Wolf-Rüdiger Schlue

Calcium–magnesium interactions in pancreatic acinar cells

Although the role of calcium (Ca2+)in the signal transduction and pathobiology of the exo¬crine pancreas is firmly established, the role of magne¬sium (Mg2+) remains unclear. We have characterized the intracellular distribution of Mg2+ in response to hormone stimulation in isolated mouse pancreatic acinar cells and studied the role of Mg2+ in modulating Ca2+ signaling using microspectrofluorometry and dig¬ital imaging of Ca2+‐orMg2+‐sensitive fluorescent dyes as well as Mg2+‐sensitive intracellular microelectrodes. Our results indicate that an increase in intracellular Mg2+ concentrations reduced the cholecystokinin (CCK) ‐induced Ca2+ oscillations by inhibiting the capacitive Ca2+ influx. An intracellular Ca2+ mobiliza¬tion, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone with¬drawal independent of the electrochemical gradients for Mg2+,Ca2+,Na+, and K+, and not caused by Mg2+ efflux from acinar cells. In an attempt to characterize possible Mg2+ stores that would explain the reversible, hormone‐induced intracellular Mg2+ movements, we ruled out mitochondria or ATP as potential Mg2+ buffers and found that the CCK‐induced [Mg2+]i de¬crease was initiated at the basolateral part of the acinar cells, where most of the endoplasmic reticulum (ER) is located, and progressed from there toward the apical pole of the acinar cells in an antiparallel fashion to Ca2+ waves. These experiments represent the first characterization of intracellular Mg2+ movements in the exocrine pancreas, provide evidence for possible Mg2+ stores in the ER, and indicate that the spatial and temporal distribution of intracellular Mg concentra¬tions profoundly affects acinar cell Ca2+ signaling.— Mooren, F. C., Turi, S., Günzel, D., Schlue, W.‐R., Domschke, W., Singh, J., Lerch, M. M. Calcium‐mag¬nesium interactions in pancreatic acinar cells. FASEB J. 15, 659‐672 (2001)

Sodium‐magnesium antiport in Retzius neurones of the leech Hirudo medicinalis.

1. Intracellular free magnesium ([Mg2+]i) and sodium ([Na+]i) concentrations were measured in Retzius neurones of the leech Hirudo medicinalis using ion‐sensitive microelectrodes. 2. The mean steady‐state values for [Mg2+]i and [Na+]i were 0.46 mM (pMg, 3.34 +/‐ 0.23; range, 0.1‐1.2 mM; n = 32) and 8.95 mM (pNa, 2.05 +/‐ 0.15; range, 5.1‐15.5 mM, n = 21), respectively, at a mean membrane potential (Em) of ‐35.6 +/‐ 6.1 mV (n = 32). Thus, [Mg2+]i is far below the value calculated for a passive distribution (16.9 mM) but close to the equilibrium value calculated for a hypothetical 1 Na(+)‐1 Mg2+ antiport (0.41 mM). 3. Simultaneous measurements of [Mg2+]i, [Na+]i and Em in Retzius neurones showed that an increase in the extracellular Mg2+ concentration ([Mg2+]o) resulted in an increase in [Mg2+]i, a parallel decrease in [Na+]i and a membrane depolarization, while a decrease in [Mg2+]o had opposite effects. These results are compatible with calculations based on a 1 Na(+)‐1 Mg2+ antiport. 4. Na+ efflux at high [Mg2+]o still occurred when the Na(+)‐K+ pump was inhibited by the application of ouabain or in K(+)‐free solutions. This efflux was blocked by amiloride. 5. In the absence of extracellular Na+ ([Na+]o), no Mg2+ influx occurred. Mg2+ influx at high [Mg2+]o was even lower than in the presence of [Na+]o. Mg2+ efflux was blocked in the absence of [Na+]o. 6. The rate of Mg2+ extrusion was reduced by lowering [Na+]o, even if the Na+ gradient across the membrane remained almost unchanged. 7. Mg2+ efflux was blocked by amiloride (half‐maximal effect at 0.25 mM amiloride; Hill coefficient, 1.3) but not by 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA). 8. No changes in intracellular Ca2+ and pH (pHi) could be detected when [Mg2+]o was varied between 1 and 30 mM. 9. Changing pHi by up to 0.4 pH units had no effect on [Mg2+]i. 10. The results suggest the presence of an electrogenic 1 Na(+)‐1 Mg2+ antiport in leech Retzius neurones. This antiport can be reversed and is inhibited by low extracellular and/or intracellular Na+ and by amiloride.

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis.

