alpha 1-Adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na+-dependent transport mechanism

Theresa E. Fagan and Andrea Romani

Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970


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The stimulation of the alpha 1-adrenergic receptor by phenylephrine results in a sizable extrusion of Mg2+ from liver cells. Phenylephrine-induced Mg2+ extrusion is almost completely abolished by the removal of extracellular Ca2+ or in the presence of SKF-96365, an inhibitor of capacitative Ca2+ entry. In contrast, Mg2+ extrusion is only partially inhibited by the Ca2+-channel blockers verapamil, nifedipine, or (+)BAY-K8644. Furthermore, Mg2+ extrusion is almost completely prevented by TMB-8 (a cell-permeant inhibitor of the inositol trisphosphate receptor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (an intracellular Ca2+-chelating agent), or W-7 (a calmodulin inhibitor) Thapsigargin can mimic the effect of phenylephrine, and the coaddition of thapsigargin and phenylephrine does not result in an enlarged extrusion of Mg2+ from the hepatocytes. Regardless of the agonist used, Mg2+ extrusion is inhibited by >90% when hepatocytes are incubated in the presence of physiological Ca2+ but in the absence of extracellular Na+. Together, these data suggest that the stimulation of the hepatic alpha 1-adrenergic receptor by phenylephrine results in an extrusion of Mg2+ through a Na+-dependent pathway and a Na+-independent pathway, both activated by changes in cellular Ca2+.

alpha 1-adrenergic receptor; magnesium homeostasis


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TOTAL CELLULAR MG2+ content ranges between 16 and 20 mM in the majority of mammalian cell types (9, 38). The observation (30) that under resting conditions Mg2+ crosses the cell membrane at a very slow rate has supported for a long time the idea that total and free Mg2+ concentrations remain relatively stable within the cell and play no major role in regulating cellular enzymes or metabolism. During the last decade, however, increasing experimental evidence (14, 28, 36, 46, 48) indicated that ~5-10% of total cellular Mg2+ content can be extruded from a variety of mammalian cell types, including hepatocytes (17, 37), into the extracellular compartment and the circulation (15, 22) after stimulation of the beta -adrenergic receptor by isoproterenol or catecholamine. The same cell types extrude a quantitatively similar amount of Mg2+ when stimulated by forskolin (36, 37), an agent that activates adenylyl cyclase, or by cell-permeant cAMP analogs (e.g., dibutyryl cAMP, 8-chloro-cAMP, or 8-bromo-cAMP) (36, 37). Hence, the general consensus is that the increase in cytosolic cAMP subsequent to the stimulation of beta -adrenergic receptors or the addition of forskolin or cell-permeable cAMP analogs activates Mg2+ extrusion mechanisms located in the plasma membrane (14). Although the Mg2+ transport mechanisms have not been isolated or purified, experimental evidence supports the idea that Mg2+ extrusion occurs via two distinct routes: a Na+-dependent pathway and a Na+-independent pathway (10).

The Mg2+ extrusion mechanism activated by isoproterenol or catecholamine via cellular cAMP increase is blocked by the removal of Na+ from the extracellular medium or by the Na+ transport inhibitors amiloride (12) or imipramine (7). Hence, this pathway has been tentatively identified as a cAMP-phosphorylated (14) Na+/Mg2+ exchanger (8). The operation of a Mg2+ extrusion pathway distinct from the Na+-dependent route has also been observed in several cell types (11, 13) and in isolated liver plasma membrane (1) after hormonal (34, 35) and nonhormonal stimuli (1, 6, 11, 13, 16). This Na+-independent pathway appears to extrude Mg2+ in exchange for extracellular Ca2+ (34, 35) or Mn2+ (6, 16) or in cotransport with anion (11). Yet, the modality of activation of this pathway remains largely uncharacterized.

Our (23) laboratory has reported that insulin pretreatment of liver cells completely abolishes the Mg2+ extrusion induced by isoproterenol or cAMP analogs, leaving unaffected that mediated by phenylephrine. As insulin decreases cellular cAMP (21, 42), this observation supports the notion that the stimulation of the alpha 1-adrenoceptor induces Mg2+ extrusion via a cAMP-independent process.

The stimulation of alpha 1-adrenergic receptors activates phospholipase C (4) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol and inositol trisphosphate (IP3). Diacylglycerol activates the protein kinase C signaling pathway, whereas the interaction of IP3 with a specific receptor in the endoplasmic reticulum elicits a release of Ca2+ from this intracellular organelle (43), which in turn increases cytosolic free Ca2+ and triggers a capacitative Ca2+ entry across the plasma membrane (31).

We (33) have previously reported that activation of protein kinase C elicits a Mg2+ accumulation into liver cells rather than an extrusion. Therefore, in the present study we investigated the possibility that stimulation of the alpha 1-adrenoceptor mediates Mg2+ extrusion from liver cells via the IP3-Ca2+ signaling pathway. The results reported here indicate that the changes in cellular Ca2+ content induced by stimulation of the alpha 1-adrenoceptor result in the activation of both Na+-dependent and Na+-independent Mg2+ extrusion mechanisms in the hepatocyte cell membrane. This observation suggests that stimulation of the alpha 1-adrenergic receptor can induce Mg2+ extrusion from liver cells via Ca2+ signaling in alternative or addition to the operation of the Mg2+ efflux pathway activated via cAMP and the beta -adrenergic receptor.


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MATERIALS AND METHODS
RESULTS
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Materials

Collagenase type Cls-2 (235-280 U/mg) was from Worthington (Freehold, NJ). SKF-96365, (+)BAY-K8644 and (-)BAY-K8644, ryanodine, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) were from Calbiochem (La Jolla, CA). Percoll was from Pharmacia (Piskataway, NJ). 125I-labeled cAMP RIA was from Amersham (Arlington Heights, IL). All other agents and chemicals were from Sigma Chemical (St. Louis, MO).

