SUMMARY1. We have used double-barrelled ion-sensitive microelectrodes to measure the intracellular pH, pHi, the intracellular Na+ activity, aiNa, and the membrane potential in identified glial cells of the central nervous system of the leech Hirudo medicinalis to study the effect of CO2-HCO3-.2. When a HEPES-buffered saline was exchanged for a saline buffered with 2 % CO2 +11 mM-HCO3-, keeping the pH constant at 7 4, the mean steady-state pHi ofthe glial cells increased from 6-85+0-06 to 7418+0-13 (mean+S.D., n = 25).3. This C02-HC03--dependent alkalinization was inhibited in the absence of external Na+ (exchanged by N-methyl-D-glucamine), but was unaffected by the inhibitor of Na+-H+ exchange, amiloride (2 mM).4. The a' of the glial cells increased by 2-4 mm from a mean steady state of 7-2 + 2 mm (mean + S.D., n = 6) upon introduction ofC02-HC03--buffered saline. This C02-HC03--dependent rise in a' a increased to about double when the pHi had been decreased by acid loading the cells (addition and subsequent removal of NH4+).5. The CO2-HCO3--dependent increases of pHi and aia were inhibited by the stilbene 4,4-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 05-1 0 mm).6. Removal of external Cl-and depletion of intracellular Cl-did not inhibit the C02-HC03--dependent alkalinization.7. The C02-HC03--dependent alkalinization was unaffected by inhibitors of the carbonic anhydrase, acetazolamide (0-2 mM) or ethoxzolamide (2 ,UM).8. The membrane potential became more negative by 3-20 mV upon addition of C02-HCO3-. This hyperpolarization was even further enlarged in the presence of Ba2+ (which reduces the K+ permeability) or at increased external K+ concentration (which depolarizes the membrane and brings the membrane potential to the K+ equilibrium potential). The C02-HCO3 -induced membrane hyperpolarization was inhibited in Na+-free saline and in the presence of DIDS. Ouabain (0 5 mM) sometimes reduced, but never abolished, the hyperpolarization.9. The stoichiometry of the co-transport is suggested to be 2 HC03-: 1 Na+ with an equilibrium potential of -90 mV calculated for this coupling ratio in the steady state.10. It is concluded that in the presence of C02-HC03-an inwardly directed electrogenic Na+-HCO3-co-transport is stimulated across the glial membrane, which J. W. DEITMER AND W.-R. SCHLUE greatly determines the pHi and thereby affects the intracellular buffering power of the glial cells.

SUMMARY1. Neutral-carrier pH-sensitive micro-electrodes were used to investigate intracellular pH (pHj) 4. In HC03--free solutions, pHi recovery from acidification was blocked by removing external Na or by amiloride (2 mM).5. In solutions buffered with 20 CO2 and 11 mM-HCO3-, amiloride slowed but did not block pHi recovery. The anion exchange inhibitor SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) also slowed pHi recovery in the presence of HCO3-.6. In CO2/HCO3-solution the removal of external Na either slowed or blocked pH1recovery, and blocked it completely in the presence of amiloride.7. We conclude that in HCO3--free solutions pHi regulation is by a Na-H exchange system; but in the presence of HCO3-there is an additional mechanism which is probably a Na-dependent Cl-HCO3 exchanger.

Free [Ca] within organelles of permeabilized BHK-21 cells was measured using ratio imaging of compartmentalized mag-fura-2. In BHK-21 cells, this dye monitors free [Ca] in principally one type of ATP-dependent Ca-sequestering organelle in which intrastore Ca was released uniformly and entirely by 100 nM thapsigargin or removal of ATP or Ca from the bath, and was reduced by 85% upon treatment with a supramaximal dose of InsP3 (6 microM). Examination of the spatial distribution of InsP3-sensitive Ca stores showed that InsP3 released Ca throughout all regions of the cell, although we often noted a perinuclear region (which we speculate may correspond to the Golgi apparatus) with reduced responsiveness to InsP3. InsP3-induced changes of intraluminal Mg could not be detected. Cyclic ADP-ribose, ryanodine, caffeine, mitochondrial inhibitors, and GTP, agents known to influence intraorganellar Ca sequestration in other cell types, were all without effect on the mag-fura-2 ratio. In situ calibration of the mag-fura-2 ratio with Ca ionophores revealed that the average free intraorganellar [Ca] was initially 188 +/- 21 microM in the presence of 170 nM free Ca and 3 mM ATP, and was reduced to 25 +/- 5 microM upon stimulation with 6 microM InsP3. The ionic dependence of the release and reloading process was also investigated. The presence of either K, Na, or Cl could consistently support both InsP3-induced release and the refilling of stores with Ca, but physiological concentrations of HCO3 were effective in sustaining the response in only 24% of cells examined.

Although the role of calcium (Ca2+) in the signal transduction and pathobiology of the exocrine pancreas is firmly established, the role of magnesium (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 digital imaging of Ca2+- or Mg2+-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+ mobilization, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone withdrawal 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 decrease 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 concentrations profoundly affects acinar cell Ca2+ signaling.

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