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  • 1
    ISSN: 1573-9007
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In this short review, we will first discuss localized cytoplasmic calcium signals in pancreatic acinar cells. In the second part of the review, we will describe recently discovered polarized calcium efflux and calcium propagation through the lumen of the endoplasmic reticulum — ER (a phenomenon we have termed “calcium tunnelling”). Finally, we will present a hypothesis concerning the roles that these mechanisms could play in transcellular calcium flux.
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  • 2
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 3
    ISSN: 1432-1424
    Keywords: Ca2+ channels ; vasopressin ; single-channel currents ; whole-cell current ; insulin secreting cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The effect of vasopressin on voltage-sensitive Ca2+ currents in the rat insulinoma cell line RINm5F has been investigated in patch-clamp whole-cell and single-channel current recording experiments. In the whole-cell recording configuration the dominant inward current in the presence of tetrodotoxin was noninactivating and had a high voltage threshold. This current was much enhanced when external Ca2+ was replaced by Ba2+ and was blocked by 1 μm nifedipine. It can therefore be classified as an L-current. Vasopressin enhanced the L-current without changing the voltage threshold of activation or the voltage at which the peak current was observed. Vasopressin effects were seen at concentrations as low as 0.01nm, and the maximal effect was observed at about 1nm. In higher concentrations the vasopressin effects were weaker, with effects at 50nm of about the same magnitude as at 0.01nm. In single-channel current recording experiments carried out with the cell-attached configuration there were no effects on single L-channel currents when vasopressin was added to the bath solution, but in experiments in which vasopressin (5nm) was infused into the patch pipette a marked increase in the apparent channel open state probability was observed. We conclude that vasopressin, a peptide that is known to markedly enhance glucose-evoked insulin secretion, stimulates opening of the voltage-sensitive Ca2+ channels in insulin-secreting cells.
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  • 4
    ISSN: 1432-1424
    Keywords: cholecytokinin ; Ca2+ signal ; caffeine ; heparin ; G protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The effects on the cytosolic Ca2+ concentration of activating cholecystokinin receptors on single mouse pancreatic acinar cells have been investigated using patch-clamp whole-cell recording of Ca2+-dependent Cl− current. We used the nonsulphated octapeptide of cholecystokinin (CCK8-NS) since the effects of even high concentrations were rapidly reversible which was not the case for the sulphated octapeptide. A submaximal concentration of CCK8-NS (10nm) evoked a current response consisting of short-lasting (a few seconds) spikes, and some of these spikes were seen to trigger larger and longer (about half a minute) current pulses. At a higher concentration (100nm) CCK8-NS evoked smooth and sustained responses. The effect of CCK8-NS was almost abolished when the internal perfusion solution contained a high concentration of the Ca2+ chelator EGTA (5mm). The responses evoked by CCK8-NS were independent of the presence of Ca2+ in the external solution at least for the first 5 min of stimulation. Internal perfusion with GTP-γ-S markedly potentiated the effect of CCK8-NS or at a higher concentration itself induced responses very similar to those normally evoked by CCK8-NS. Caffeine added to the external solution at a low concentration (0.2–1mm) enhanced weak CCK8-NS responses, whereas high caffeine concentrations always inhibited the CCK8-NS-evoked responses. These inhibitory caffeine effects were quickly reversible. Forskolin evoked a similar inhibitory effect. Intracellular heparin (200 μg/ml) infusion markedly inhibited the response to CCK8-NS stimulation. We conclude that the primary effect of activating CCK receptors is to induced inositoltrisphosphate (IP3) production. IP3 evokes a small and steady Ca2+ release, and this in turn evokes pulsatile release of a larger magnitude from a caffeine-sensitive Ca2+ pool. The action of CCK is thus very similar to that previously established for muscarinic receptor activation in the same cells. Nevertheless, the pattern of the cytosolic Ca2+ fluctuations are different, and the basic process of Ca2+-induced Ca2+ release and Ca2+ signal spreading must therefore be modulated by a messenger yet unknown.
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  • 5
    ISSN: 1432-1424
    Keywords: pancreatic islet cells ; K+ channel ; patchclamp ; single-channel recording ; Ca2+ activation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The Ca2+-activated K+ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na+ outside, K+ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than −40 mV. In symmetrical K+-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca2+-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca2+ concentration in contact with the membrane inside ([Ca2+]i) to 1.5×10−7 m had little effect on the relationship between membrane potential andP. When [Ca2+]i was increased to 3×10−7 m and 6×10−7 m smaller potential changes were required to open the channels. Increasing [Ca2+]i further to 8×10−7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from −50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca2+]i=1 and 2 μm. In this situation a membrane potential change from −70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K+ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca2+]i within the range 0.1 to 1 μm which suggests a physiological role for this channel in regulating the membrane potential and Ca2+ influx through voltage-activated Ca2+ channels.