Methods

Liver cell isolation. Fed male Sprague-Dawley rats (230-250 g body wt) were anesthetized by intraperitoneal injection of saturated pentobarbital sodium solution. The portal vein was cannulated, and collagenase-dispersed hepatocytes were isolated according to the procedure of Seglen (41). The hepatocytes were resuspended in a medium (resuspension medium) having the following composition (in mM): 120 NaCl, 3 KCl, 10 mM HEPES, 12 mM NaHCO3, 1.2 mM KH2PO4, 1.2 CaCl2, 1.2 MgCl2, and 15 mM glucose, pH 7.2, equilibrated under slow flow of O2-CO2 (95:5 vol/vol) at room temperature. Cells not viable were removed by sedimenting the hepatocytes through a Percoll gradient. Briefly, 45 ml Percoll were mixed with 30 ml of a 2.5-fold concentrated resuspension medium to achieve the final ion concentration reported above, and pH was adjusted to 7.2. Five milliliters of cell suspension were layered onto 5 ml of Percoll gradient and sedimented at 3,500 g for 1 min in a bench centrifuge. The layer of damaged cells at the interface with Percoll gradient was removed, and the pellet of viable hepatocytes was washed three times (at 600 g for 1 min) with 5 ml of resuspension medium and resuspended therein at a final concentration of ~2 × 106 cell/ml. Cell viability, assessed by trypan blue exclusion test, was 92 ± 2% (n = 14) and did not change significantly during the following 3 h.

Mg2+ determination. To evaluate the amount of Mg2+ extruded from the cells, aliquots of hepatocytes were withdrawn from the cell suspension and gently washed (at 600 g for 30 s) in a Microfuge tube with a medium having a composition similar to that described above but devoid of Mg2+ (incubation medium). The Mg2+ present as a contaminant in the incubation medium was measured by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100 and found to be ~5 µM. The cells were incubated in the incubation medium at 37°C under continuous stirring and O2-CO2 (95:5 vol/vol) flow. After 5 min of equilibration, various stimulatory agents were added to the incubation mixture (see Fig. 1). In contrast, inhibitory agents were added together with the cells. At the time points reported in Figs. 1-9, aliquots of the cell mixture were withdrawn in duplicate, and the hepatocytes were sedimented in a Microfuge tube (at 3,500 g for 30 s). The supernatants were removed, and the Mg2+ content was measured by AAS. The first two time points before the addition of any stimulatory agent were used to establish the Mg2+ baseline. After the agonist addition, net Mg2+ extrusion was calculated as follows: the extracellular Mg2+ content at the first two time points was calculated, averaged, and subtracted from each of the subsequent time points.


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Fig. 1.   Mg2+ extrusion from isolated hepatocytes. Collagenase-dispersed rat hepatocytes were incubated in a medium devoid of extracellular Mg2+ and stimulated by administration of 5 µM phenylephrine. Mg2+ extrusion over time was determined as described in MATERIALS AND METHODS. open circle , Control; black-triangle, phenylephrine. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. control sample.



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Fig. 2.   Net Mg2+ extrusion from hepatocytes stimulated by phenylephrine (Phe) in the presence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and stimulated by 5 µM phenylephrine after being loaded for 25 min with 10 µM BAPTA-AM or in the presence of 50 µM TMB-8. TMB-8 was added to the incubation system together with the cells (see MATERIALS AND METHODS). A: a typical experiment. black-triangle, Phenylephrine; , BAPTA-AM; , BAPTA-AM + phenylephrine; open circle , TMB-8; , TMB-8 + phenylephrine. B: net Mg2+ extrusion at 6 min after phenylephrine administration. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. phenylephrine in the absence of inhibitors.



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Fig. 3.   Mg2+ extrusion from hepatocytes stimulated by thapsigargin (Thaps). Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and sequentially stimulated by addition of 5 µM phenylephrine and 1 µM thapsigargin. The agonists were added where indicated by the arrows following sample withdrawal. Data for net Mg2+ extrusion are given in RESULTS. open circle , Control; , phenylephrine; , phenylephrine + thapsigargin. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. control sample.



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Fig. 4.   Net Mg2+ extrusion from hepatocytes stimulated by phenylephrine in the presence of SKF-96365 (SKF) as an inhibitor of Ca2+ entry. Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and stimulated by 5 µM phenylephrine in the presence of 10, 30, or 50 µM SKF-96365. SKF-96365 was added to the incubation system together with the cells (see MATERIALS AND METHODS). A: a typical experiment. black-triangle, Phenylephrine; open circle , SKF-96365; , SKF-96365 + phenylephrine. B: net Mg2+ extrusion elicited by phenylephrine in the presence and in the absence of varying doses of SKF-96365. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. phenylephrine in the absence of inhibitor.



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Fig. 5.   Net Mg2+ extrusion from hepatocytes stimulated by phenylephrine in the presence of various Ca2+-channel blockers. Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and stimulated by 5 µM phenylephrine in the presence of 25 µM verapamil (Vera), 15 µM nifedipine (Nife), or 5 µM (+)BAY-K8664 or (-)BAY-K8664 (Bay). The doses of various Ca2+-channel blockers were added to the incubation system together with the cells (see MATERIALS AND METHODS). Values represent net Mg2+ extrusion at 6 min after phenylephrine addition in the presence of the mentioned inhibitors and are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. phenylephrine in the absence of Ca2+-channel blockers.



