| IntroductionPancreatic β cells maintain glucose homeostasis by their regulated Ca2+-dependent secretion of insulin. Cytosolic free Ca2+ ([Ca2+]i) is the key mediator in glucose-insulin-secretion coupling. Calcium influx, calcium release, calcium sequestration and calcium buffering are important factors in affecting the concentration of free intracellular calcium [Ca2+]i. It's known that only about less than 5% of calcium entering a cell shows up as free calcium; the majority is rapidly bound to cellular binding sites, and later on sequestered into calcium storing organelles or extruded by calcium transport mechanisms.It's shown that calcium buffer is a very important aspect of calcium signaling. Many calcium rises are restricted, at least for short times, to the vicinity of calcium entry or release sites and then provoke [Ca2+]i-dependent vital events such as contraction, neurotransmitter or insulin release. Thus, it is important to obtain experimental data on cellular Ca2+ buffers.In the present studies, the Ca2+ buffers in pancreatic β cells were measured by a combination of fura-2 fluorescence and Ca2+ current measurement, and the effects of Ca2+ buffers on insulin secretion were also explored. MethodsCell: Pancreatic islet β cells of adult Wistar rats (150-200g) were isolated and then cultured for 7 days.Solutions: The bath solution contained (mM): NMG 138, KCl 5.6, MgCl2 1.2, CaCl2 2.6, glucose 3, FPL 4μM, Hepes 10 (pH 7.4). The internal solution contained (mM): CsCl 125, NaCl 10, MgCl2 1.0, MgATP 4, Hepes 5. Fura-2 fluorescence measurement were combined with whole cell patch-clamp to monitor membrane currents, fura-2 F340 and F380.Theory: Ca2+ buffers are calculated by following equations:(1) △[Ca2+]t = △[Ca2+]i + △[BCa]+ △[SCa]Where △ represents a change, B is fura-2, and S is endogenous Ca2+ buffers. [Ca2+]t is total [Ca2+]. The Ca2+ binding capacity of cytoplasm((S), can be calculated from the equations as following(Zhou & Neher, J Physiol, 469:245, 1993):(2) (S = (B (fmax/f-1)-1(3)f = △Fd /∫IcadtWhere (B is Ca2+ buffer capacity of fura-2, f is the ratio of fluorescence change over Ca2+ current integral during a short pulse of Ca2+ influx, i.e. "F/Q" ratio, Fd is Ca2+ sensitive fluorescence of fura-2, Ica is Ca2+ current. Membrane capacitance (Cm) measurements: Exocytosis was detected as changes in membrane capacitance, which was estimated by the built-in lock-in amplifier of the EPC-9 in perforated or conventional whole cell configuration. The capacitance measurements were performed at 33℃.Results and DiscussionPatch clamp and rapid microfluorometric calcium measurements were simultaneously performed on β cells. During patch clamp measurements, endogenous calcium buffering properties can be determined by the 'added buffer' method based on loading cells with fluorescent indicator dyes whose buffering properties are known. For a certain amount of calcium flowing into the cell, combination competition between exogenous (e.g. fura-2) and endogenous calcium buffers occur, and the calcium buffers capacity ((S) of combined calcium to free calcium in physiological conditions can be calculated. The results show that dynamics of endogenous calcium buffers capacity progressively decreased from ~1454 to steady value of ~79. This decrease in buffers capacity could be ascribes to the loss of mobile buffers, which exchanged with the contents of the patch pipette in the conventional whole cell patch configuration. Our data indicate that only 0.7‰ Ca2+ entering the β cell remained free on the time scale required to reach equilibrium with the buffers.The main role of β cells is to secret insulin. In this study, we explored the effect of calcium buffers on insulin secretion in β cell via the techniques of patch clamp and membrane capacitance (Cm) measurements. We found in the intact cell without loading of fura-2, a depolarization pulse (200ms) in the perforated whole cell patch configuration evoked a significant Cm increase, whereas the same pulse...
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