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  • 6
    ISSN: 1432-1424
    Keywords: K+ channel ; ATP ; glyceraldehyde ; RINm5F cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The control of K+ channels in the insulin-secreting cell line RINm5F has been investigated by patch-clamp singlechannel current recording experiments. The unitary current events recorded from cell-attached patches are due to large and small inwardly rectifying ATP-sensitive K+ channels with conductance properties similar to the two channels previously identified in primary cultured rat islet cells (Findlay, I., Dunne, M.J., & Petersen, O. H.J. Membrane Biol. 88:165–172, 1985). Cell permeabilization through brief exposure to 10 μm digitonin or 0.05% saponin (outside the isolated membrane patch area) results in a dramatic increase in current through the cell-attached patch due to opening of many large and small K+-selective channels. These channels are inhibited in a dose-dependent manner by ATP applied to the bath (near-complete inhibition by 5mm ATP). During prolonged ATP exposure (1–5 min) the initial inhibition is followed by partial recovery of channel activity, although further activation does occur when ATP is subsequently removed. From the maximal number of coincident channel openings in the permeabilized cells (in the absence of ATP), it is estimated that there are on average 12 large ATP-sensitive K+ channels per membrane patch, but in the intact cells less than 5% of the membrane patches exhibited three or more coincident K+ channel openings, indicating the degree to which the channels are inhibited in the resting condition by endogenous ATP. Stimulation of RINm5F cells to secrete insulin was carried out by challenging intact cells with 10mm d-glyceraldehyde.d-glyceraldehyde induced depolarization of the membrane from about −70 to −20 mV and evoked a marked reduction in the open-state probability of both the large and small ATP-sensitive channels.d-glyceraldehyde also induced action potentials in a number of cases. All effects of stimulation were largely transient, lasting about 100 sec. The two ATP-sensitive K+ channels are probably responsible for the resting potential and play a crucial role in coupling metabolism to membrane depolarization.
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  • 7
    ISSN: 1432-1424
    Keywords: K+ channel ; ATP ; diazoxide ; tolbutamide ; RINm5F cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The single-channel current recording technique has been used to study the effects of diazoxide, tolbutamide and ATP, separately and combined, on the gating of nucleotide-regulated K+ channels in the insulin-secreting cell line RINm5F. The effects of diazoxide, tolbutamide and ATP4− were studied at the intracellular membrane surface, using, the open-cell membrane patch configuration. Alone diazoxide was found only inconsistently to evoke channel stimulation, 57% of all applications of the drug (72 times in 48 separate patches) having no effect at concentrations between 0.02 and 0.4mm. In the presence of ATP, however, diazoxide consistently evoked channel activation (seen 87 times in 49 patches, 95% of all applications). The interactions of diazoxide and ATP seemed competitive. Stimulation of channels by diazoxide in the presence of 1mm ATP was suppressed if the concentration of ATP was elevated to 2 or 5mm. In solutions in which Mg2+ had been chelated with EDTA, diazoxide failed to activate channels closed by 1mm ATP; however, this was not due to a direct effect on the channels caused by the absence of Mg2+, but could be explained by the enhanced ATP4− concentration after Mg2+ removal. When the total ATP concentration was lowered to give the same [ATP4−] in the absence of Mg2+ to that present in the control experiments, diazoxide was able to evoke full activation. Channel inhibition evoked by tolbutamide, 0.01 to 1.0mm, did not require the presence of either ATP or Mg2+. In the presence of ATP tolbutamide further reduced the number of channel openings. Diazoxide was able to compete with tolbutamide for control of channel activity, an effect that was augmented by the presence of ATP. In the presence of 0.1mm tolbutamide, diazoxide was unable to stimulate channel openings; however, if the dose of tolbutamide was lowered or ATP made available to the inside of the membrane, channel stimulation occurred.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Virchows Archiv 201 (1910), S. 96-107 
    ISSN: 1432-2307
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
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  • 9
    ISSN: 1432-2013
    Keywords: Parotid ; Amylase secretion ; Potassium release ; α- and β-Adrenoceptor ; Acetylcholine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 1. The output of amylase from superfused mouse parotid segments in response to stimulation with acetylcholine (ACh), phenylephrine and isoprenaline during exposure to solutions with varying potassium concentrations was monitored by an on line automated fluorometric method. 2. During stimulation with ACh or phenylephrine a 10-fold increase in superfusion fluid potassium concentration caused an immediate very marked reduction in amylase output which was fully reversible. A 10-fold reduction in potassium concentration resulted in a prominent rise in amylase output. During stimulation with isoprenaline there was no effect on the amylase output of varying the extracellular potassium concentration. Acetylcholine and phenylephrine caused potassium release from the mouse parotid whereas isoprenaline had no such effect. 3. It appears that under conditions where stimulation-induced potassium release is enhanced there is also an enhanced amylase secretion and vice versa. There may therefore be a link between passive potassium transport and amylase secretion.
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  • 10
    ISSN: 1432-0878
    Keywords: Intercellular communication ; Acinar cells ; Lucifer Yellow ; Uncoupling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Direct cell to cell movement of the fluorescent dye Lucifer Yellow CH (457 daltons) in exocrine acinar tissue is demonstrated by direct observation of living mouse pancreatic segments. Electrical uncoupling of pancreatic acinar cells by local application of a high concentration of acetylcholine significantly restricts cell to cell passage of the fluorescent dye. This result shows that a secretagogue can control direct movement of organic molecules between cells through junctional channels.
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