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Fig. 6.   Net Mg2+ extrusion from hepatocytes stimulated by phenylephrine or thapsigargin in the absence of extracellular Ca2+. Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and Ca2+ (see MATERIALS AND METHODS) and stimulated by the addition of 1 µM thapsigargin or 5 µM phenylephrine. Net Mg2+ extrusion at 6 min was calculated as reported in MATERIALS AND METHODS. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. samples stimulated with phenylephrine or thapsigargin (Fig. 3) in the presence of extracellular Ca2+.



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Fig. 7.   Net Mg2+ extrusion from hepatocytes stimulated by phenylephrine or thapsigargin in the presence of the calmodulin inhibitor W-7. Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and stimulated by 5 µM phenylephrine or 1 µM thapsigargin in the presence of 50 µM W-7 as a calmodulin inhibitor. W-7 was added to the incubation system together with the cells (see MATERIALS AND METHODS). A: a typical experiment. black-triangle, Phenylephrine; black-lozenge , W-7; , W-7 + phenylephrine; open circle , W-7 + thapsigargin. B: net Mg2+ extrusion induced by phenylephrine or thapsigargin at 6 min. Values are means ± SE of 6 different preparations (each performed in triplicate). * Statistically significant vs. samples stimulated by phenylephrine or thapsigargin in the absence of W-7.



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Fig. 8.   Net Mg2+ extrusion from hepatocytes stimulated by various agonists in the absence of extracellular Na+. Hepatocytes were incubated in a Na+-free medium (see MATERIALS AND METHODS) devoid of extracellular Mg2+ and stimulated by 5 µM phenylephrine or 1 µM thapsigargin. A: a typical experiment. black-triangle, Phenylephrine; open circle , Na+-free medium; , Na+-free medium + phenylephrine. B: net Mg2+ extrusion elicited by these agents at 6 min after the addition. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. samples stimulated by phenylephrine or thapsigargin in the presence of extracellular Na+.



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Fig. 9.   Net Mg2+ extrusion from hepatocytes stimulated by various agonists in the presence of Na+ transport inhibitors. Hepatocytes were incubated in a medium devoid of extracellular Mg2+ and stimulated by phenylephrine in the presence of 1 mM amiloride (Ami), 0.5 mM imipramine (Imi), or 0.5 mM quinidine (Quin) as inhibitors of Na+ transport. The doses of Na+ transport inhibitors were added to the incubation system together with the cells (see MATERIALS AND METHODS). A: a typical experiment. black-triangle, Phenylephrine; , amiloride; open circle , amiloride + phenylephrine; ×, imipramine; , imipramine + phenylephrine. B: net Mg2+ extrusion at 6 min after phenylephrine addition. Values are means ± SE of 8 different preparations (each performed in quadruplicate). * Statistically significant vs. samples stimulated by phenylephrine in the absence of Na+ transport inhibitors.

Na+- or Ca2+-free medium. To investigate the role of extracellular Na+ or Ca2+ on Mg2+ extrusion, hepatocytes were incubated in medium devoid of one of these two cations. For the Na+-free medium, NaCl and NaHCO3 were replaced with equiosmolar concentrations of choline chloride and KHCO3, respectively (pH 7.2 with KOH). The increase in K+ content subsequent to the replacement of NaHCO3 with KHCO3 did not appear to affect basal cellular Mg2+ homeostasis, consistent with previous reports from this laboratory (34, 35). For the Ca2+-free medium, no CaCl2 was added to the medium or, alternatively, a final extracellular Ca2+ concentration <50 nM was obtained by EGTA addition [calculated according to Fabiato's program (5)].

The incubation of hepatocytes in any of these media for 20 min did not result in a significant change in cell viability (assessed by trypan blue exclusion test and lactate dehydrogenase release). The removal of extracellular Na+ or Ca2+ from the medium did not affect hepatocyte basal extracellular Mg2+ content (i.e., in the absence of any stimulatory agent).

To exclude that a reduced Mg2+ extrusion in the absence of extracellular Na+ or Ca2+ could be ascribed to an altered responsiveness of cellular signaling mechanism, including adrenergic receptors, pharmacological doses of adrenergic agonist or other stimulatory agents were used under all experimental conditions.

45Ca accumulation. To quantify the amount of Ca2+ accumulated by the hepatocytes under the experimental conditions described above, the cells were incubated in the presence of 1.2 mM CaCl2 labeled with 0.5 µCi/ml. After 3 min equilibration, 0.5 ml of the incubation mixture was withdrawn in duplicate before and 6 min after adrenergic agonist administration and filtered onto glass fiber filters (Whatman, 0.25-µm pore size) under vacuum suction (32). The filters were rapidly washed with 5 ml "ice-cold" sucrose (250 mM) (32). The radioactivity retained onto the filters was measured by beta -scintillation counting in a Beckman LS 7000.

Other procedures. Protein was measured according to the method of Lowry et al. (26), using BSA as a standard.

Statistical analysis. Values were reported as means ± SE. Data were first analyzed by one-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance of P < 0.05.


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Figure 1 shows that in the absence of a stimulatory agent isolated hepatocytes did not extrude a significant amount of cellular Mg2+ for the entire period of incubation. The administration of the alpha 1-adrenergic agonist phenylephrine (5 µM) to hepatocyte suspensions resulted in a time-dependent extrusion of Mg2+ that reached the maximum (net extrusion ~4 nmol Mg2+/106 cells) within 6 min from the agonist addition (8 min, Fig. 1). Therefore, several values are expressed as the net amount of Mg2+ released from the hepatocytes at 6 min after agonist addition (see MATERIALS AND METHODS for details).

The activation of alpha 1-adrenergic receptor by phenylephrine is coupled to the activation of phospholipase C and the production of IP3 and diacylglycerol. Whereas diacylglycerol production directly activates the protein kinase C signaling pathway, IP3 production mediates a release of Ca2+ from the endoplasmic reticulum; increases in cytosolic Ca2+ ultimately result in entry of Ca2+ across the cell membrane (31). We (33) have previously reported that the activation of the protein kinase C signaling pathway by diacylglycerol analogs mediates an accumulation of Mg2+ into cardiac or liver cells rather than an extrusion. Thus we investigated the possibility that the alpha 1-agonist-induced Mg2+ extrusion occurs as a consequence of the changes in cytosolic Ca2+ via IP3 and/or Ca2+ entry across the cell membrane.

We started by investigating whether inhibition of the IP3-mediated Ca2+ release would abolish Mg2+ extrusion. To this purpose, hepatocytes were incubated in the presence of 50 µM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a cell-permeant inhibitor of the IP3 receptor (48). Alternatively, hepatocytes were loaded for 25 min at room temperature with 10 µM BAPTA-AM (35), an intracellular chelator for Ca2+, before being washed and transferred to the incubation system for phenylephrine administration. As Fig. 2A shows, the administration of any of these agents per se did not change basal extracellular Mg2+ compared with untreated cells. However, both TMB-8 and BAPTA almost completely abolished the extrusion of Mg2+ induced by phenylephrine (Fig. 2B). Doses of agonist two to three times larger than those reported in Fig. 2 were also ineffective at eliciting Mg2+ extrusion (not shown).

Figure 3 provides additional evidence that the release of Ca2+ from the endoplasmic reticulum plays a key role in inducing Mg2+ extrusion. The addition of 1 µM thapsigargin to hepatocyte suspension, which selectively inhibits reticular Ca2+-ATPase and increases cytosolic Ca2+ (35), resulted in an extrusion of Mg2+ from the cells. In terms of time course and amplitude, this extrusion resembled that elicited by phenylephrine (Fig. 3). The addition of phenylephrine at 6 min, at which time maximal extrusion of Mg2+ by thapsigargin was attained (Fig. 3) or the coaddition of phenylephrine and thapsigargin (at 2 min) did not result in an enlarged mobilization of Mg2+ from the cells. Net Mg2+ extrusion was 3.35 ± 0.58 and 3.81 ± 0.49 nmol · 106 cells-1 · 6 min-1 for hepatocytes stimulated by thapsigargin and thapsigargin plus phenylephrine, respectively (n = 8 for both experimental conditions) compared with 4.05 ± 0.46 nmol Mg2+ · 106 cells-1 · 6 min-1 for hepatocytes stimulated by phenylephrine alone (Fig. 1). Consistent with the results reported in Fig. 2A, the addition of thapsigargin to hepatocytes loaded with BAPTA-AM did not result in Mg2+ extrusion (not shown).

We next determined whether the rise in cytosolic Ca2+ elicited by IP3 or thapsigargin was sufficient or if an entry of Ca2+ across the plasma membrane was required to induce Mg2+ extrusion. For this purpose, hepatocytes were incubated in the presence of SKF-96365 (Fig. 4), an agent reported to inhibit capacitative Ca2+ entry (27). Although SKF-96365 alone at concentrations up to 50 µM did not affect Mg2+ baseline (Fig. 4A), the results reported in Fig. 4B indicate that the presence of 10 µM SKF-96365 in the incubation system was sufficient to reduce phenylephrine-induced Mg2+ mobilization by ~80%. Larger concentrations of SKF-96365 completely abolished phenylephrine-induced Mg2+ efflux (Fig. 4B). In contrast, when verapamil (25 µM), nifedipine (15 µM), (+)BAY-K8644 (5 µM), or (-)BAY-K8644 (5 µM) was administered to suspensions of hepatocytes stimulated in vitro by phenylephrine, Mg2+ extrusion was affected only partially (Fig. 5). Verapamil and nifedipine reduced Mg2+ extrusion at 6 min by ~35% and 45%, respectively, whereas (+)BAY-K8644 was less effective, inhibiting the extrusion by only 25% (Fig. 5). Furthermore, the Ca2+-channel agonist (-)BAY-K8644 alone did not mobilize Mg2+ (not shown) or increase the amplitude of phenylephrine-induced Mg2+ extrusion when administered together with the alpha 1-agonist but rather decreased alpha 1-agonist-induced Mg2+ extrusion by ~25% (Fig. 5). Doses of inhibitor larger than those reported in Fig. 5 did not result in an increased inhibitory effect (not shown). Overall, these data would suggest that capacitative Ca2+ entry, and not activation of L-type Ca2+ channels, is involved in Mg2+ mobilization and support a specific effect of SKF-96365 in inhibition of Mg2+ extrusion from the hepatocytes.

A role for extracellular Ca2+ in favoring Mg2+ extrusion by phenylephrine is further supported by the results shown in Fig. 6, which indicate that phenylephrine-induced Mg2+ mobilization from hepatocytes incubated in the absence of extracellular Ca2+ (i.e., no Ca2+ added to the incubation system) was decreased by ~80%. Qualitatively similar results were obtained in hepatocytes incubated in the presence of EGTA to chelate extracellular Ca2+ to a submicromolar level (data not shown). In contrast, the Mg2+ extrusion elicited by thapsigargin (Fig. 6) was unaffected under similar experimental conditions (3.65 ± 0.22 vs. 3.35 ± 0.58 nmol Mg2+ · 106 cells-1 · 6 min-1 in the absence or in the presence of extracellular Ca2+, respectively).

To quantify the amount of extracellular Ca2+ entering the hepatocytes after phenylephrine stimulation, the cells were incubated in the presence of CaCl2 labeled with 45Ca2+, as described in MATERIALS AND METHODS. After the addition of phenylephrine, a net accumulation of 9.9 ± 2.1 nmol 45Ca2+/106 cells within 6 min of stimulation was observed. This uptake is approximately twofold larger than the amount of Mg2+ extruded from the cells under the same experimental conditions, thus not fully supporting a molar exchange ratio of extracellular Ca2+ for intracellular Mg2+.

The possibility that the increase in cytosolic Ca2+ elicited via IP3 and capacitative Ca2+ entry induces Mg2+ extrusion by activating via calmodulin a specific Mg2+ transport pathway was addressed by pretreating the hepatocytes with the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) for 3 min before phenylephrine administration. As Fig. 7 illustrates, W-7 at the dose of 50 µM reduced the phenylephrine-induced Mg2+ extrusion by ~80%. Qualitatively similar results were observed when thapsigargin was used instead of phenylephrine to induce Mg2+ extrusion (Fig. 7B). Larger doses of W-7 did not result in a more pronounced inhibition of the agonist-induced Mg2+ extrusion from the cells, nor were larger doses of any of the agonists able to mobilize Mg2+ in the presence of 50 µM W-7 (not shown).

As mentioned previously, the administration of catecholamine or the beta -adrenergic agonist isoproterenol to liver cells results in an extrusion of Mg2+ through a mechanism that, in the absence of more precise structural information, has been tentatively identified as a Na+/Mg2+ exchanger (8). To ascertain whether this exchange mechanism mediated the residual Mg2+ extrusion observed in the absence of extracellular Ca2+ (Fig. 6) after phenylephrine administration, hepatocytes were incubated in a medium devoid of extracellular Na+. Under these experimental conditions (Fig. 8), phenylephrine administration elicited an extrusion of Mg2+ that accounted for <20% (~0.7 nmol Mg2+ · 106 cells-1 · 6 min-1, n = 8) of that reported in Fig. 1. A quantitatively similar reduction in Mg2+ extrusion was observed when thapsigargin was used instead of phenylephrine (Fig. 8B). Consistent with this result, a ~75-80% reduction in Mg2+ extrusion was also observed in hepatocytes incubated in a medium containing a physiological concentration of extracellular Na+ and stimulated by phenylephrine in the presence of 1 mM amiloride or 0.5 mM imipramine or quinidine (Fig. 9). These are all agents that inhibit, although in a nonspecific manner, the putative Na+/Mg2+ exchanger (7, 12).


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Several laboratories (14, 17, 22, 28, 36, 46-48) have provided compelling evidence about the ability of various mammalian cell types to extrude 5-10% of their total cellular Mg2+ content after various hormonal and nonhormonal stimuli (18, 44, 45). In the majority of cases, Mg2+ extrusion across the cell membrane occurs via a Na+-dependent mechanism (17, 34, 35, 46). Although not structurally defined, experimental data suggest that this transporter is a Na+/Mg2+ exchanger (8), with a stoichiometry varying from 1 Na+ for 1 Mg2+ to 3 Na+ for 1 Mg2+, according to the cell type or the experimental conditions considered (8, 40, 44). The operation of a Na+-independent Mg2+ extrusion mechanism has also been observed, but the ion(s) co- or countertransported for Mg2+ and the modality of activation have not been fully characterized (11, 13). By using purified rat liver plasma membrane vesicles, our laboratory (1, 2) has provided evidence for the presence of two distinct Mg2+ transport mechanisms (1) operating as a Na+/Mg2+ and Ca2+/Mg2+ exchanger in the basolateral and apical domains, respectively, of the hepatocyte plasma membrane.

In intact cells, the Na+/Mg2+ exchanger appears to be activated by the increase in cytosolic cAMP level that follows the stimulation of beta -adrenoceptor by catecholamine or isoproterenol (14, 17, 22, 28, 36, 37, 46, 48) or the administration of forskolin (36, 37) or cell-permeant cAMP analogs (36, 37). This observation and the ability of the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate isomer to prevent Mg2+ extrusion by inhibiting protein kinase A (47) largely support the hypothesis that the Na+/Mg2+ exchanger is phosphorylated by cAMP. The Na+-independent mechanism, whether it is the Ca2+/Mg2+ exchanger observed in liver plasma membranes (1, 2) or a transporter using alternative ions (6, 11, 13), does not appear to be modulated in a similar manner. The pretreatment of liver cells with insulin, which hampers the ability of the beta -adrenergic receptor to respond to specific agonists (21) and decreases cellular cAMP level via phosphodiesterase activation (42), completely blocks the Mg2+ extrusion induced by isoproterenol via cAMP, leaving unaffected that elicited by phenylephrine (23). This observation suggests that catecholamine induces Mg2+ extrusion from liver cells by activating a cAMP-dependent and -independent mechanism via beta - and alpha 1-adrenergic-mediated signaling pathways, respectively (23). The ability of phenylephrine to induce a Mg2+ extrusion from liver cells via alpha 1-adrenoceptor stimulation has been previously reported by Jakob et al. (20), but the modality by which the agonist induces Mg2+ extrusion was not elucidated. The present study was aimed at characterizing the signaling pathway that elicits Mg2+ extrusion from liver cells after stimulation of the alpha 1-adrenoceptor.

Activation of Mg2+ Extrusion Pathway by Phenylephrine

The stimulation of the alpha 1-adrenergic receptor by phenylephrine activates phospholipase C and results in the hydrolysis of PIP2 to diacylglycerol and IP3 (4). Diacylglycerol specifically activates protein kinase C, a condition that results in Mg2+ accumulation in a variety of cell types, including hepatocytes (33). In contrast, IP3 activates a specific receptor in the endoplasmic reticulum and induces a release of reticular Ca2+ into the cytosol (43). The increase in cytosolic Ca2+ is further sustained by Ca2+ entry across the plasma membrane (36). The data reported here indicate that the latter sequence of events is involved in phenylephrine-induced Mg2+ extrusion. The block of reticular Ca2+ release by the IP3 receptor inhibitor TMB-8, the chelation of cytosolic Ca2+ by BAPTA, and the inhibition of Ca2+ entry across the plasma membrane by SKF-96365 all abolish the extrusion of Mg2+ induced by phenylephrine. The possibility that the inhibitory effect of BAPTA is attributable to chelation of cytosolic Mg2+ is inconsistent with the discrepancy between the concentration of BAPTA-AM used in our experimental protocol (10 µM) and the cytosolic free Mg2+ concentration present in the hepatocyte (0.7-1 mM; Refs. 3, 9, and 38). Assuming a cell volume of 10 pl (19), the amount of Mg2+ extruded from the hepatocyte is quantitatively identical to, if not larger than, the cytosolic free Mg2+ concentration, thus making it highly improbable that BAPTA blocks Mg2+ extrusion by chelating cytosolic Mg2+. The inhibition of Mg2+ extrusion by SKF-96365, compared with verapamil, nifedipine, or (+)BAY-K8644, or the absence of extracellular Ca2+ suggest that Ca2+ entry across the cell membrane via capacitative Ca2+ rather than L-type Ca2+ channel is required to activate the Mg2+ extrusion mechanism. However, because the Mg2+ extrusion mechanism is not structurally characterized and SKF-96365 may also inhibit other transport mechanisms (29), the possibility that this compound has a direct or indirect inhibitory effect on the Mg2+ extrusion pathway cannot be excluded altogether.

A role of cellular Ca2+ in mediating Mg2+ extrusion is further supported by the ability of thapsigargin to mimic the phenylephrine effect. The similarity of these results would suggest that the increase in cytosolic Ca2+ achieved by either of these two agents is sufficient to activate Mg2+ extrusion. Furthermore, the inability of phenylephrine, added subsequently or concomitantly to thapsigargin, to mobilize additional Mg2+ from the cell would indicate that phenylephrine operates via the same mechanism. Finally, the inhibitory effect of W-7 (Fig. 7) would imply that calmodulin may be the final activator of the Mg2+ extrusion mechanism after changes in cellular Ca2+ induced by phenylephrine or thapsigargin. At the present time, we do not have a clear explanation for why thapsigargin, but not phenylephrine, can still induce Mg2+ extrusion in the absence of extracellular Ca2+. One possible explanation could be that, even in the absence of Ca2+ entry from the extracellular compartment, the inhibition of reticular Ca2+-ATPase by thapsigargin achieves a persistent increase in cytosolic Ca2+ (35) that is sufficient to activate the Mg2+ transporter or to displace Mg2+ from intracellular binding sites (25), thus making it available for the subsequent extrusion. In contrast, in the absence of extracellular Ca2+ the quantum amount of Ca2+ released via IP3 from the endoplasmic reticulum is rapidly reaccumulated within the organelle via Ca2+-ATPase and does not attain a level of cytosolic Ca2+ sufficient to effectively or persistently activate the Mg2+ extrusion mechanism. Consistent with this hypothesis, it has been reported (24) that IP3 induces Ca2+ entry by directly activating the htrp channel in the plasma membrane. Thus it can be speculated that phenylephrine and IP3 signaling, but not thapsigargin, requires the operation of this auxiliary mechanism to guarantee a sufficient increase in cytosolic Ca2+ and the subsequent activation of the Mg2+ extrusion mechanism.

The Mg2+ extrusion elicited via stimulation of the alpha 1-adrenoceptor by phenylephrine has to be reconciled with the observation that involvement of protein kinase C, which also occurs after activation of the alpha 1-adrenoceptor (4), instead induces Mg2+ accumulation in liver cells and other cell types as well. The Mg2+ accumulation induced by vasopressin, diacylglycerol analogs, or phorbol myristate derivatives vs. the Mg2+ extrusion induced by the alpha 1-adrenoceptor agonist may be explained by the activation of different protein kinase C isoforms (e.g., activation of the Ca2+-sensitive protein kinase C isoform via alpha 1-adrenoceptor), which, in turn, may dock to different receptors for activated C kinase and activate different transport mechanisms. Alternatively, or additionally, the Mg2+ extrusion elicited via the alpha 1-adrenoceptor may occur primarily through Ca2+ signaling, whereas Mg2+ entry may be essentially controlled by the protein kinase C signaling pathway. Clearly, this apparent discrepancy needs further elucidation.