Abstract To investigate the Mg2+ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg2+ ([Mg2+]i) and Na+ ([Na+]i) concentrations, as well as the membrane potential (Em), were measured using Mg2+- and Na+-selective microelectrodes. The mean steady-state values of [Mg2+]i were found to be 0.91 mM (mean Em=–63.6 mV) in NG cells and 0.20 mM (mean Em=–40.6 mV) in P neurones with a [Na+]i of 6.92 mM (mean Em=–61.6 mV) and 7.76 mM (mean Em=–38.5 mV), respectively. When the extracellular Mg2+ concentration ([Mg2+]o) was elevated, [Mg2+]i in P neurones increased within 5–20 min whereas in NG cells a [Mg2+]i increase occurred only after long-term exposure (6 h). After [Mg2+]o was reduced back to 1 mM, a reduction of the extracellular Na+ concentration ([Na+]o) decreased the inwardly directed Na+ gradient and reduced the rate of Mg2+ extrusion considerably in both NG cells and P neurones. In P neurones Mg2+ extrusion was reduced to 15.4% in Na+-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg2+ extrusion from NG cells was reduced to 6.2% in Na+-free solutions. The results suggest that the major [Mg2+]i-regulating mechanism in both cell types is Na+/ Mg2+ antiport. In P neurones a second, Na+-independent Mg2+ extrusion system may exist.

Determination of [Mg2+]i – an update on the use of Mg2+-selective electrodes

Since their invention, ion-selective microelectrodes have become an indispensable tool for investigations of intracellular ion regulation and transport. While highly selective sensors for all major intracellular monovalent ions have been available for decades, the development of sensors for divalent cations seems to have presented more difficulties. As ion-selective microelectrodes typically have time-constants in the range of 0.5 to several seconds they turned out to be inapt for the investigation of intracellular Ca2+. The development of sensors for Mg2+-selective electrodes has made its most striking progress only over the past few years. While the first Mg2+ sensor, ETH 1117, was barely able to detect physiological Mg2+ concentrations in the presence of other mono- and divalent cations, the newest sensors allow measurements in the micromolar range. When used in macroelectrodes, the most recent developments, ETH 5506 and ETH 5504, have even been reported to do so in the presence of millimolar Ca2+ concentrations. Although there is still room for improvement to make these sensors applicable in microelectrodes, some preliminary data look extremely promising and indicate that a new era for Mg2+-selective microelectrodes is about to start.

Apparent Intracellular Mg2+ Buffering in Neurons of the Leech Hirudo medicinalis

The apparent intracellular Mg2+ buffering, or muffling (sum of processes that damp changes in the free intracellular Mg2+ concentration, [Mg2+](i), e.g., buffering, extrusion, and sequestration), was investigated in Retzius neurons of the leech Hirudo medicinalis by iontophoretic injection of H+, OH-, or Mg2+. Simultaneously, changes in intracellular pH and the intracellular Mg2+, Na+, or K+ concentration were recorded with triple-barreled ion-selective microelectrodes. Cell volume changes were monitored measuring the tetramethylammonium (TMA) concentration in TMA-loaded neurons. Control measurements were carried out in electrolyte droplets (diameter 100-200 microm) placed on a silver wire under paraffin oil. Droplets with or without ATP, the presumed major intracellular Mg2+ buffer, were used to quantify the pH dependence of Mg2+ buffering and to determine the transport index of Mg2+ during iontophoretic injection. The observed pH dependence of [Mg2+](i) corresponded to what would be expected from Mg2+ buffering through ATP. The quantity of Mg2+ muffling, however, was considerably larger than what would be expected if ATP were the sole Mg2+ buffer. From the decrease in Mg2+ muffling in the nominal absence of extracellular Na+ it was estimated that almost 50% of the ATP-independent muffling is due to the action of Na+/Mg2+ antiport.

Ultramicroelectrodes for membrane research

Abstract This article describes the construction, testing, calibration and application of ion-selective double-barrelled microelectrodes based on neutral carrier or ion-exchanger liquid membranes. At present the use of ion-selective microelectrodes is the only method available for the direct and continuous measurement of ion activities in the intra- and extracellular spaces, on membrane surfaces, and even in subcellular organelles of a variety of intact cells. Intracellular ion activities in identified neurones of the central nervous system of the leech (Hirudo medicinalis L.) have been measured with K+-, Na+-, H+-, Mg2+- and Ca2+-selective double-barrelled microelectrodes under various experimental conditions, such as changes in ion composition of the external medium, application of neurotransmitters and exposure to drugs. This approach has provided significant information about electrochemical potentials of ions, properties of receptor-coupled ion channels, and membrane transport related to ion activities in this nervous system.

pH recovery from intracellular alkalinization in Retzius neurones of the leech central nervous system.

