a 3-μm tip diameter was developed for monitoring oxygen levels at single cells and mouse pancreatic islets. The sensor was fabricated by electrochemically recessing an etched Pt wire inside a pulled glass micropipet and then coating with cellulose acetate. This fabrication process was found to be simpler than previous oxygen electrode designs of comparable size. The microsensors had a average sensitivity of 0.59 ± 0.29 pA/mmHg (mean ± SD, n = 42), signals that were minimally perturbed by convection, and response times of 1 s. The electrode was used to measure the oxygen gradient around and inside single mouse islets. The measurements demonstrate that oxygen levels within even the largest islets at maximal glucose stimulation are 67 ± 1.6 mmHg (mean ± SD, n = 5), indicating that islets have adequate oxygen supplies by diffusion under tissue culture conditions to support insulin secretion. The electrode was also used to record the dynamics of oxygen level at single islets as a function of glucose concentration. As glucose level was changed from 3 to 10 mM, oxygen level decreased by 15.8 ± 2.3 mmHg (mean ± SEM, n = 6) and oscillations with a period of 3.3 ± 0.6 min (mean ± SEM, n = 6) appeared in the oxygen level. In islets bathed in quiescent solutions containing 10 mM glucose, similar oscillations could be observed. In addition, in the quiet solutions it was possible to detect faster oscillations with a period of 12.1 ± 1.7 s (mean ± SEM, n = 6) superimposed on the slower oscillations. Oxygen consumption could also be observed at single insulinoma cells using the electrode. Individual cells also showed oscillations in oxygen consumption with a period of a few seconds. The results demonstrate that the electrode can be used for dynamic oxygen level recordings in biological microenvironments.

A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6xhistidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed in Escherichia coli and purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, betaTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both betaTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 +/- 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 +/- 4%) in betaTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively).

PMID: 10764813;Abstract:

The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca²⁺ concentration ([Ca²⁺](i)) in isolated mouse pancreatic β-cells and clonal β-cells was investigated. Application of insulin to single β-cells resulted in increases in [Ca²⁺](i) that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca²⁺](i) originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca²⁺](i) changes and insulin secretion at single β-cells were abolished by treatment with 100 nM wortmannin or 1 μM thapsigargin; however, they were unaffected by 10 μM U73122, 20 μM nifedipine, or removal of Ca²⁺ from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 μM bisindolylmaleimide I, but [Ca²⁺](i) changes were unaffected. In an insulin receptor substrate-1 gene disrupted β-cell tumor line, insulin did not evoke either [Ca²⁺](i) changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca²⁺](i) are due to release of intracellular Ca²⁺ stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca²⁺](i) and activation of protein kinase C.