Role of Extracellular Na+ in Phenylephrine-Induced Mg2+ Extrusion

Surprisingly, the data reported here indicate that the Mg2+ extrusion elicited by the alpha 1-adrenergic agonist or thapsigargin requires extracellular Na+ in addition to Ca2+. In the absence of external Na+ (Fig. 8) or under conditions in which Na+ transport is inhibited by the presence of amiloride, imipramine, or quinidine (Fig. 9), the amplitude of Mg2+ extrusion elicited by phenylephrine or thapsigargin is reduced by 70-80% compared with that occurring under normal conditions. Because the amount of 45Ca2+ entering phenylephrine-stimulated hepatocytes in the absence or presence of extracellular Na+ is not significantly different (10.5 ± 2.3 vs. 9.9 ± 2.1 nmol 45Ca2+/mg protein, respectively), it can reasonably be excluded that the reduced Mg2+ extrusion observed under these conditions is due to an altered Ca2+ signaling or transport. The most likely explanation at hand is that phenylephrine and thapsigargin induce Mg2+ extrusion across the plasma membrane by changing cellular Ca2+ content and activating a Na+-dependent mechanism via Ca2+ and calmodulin. Hence, from our data it appears that hepatocytes primarily use the Na+ gradient across the plasma membrane as the driving force for Mg2+ extrusion, relegating a Ca2+-dependent Mg2+ extrusion mechanism [the putative Ca2+/Mg2+ exchanger observed in liver plasma membrane (1, 2)] to a subsidiary role. The predominance of a Na+-dependent Mg2+ extrusion over the Ca2+-dependent Mg2+ mobilization would be consistent with the observation (2) that the Na+/Mg2+ exchanger is mainly localized in the basolateral portion of the hepatocyte membrane, a domain that accounts for ~85-90% of the hepatocyte plasma membrane vs. a mere 10-15% of the apical domain in which the Ca2+/Mg2+ exchanger appears to be localized. Therefore, the twofold difference between the amounts of accumulated Ca2+ and extruded Mg2+ measured in intact hepatocytes may indicate that cellular Mg2+ is exchanged for extracellular Ca2+ with a different ratio in intact cells, as other ions (e.g., Na+) can cross the hepatocyte plasma membrane for charge compensation. Lastly, the data reported in this study do not allow us to determine whether the extruded Mg2+ is mobilized from the endoplasmic reticulum (concomitantly to Ca2+ mobilization) or another intracellular compartment such as mitochondria or cytosol, where Mg2+ is highly represented as both free (0.7-1 mM; Refs. 3, 9, and 38) or in a complex with ATP and other phosphonucleotides (39). The different Mg2+ transport mechanisms and the potential intracellular sources from which Mg2+ can be mobilized are depicted in Fig. 10.


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Fig. 10.   Different Mg2+ extrusion mechanisms in liver cells activated by the stimulation of alpha 1- and beta -adrenergic receptors (alpha 1-AR and beta -AR, respectively). The modality of activation of the Na+- and Ca2+-dependent Mg2+ extrusion mechanisms in liver cells after the stimulation of alpha 1- and beta -adrenergic receptors and the sites of action of some of the Ca2+-signaling inhibitors used in this study (1, TMB-8; 2, BAPTA; 3, SKF-96365; 4, W-7) are depicted. Thapsigargin, which inhibits the reticular Ca2+-ATPase, is not shown. Dotted lines, the possible pools [mitochondria (Mito), endoplasmic reticulum (ER), and cytosolic ATP*Mg2+ complex] from which Mg2+ could be mobilized after alpha 1- or beta -adrenergic stimulation. It is presently unknown whether the cAMP effect is mediated through an intermediate effecting protein (?). Note that alpha 1- and beta -adrenergic receptors have been depicted in the apical and basolateral portion of the hepatocyte membrane, respectively, only for graphical reasons [i.e., to be in proximity to the Ca2+/Mg2+ exchanger (apical domain) and Na+/Mg2+ antiporter (basolateral domain) mentioned in DISCUSSION]. IP3, inositol trisphosphate; PLC, phospholipase C; DG, diacylglycerol; CaM, calmodulin.

In conclusion, the data reported here indicate that the increase in cytosolic Ca2+ elicited via stimulation of the alpha 1-adrenoceptor by phenylephrine or by administration of thapsigargin or ryanodine induces an extrusion of Mg2+ from liver cells primarily via a Ca2+-activated, Na+-dependent transport mechanism (>90%). An auxiliary Mg2+ extrusion pathway requiring extracellular Ca2+ appears to contribute to a lesser extent (<= 10%) to Mg2+ efflux. The physiological significance for such a redundancy of Mg2+ transport pathways and activating signaling mechanisms is presently undefined, although it could be explained with the necessity of modulating Mg2+ concentration in both bile (apical domain) and plasma (basolateral domain of the hepatocyte). In addition, it can be speculated that Ca2+ signaling may represent an alternative mechanism by which cellular Mg2+ can be extruded from liver cells under conditions (e.g., insulin administration; Ref. 23) in which the cAMP-signaling pathway is inhibited.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-18708, National Institute on Alcohol Abuse and Alcoholism Grant R9-AA-11593A1, and Diabetes Association of Greater Cleveland Grant 397-A-97.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Romani, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, Ohio 44106-4970 (E-mail: amr5{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 July 2000; accepted in final form 4 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Cefaratti, C, Romani A, and Scarpa A. Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver. Am J Physiol Cell Physiol 275: C995-C1008, 1998[Abstract/Free Full Text].

2.   Cefaratti, C, Romani A, and Scarpa A. Differential localization and operation of distinct Mg2+ transporters in apical and basolateral sides of rat liver plasma membrane. J Biol Chem 275: 3772-3780, 2000[Abstract/Free Full Text].

3.   Corkey, BE, Duszynsky J, Rich TL, Matschinsky B, and Williamson JR. Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria. J Biol Chem 261: 2567-2574, 1986[Abstract/Free Full Text].

4.   Exton, JH. Molecular mechanisms involved in alpha -adrenergic responses. Trends Pharmacol Sci 3: 111-115, 1982[ISI].

5.   Fabiato, A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378-417, 1988[ISI][Medline].

6.   Feray, JC, and Garay R. A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes. J Biol Chem 262: 5764-5768, 1987.

7.   Feray, JC, and Garay R. Demonstration of a Na+:Mg2+ exchange in red cells by its sensitivity to tricyclic antidepressant drugs. Naunyn Schmiedebergs Arch Pharmacol 338: 332-337, 1988[ISI][Medline].

8.   Flatman, PW. Mechanisms of magnesium transport. Annu Rev Physiol 53: 259-271, 1991[ISI][Medline].

9.   Grubbs, RD, and Maguire ME. Magnesium as a regulatory cation: criteria and evaluation. Magnesium 6: 113-127, 1987[ISI][Medline].

