Thomas Nilsson

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells.

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration ([Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent β‐cells.

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

SummaryPlasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca2+]i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca2+]i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca2+]i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca2+]i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca2+]i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca2+]i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.

Glucose-stimulated efflux of fura-2 in pancreatic β-cells is prevented by probenecid

Fura-2 loaded pancreatic beta-cells, isolated from obese hyperglycemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), insulin release and efflux of indicator. In the absence of glucose there was a continuous efflux of fura-2, which was markedly increased by stimulation with a high concentration of the sugar. Probenecid both reduced basal efflux of fura-2 and prevented that promoted by glucose. There was no interference of the drug with glucose-induced either insulin release or rise in [Ca2+]i. When applying fura-2 in pancreatic beta-cells, the use of probenecid markedly improves the measurements of [Ca2+]i.

Glucose-stimulated efflux of indo-1 from pancreatic β-cells is reduced by probenecid

Indo‐1 loaded pancreatic β‐cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo‐1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo‐1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin‐2 and fura‐2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo‐1 probenecid and thereby improve the measurements of [Ca2+]iin pancreatic β‐cells.

Activation of protein kinase C assists insulin producing cells in recovery from raised cytoplasmic Ca2+ by stimulating Ca2+ efflux

The effects of protein kinase C (PKC) activation on the cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in clonal insulin-producing RINm5F cells, using the fluorescent Ca2+ indicators quin-2 and fura-2. Both under basal and stimulatory conditions PKC activation lowered [Ca2+]i in these cells by promoting an active extrusion of Ca2+ to the extracellular space. PKC activation therefore assists insulin-producing cells in recovery from raised [Ca2+]i. Such an effect might be part of the signal regulating the insulin secretory process.

Regulation of cytoplasmic free Ca2+ in insulin-secreting cells

The cytoplasmic free Ca2+ concentration([Ca2+]i) has a fundamental role in the β-cell stimulus-secretion coupling and is regulated by a sophisticated interplay between nutrients, hormones and neurotransmitters. Metabolism of glucose and other nutrients leads to ATP generation, closure of ATP-regulated K+-channels, depolarization, opening of voltage-activated L-type Ca2+-channels, increase in [Ca2+]i and insulin release (1,2). Hormones and neurotransmitters affect the β-cell through the activation of receptors coupled to various effector systems, such as the adenylate cyclase (AC) or phospholipase C (PLC) system (2). Upon activation of these systems, cAMP is formed or phosphatidyl inositol 4,5-bisphosphate is hydrolysed, resulting in the formation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). Whereas InsP3 mobilizes intracellularly bound Ca2+, most probably from the endoplasmic reticulum, DAG activates protein kinase C (PKC) (1–3). Although InsP3 increases [Ca2+]i, there is little effect on insulin release, suggesting that the trisphosphate is not primarily involved as a signal for exocytosis in the β-cell (3). With regard to PKC, the physiological role is more clear and this enzyme is involved as a modulator of multiple steps in the β-cell signal-transduction pathway (1–3).

The determination of cadmium in microgram amounts of pancreatic tissue by electrothermal atomic absorption spectometry

Abstract Cadmium was determined either by direct insertion of freeze-dried biological samples (1–13 μg) or by injection of 2-μl samples of perifusion medium into the graphite furnace. At 228.8 nm ⩾ 10 fmol of cadmium could be measured. The endogenous cadmium contents in the endocrine and exocrine parts of the pancreas were 8.5 ± 2.0 and 15.1 ± 1.4 μ mol (dry wt.), respectively. A less sensitive wavelength (326.1 nm) was employed for measuring the larger amounts obtained after specimens had been incubated in the presence of cadmium.

Interaction with the inositol 1,4,5-trisphosphate receptor promotes Ca2+ sequestration in permeabilised insulin-secreting cells

Electropermeabilised insulin‐secreting RINm5F cells sequestered Ca2+, resulting in a steady‐state level of the ambient free Ca2+ concentration corresponding to 723 ± 127 nM (mean ± SEM, n = 10), as monitored by a Ca2+‐selective minielectrode. Inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) promoted a rapid and pronounced release of Ca2+. This Ca2+ was resequestered and a new steady‐state Ca2+ level was attained, which was always lower (460 ± 102 nM, n = 10, P < 0.001) than the steady‐state Ca2+ level maintained before the addition of Ins(1,4,5)P3. Whereas the initial reuptake of Ca2+ subsequent to Ins(1,4,5,)P3 stimulation was relatively slow, the later part of reuptake was fast as compared to the reuptake phases of a pulse addition of extraneous Ca2+. In the latter case the uptake of Ca2+ resulted in a steady‐state level similar to that found in the absence of Ins(1,4,5)P3. Addition of Ins(1,4,5)P3 under this condition resulted in a further Ca2+ uptake and thus a lower steady‐state Ca2+ level, Heparin, which binds to the Ins(1,4,5)P3 receptor, also lowered the steady‐state free Ca2+ concentration, In contrast to Ins(1,4,5)P3 inositol 1,3,4,5‐tetrakis‐phosphate was without effect on Ca2+ sequestration. These findings are consistent with the presence of a high‐affinity Ins(1,4,5)P3 receptor promoting continuous release of Ca2+ under basal conditions and/or the Ins(1,4,5)P3 receptor being actively involved in Ca2+ sequestration.