1. Neutral‐carrier pH‐sensitive microelectrodes were used to investigate intracellular pH (pHi) recovery from alkalinization in leech Retzius neurones in Hepes‐ and in CO2‐HCO3(‐)‐buffered solution. The Retzius neurones were alkaline loaded by the addition and subsequent removal of 16 mM acetate, by changing from 5% CO2‐27 mM HCO3‐ to 2% CO2‐11 mM HCO3‐ or by changing from CO2‐HCO3(‐)‐ to Hepes‐buffered solution. 2. In Hepes‐buffered solution (pH 7.4) the mean pHi was 7.29 +/‐ 0.11 and the mean membrane potential ‐44.7 +/‐ 5.9 mV (mean +/‐ S.D.; n = 83). 3. The rate of pHi recovery from alkalinization increased with decreasing pH of the bathing medium (pHb). pHi changed about 0.30 pH units for a pHb unit change. 4. A decrease of extracellular buffer concentration (Hepes concentration lowered from 20 to 5 mM) caused an acidification of extracellular and intracellular pH and an acceleration of pHi recovery from alkalinization. 5. A depolarization of the Retzius cell membrane‐induced by increasing the K+ concentration of the bathing medium from 4 to 20 mM (delta Em = 16.5 +/‐ 5.5 mV) or from 4 to 40 mM (delta Em = 24.8 +/‐ 3.5 mV)‐‐evoked a decrease of pHi and an acceleration of pHi recovery from alkalinization. 6. The H+ current blocker Zn2+ (0.5 mM) inhibited pHi recovery from alkalinization at resting membrane potential as well as during depolarization. The inhibition was more pronounced during depolarization. 7. In Cl(‐)‐free, CO2‐HCO3(‐)‐buffered solution pHi recovery from an alkaline load by changing from 5% CO2‐27 mM HCO3‐ to 2% CO2‐11 mM HCO3‐ was slowed by 48‐71%. The rate of pHi recovery from an alkaline load induced by changing from CO2‐HCO3‐ to Hepes buffer was reduced by 33‐56% in Cl(‐)‐free solution. The removal of external Cl‐ did not affect pHi recovery in Hepes‐buffered solution. 8. The pHi recovery from alkalinization was DIDS‐insensitive in CO2‐HCO3(‐)‐ as in Hepes‐buffered solutions and was not slowed in the absence of external Na+. 9. It is concluded that in Retzius neurones pHi recovery from alkalinization is mediated by a passive voltage‐dependent H+ influx along the electrochemical proton gradient. In the presence of CO2‐HCO3‐ buffer a DIDS‐insensitive Cl(‐)‐HCO3‐ exchanger additionally regulates pHi after an intracellular alkaline load. It cannot be excluded that intracellular processes (e.g. H+ release from organelles, metabolic H+ production) are also involved in pHi recovery from alkalinization.

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system.

We investigated the effects of ATP (adenosine 5′‐triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose‐dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na+‐dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5′‐diphosphate) > AMP (adenosine 5′‐monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2‐methylthio‐ATP mimicked the ATP‐induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8‐SPT (8‐(p‐sulphophenyl)‐theophylline) also depressed ATP‐induced depolarizations and some ATP‐insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP‐induced depolarization requires activation of Na+ channels or nonselective cation channels, whereas the ATP‐induced hyperpolarization indicates activation of K+ channels. ATP also increased the intracellular Ca2+ concentration ([Ca2+]i), that is independent of Ca2+ influx but reflects intracellular Ca2+ release possibly triggered by IP3 formation. ADP and AMP also increased [Ca2+]i, but were less efficient than ATP; adenosine and 2‐methylthio‐ATP did not affect [Ca2+]i. In view of the mobilization of intracellular Ca2+, ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca2+ signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized. GLIA 29:191–201, 2000.

Ionic mechanism of 4-aminopyridine action on leech neuropile glial cells.

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K+ channels, but also induced Na+ and Ca2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was -5 mV. Application of 5 mM Ni2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K+ and Cl- channels and 4-AP-induced Ca2+ influx could activate Ca2+-sensitive K+ and Cl- channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K+ conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.

The ouabain-induced [Ca2+]i increase in leech Retzius neurones is mediated by voltage-dependent Ca2+ channels

In leech Retzius neurones the inhibition of the Na+/K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+/K+ pump in bathing solutions of different ionic composition. The results show that Na+/K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+/K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+/K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels.