Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.     

Luminal and basolateral surface membranes of secretory acinar cells: electrophysiological comparison of cationic sensitivities

Cation sensitivities (K+, Na+, and Ca2+) of luminal and basolateral membrane surfaces of secretory acinar cells were compared using a luminally perfused and externally superfused salivary gland from the aquatic snail, Helisoma trivolvis. Tight junctions delimiting the two membrane surfaces were observed near the acinar lumen suggesting that the total membrane area exposed to the superfusion solution exceeded that in contact with the luminal perfusion solution. The resting membrane potential of acinar cells was found to be dependent upon the K+ concentration in both the external superfusion and the luminal perfusion solutions. Unilateral K+ elevation at either membrane surface produced a rapid and sustained depolarization of the acinar cell. For a given K+ concentration, the level of depolarization produced by K+ elevation at the basolateral surface was significantly higher than at the luminal surface. The highest level of membrane depolarization was observed following simultaneous K+ elevation at both membrane surfaces. The ability of acinar cells to generate overshooting action potentials in response to electrical field stimulation was dependent upon both Na+ and Ca2+. Complete blockade invariably occurred following bilateral removal of either cation. The effects of unilateral removal of either Na+ or Ca2+ proved to be somewhat variable. In general, unilateral removal of Na+ was more effective in reducing the regenerative response than Ca2+ while removal of either cation from the basolateral surface was more effective in reducing the regenerative response than its removal from the luminal surface. Electrically evoked action potentials in acinar cells could also be blocked with unilateral application of the Ca2+ antagonist, cadmium (Cd2+), at either membrane surface. However, higher Cd2+ concentrations were required to achieve complete blockade when applied to the luminal than to the basolateral gland surface. This result fails to support a hypothesis of voltage-sensitive Ca2+ channels being spatially restricted to the luminal cell surface in this preparation.     

Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes

Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two-chamber culture system were used to study the postsynthetic sorting of the alpha-subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB-ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting.     

Quantitative immunoferritin localization of [Na+,K+]ATPase on canine hepatocyte cell surface

Distribution of [Na+,K+]ATPase on the cell surface of canine hepatocytes was investigated quantitatively by incubating prefixed and dissociated liver cells with ferritin antibody conjugates against canine kidney holo[Na+,K+]ATPase. We found that [Na+,K+]-ATPase exists bilaterally both on the bile canalicular and sinusoid-lateral surfaces. The particle density on the bile canalicular surface was much higher (approximately 2.5 times) than that on the sinusoid-lateral surface. In the latter region, the enzyme was detected almost equally both on the sinusoidal and lateral surfaces. On all the surfaces, the distribution of the enzyme was homogeneous and no clustering of the enzyme was detected. Total number of the enzyme on the sinusoid-lateral surface was, however, approximately three times higher than that on the bile canalicular region, because the sinusoid-lateral surface represents approximately 87% of the total cell surface of a hepatocyte. We suggest that the [Na+, K+]ATPase on the bile canalicular surface is responsible for the bile acid-independent bile flow and the other transport processes on the bile canalicular cell surface, while that on the sinusoid-lateral surface is responsible not only for the active transport of Na+ but also for the secondary active transport of various substances in this region.     

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.     

Distribution of Na+,K+-ATPase in rat exocrine pancreas as monitored by K+-NPPase cytochemistry and [3H]-ouabain binding: a plasma membrane protein found primarily to be ductal cell associated

We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts.     

Effect of cell sodium on Na+/K(+)-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status

Aldosterone increased the tubular volume in cortical collecting tubules (CCD) of rabbit kidney. It modulated the rate of cell sodium accumulation, under condition of ATPase inhibition (4 degrees C, in the absence of K+). In contrast, the relationship between Na+/K(+)-ATPase-dependent Na+ extrusion rate and intracellular Na+ concentration (Nai+) was similar in control, adrenalectomized, and aldosterone-treated adrenalectomized animals: Na+ extrusion rate increased with Nai+, up to 70 mM Nai+, and then plateaued. This indicates that aldosterone does not modify the characteristics of Nai(+)-dependent Na+ extrusion rate by the Na+/K(+)-ATPase pump in CCD.     

Increase of (Na+ + K+)-activated ATPase activity of basolateral plasma membranes from intestinal mucosa of diabetic rats

A significant increase of the (Na+ + K+)-activated ATPase was found in mucosal homogenates of rat small intestine under conditions of alloxan and streptozotocin diabetes. From studies with isolated plasma membranes it has been shown that the activity changes were caused by that part of the (Na+ + K+)-activated ATPase only which is localized in the basolateral plasma membranes, whereas the enzyme activity in the brush border region remains unchanged. In connection with the enhanced capacity of ion, nonelectrolyte and water absorption in experimental diabetes, our findings support a concept of intestinal transport mechanism which suggest that the basolateral part of the (Na+ + K+)-activated ATPase is responsible for metabolic energy supply. The luminal part of the enzyme may be involved in regulation of passive Na+ influx.     

A novel role for Bcl-2 in regulation of cellular calcium extrusion

The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

Autoradiographic and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog salivary gland (author's transl)

The distribution of Na pump sites (Na+-K+ ATPase) in the acinar cells of dog submandibular gland was demonstrated by light and electron microscopical radioautography of 3H-ouabain binding sites and electron microscopical ATPase cytochemistry. The grains of 3H-ouabain by light microscopical radioautography were localized to the basal parts of acini and/or the striated ducts, and a small quantity of the grains was also present on the luminal parts of acini. The grains of 3H-ouabain by electron microscopical radioautography and the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells, while slightly on the microvilli of the luminal plasma membranes. The present evidence that the distribution of ATPase on the acinar cells determined by the cytochemistry is well concomitant with that of 3H-ouabain binding sites on the acinar cells by the radioautography, suggests that the above mentioned ATPase is Na+-K+ ATPase, a Na pump. The relationship of the distribution of the Na+-K+ ATPase and the cation transport of the plasma membranes in the acinar cells of the dog submandibular gland are discussed.     

Ionic components of secretory cell surfaces in relation to secretory function

Synopsis Rat pancreas was examined by ultrastructural cytochemical methods for localizing cations and anions, as well as polyanions and, more specifically, sulphated mucosubstance. Exceptionally abundant antimonate-precipitable cation was demonstrated between pancreatic acinar cells and at the base of the centro-acinar and other duct epithelial cells. Precipitates of nuclear heterochromatin appeared lighter whereas those of mitochondria and cytoplasm were coarser and more conspicuous in acinar that duct cells. Stimulation with synthetic secretin at a low level diminished antimonate reactivity of nuclei as well as the precipitation at the basement membrane of centro-acinar cells. At a higher dose, secretin selectively eliminated precipitation between and below centro-acinar and other duct cells while inducing increased antimonate-reactive cation in centro-acinar cells and the acinar lumens. Pancreozymin stimulation elimated antimonate-precipitable cations between acinar cells and, to a much lesser extent, those between duct cells and increased cytoplasmic precipitates on granular reticulum of acinar cells.Silver-precipitable anions were localized on the luminal surface of the apical plasma lemma and the outer surface of the latero-basal plasmalemma of centro-acinar cells but not on acinar cell surfaces. Silver precipitates also occurred on junctional complexes of acinar and duct epithelial cells and at tight junctions of acinar cells and on the inner face of the lateral plasmalemma of acinar cells.Dialysed iron staining demonstrated the most number of sites of acid mucosubstance on the luminal surface of the plasmalemma of acinar cells. Lateral and basal plasmalemmas of centro-acinar and more distal duct cells stained lightly with dialysed iron but those of acinar cells did not. Dialysed iron visualized acid mucosubstance in the lamina lucida of the basement membrane of duct but not of acinar cells. Dialysed iron staining of the plasma membranes succumbed to prior sialidase treatment whereas that of basement membrane resisted digestion. High iron diamine staining demonstrated sulphated mucosubstance in the lamina lucida of the duct basement membrane exclusively. The cytochemical results implicate centro-acinar cells as primarily responsible for contributing fluid and electrolytes to pancreatic secretion.     

Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.     

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.     

Na+-stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells

Two ATPase activities, a Na+-ATPase and a (Na+ + K+)-ATPase, have been found associated with sheets of basolateral plasma membranes from guinea-pig small intestinal epithelial cells. The specific activity of the former is 10-15% of the latter. The two ATPase activities differ in their affinity for Na+, their optimal pH, their K+ requirement and particularly in their behaviour in the presence of some inhibitors and of Ca2+. Thus the Na+-ATPase is refractory to ouabain but it is strongly inhibited by ethacrynic acid and furosemide, whilst the (Na+ + K+)-ATPase is totally suppressed by ouabain, partially by ethacrynic acid and refractory to furosemide. In addition, the Na+-ATPase is activated by micromolar concentrations of calcium and by resuspension of the membrane preparation at pH 7.8. The Na+-ATPase is only stimulated by sodium and to a lesser extent by lithium; however, this stimulation is independent of the anion accompanying Na+. The latter rules out the participation of an anionic ATPase. The relation between the characteristics of the sodium transport mechanism in basolateral membrane vesicles (Del Castillo, J.R. and Robinson, J.W.L. (1983) Experientia 39,631) and those of the two ATPase activities present in the same membranes, allow us to postulate the existence of two separate sodium pumps in this membranes. Each pump would derive the necessary energy for active ion transport from the hydrolysis of ATP, catalyzed by different ATPase systems.     

Insulin activation of (Na+,K+)-adenosinetriphosphatase exhibits a temperature-dependent lag time. Comparison to activation of the glucose transporter

The time course of insulin activation of sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)ATPase] was studied in the rat adipocyte and was compared to activation of the glucose transporter. Under conditions in which the binding of insulin to its cell surface receptor was not rate limiting, a distinct time lag was apparent between insulin addition and stimulation of transport activity. At 37 degrees C, 40-50 s elapsed before an increase in Rb+ uptake [a measure of (Na+,K+)ATPase transport activity] or 2-deoxyglucose uptake could be observed. This lag time increased in an identical manner for both transport processes as the temperature was lowered to 23 degrees C. Addition of the insulinomimetic agent hydrogen peroxide also produced a lag time similar to that for insulin before activation of Rb+ and 2-deoxyglucose uptakes was detected. These data provide the first evidence of a discrete time lag involved during stimulation of the adipocyte (Na+,K+)ATPase. A model for the molecular mechanism of insulin activation of (Na+,K+)ATPase is presented that incorporates these results into the hypothesis of insulin mediated "translocation" of glucose transporters to the plasma membrane.     

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.     

Evidence for a high and specific concentration of (Na+,K+)ATPase in the plasma membrane of the osteoclast

During bone resorption, the osteoclast actively acidifies a limited extracellular compartment. We hypothesized that, like other cells engaged in ion transport and proton translocation, the osteoclast's membrane might be highly enriched in sodium pumps. Using monoclonal antibodies to both the alpha and the beta subunits, immunoblot analysis, and [3H]ouabain binding, we have demonstrated that the osteoclast plasma membrane is both highly and specifically enriched in (Na+,K+)ATPase, compared with other bone cells, monocytes, macrophages, and other blood and bone marrow cells. The density of binding sites on the osteoclast is equivalent to that of kidney tubule cells. This observation is consistent with the hypothesis that the (Na+,K+)ATPase plays a role in the mechanism of bone resorption, possibly coupled with secondary active calcium and/or proton transport. Monoclonal antibodies against the (Na+,K+)ATPase can therefore be used as specific markers for the osteoclast in bone and bone marrow preparations.     

Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.     

Calcium-magnesium interactions in pancreatic acinar cells.

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.