10.   Gunther, T. Mechanisms and regulation of Mg2+ efflux and Mg2+ influx. Miner Electrolyte Metab 19: 259-265, 1993[ISI][Medline].

11.   Gunther, T, and Hollriegl V. Na+- and anion-dependent Mg2+ influx in isolated hepatocytes. Biochim Biophys Acta 1149: 49-54, 1993[ISI][Medline].

12.   Gunther, T, and Vormann J. Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport. Biochem Biophys Res Commun 130: 540-545, 1985[ISI][Medline].

13.   Gunther, T, and Vormann J. Characterization of Na+-independent Mg2+ efflux from erythrocytes. FEBS Lett 271: 149-151, 1990[ISI][Medline].

14.   Gunther, T, and Vormann J. Activation of Na+/Mg2+ antiport in thymocytes by cAMP. FEBS Lett 297: 132-134, 1992[ISI][Medline].

15.   Gunther, T, and Vormann J. Mechanisms of beta -agonist-induced hypermagnesemia. Magnesium Bull 14: 122-125, 1992[ISI].

16.   Gunther, T, Vormann J, and Cragoe EJ, Jr. Species-specific Mn+:Mg2+ antiport from Mg2+-loaded erythrocytes. FEBS Lett 261: 47-51, 1990[ISI][Medline].

17.   Gunther, T, Vormann J, and Hollriegl V. Noradrenaline-induced Na+-dependent Mg2+ efflux from rat liver. Magnesium Bull 13: 122-124, 1991[ISI].

18.   Handy, RD, Gow IF, Ellis D, and Flatman PW. Na-dependent regulation of intracellular free magnesium concentration in isolated rat ventricular myocytes. J Mol Cell Cardiol 28: 1641-1651, 1996[ISI][Medline].

19.   Hubbard, AL, Barr VA, and Scott LJ. Hepatocyte surface polarity. In: The Liver: Biology and Pathobiology (3rd ed.), edited by Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter D, and Shafritz DA.. New York: Raven, 1994, p. 189-213.

20.   Jakob, A, Becker J, Schottli G, and Fritzsch G. alpha 1-Adrenergic stimulation causes Mg2+ release from perfused rat liver. FEBS Lett 246: 127-130, 1989[ISI][Medline].

21.   Karoor, V, Baltensperger K, Paull H, Czech MC, and Malbon CC. Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin. J Biol Chem 270: 25305-25308, 1995[Abstract/Free Full Text].

22.   Keenan, D, Romani A, and Scarpa A. Differential regulation of circulating Mg2+ in the rat by beta 1- and beta 2-adrenergic receptor stimulation. Circ Res 77: 973-983, 1995[Abstract/Free Full Text].

23.   Keenan, D, Romani A, and Scarpa A. Regulation of Mg2+ homeostasis by insulin in perfused rat livers and isolated hepatocytes. FEBS Lett 395: 241-244, 1996[ISI][Medline].

24.   Kiselyov, K, Xu X, Mozhayeva G, Kuo T, Pessah I, Mignery G, Zhu X, Birnbaumer L, and Muallem S. Functional interaction between IP3 receptors and store-operated Htrp3 channels. Nature 396: 478-482, 1998[ISI][Medline].

25.   Koss, K, Putnam RW, and Grubbs RD. Mg2+ buffering in cultured chick ventricular myocytes: quantitation and modulation by Ca2+. Am J Physiol Cell Physiol 264: C1259-C1269, 1993[Abstract/Free Full Text].

26.   Lowry, OH, Rosenbrough NJ, Farr AL, and Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

27.   Mason, MJ, Mayer B, and Hymell LJ. Inhibition of Ca2+ transport pathways in thymic lymphocytes by econazole, miconazole, and SKF 96365. Am J Physiol Cell Physiol 264: C654-C662, 1993[Abstract/Free Full Text].

28.   Matsuura, T, Kanayama Y, Inoue T, Takeda T, and Morishima I. cAMP-induced changes of intracellular free Mg2+ levels in human erythrocytes. Biochim Biophys Acta 1220: 31-36, 1993[ISI][Medline].

29.   Parekh, AB, and Penner R. Store depletion and Ca2+ influx. Physiol Rev 77: 901-930, 1997[Abstract/Free Full Text].

30.   Polimeni, PI, and Page E. Magnesium in heart muscle. Circ Res 33: 367-374, 1973[ISI][Medline].

31.   Putney, JW, Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 1-12, 1986[ISI][Medline].

32.   Romani, A, Fulceri R, Pompella A, and Benedetti A. MgATP-dependent, glucose 6-phosphate-stimulated liver microsomal Ca2+ accumulation: difference between rough and smooth microsomes. Arch Biochem Biophys 266: 1-9, 1988[ISI][Medline].

33.   Romani, A, Marfella C, and Scarpa A. Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C. FEBS Lett 296: 135-140, 1992[ISI][Medline].

34.   Romani, A, Marfella C, and Scarpa A. Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes. Circ Res 72: 1139-1148, 1993[Abstract].

35.   Romani, A, Marfella C, and Scarpa A. Hormonal stimulation of Mg2+ uptake in hepatocytes. J Biol Chem 268: 15489-15495, 1993[Abstract/Free Full Text].

36.   Romani, A, and Scarpa A. Hormonal control of Mg2+ in the heart. Nature 346: 841-844, 1990[ISI][Medline].

37.   Romani, A, and Scarpa A. Norepinephrine evokes a marked Mg2+ efflux from liver cells. FEBS Lett 269: 37-40, 1990[ISI][Medline].

38.   Romani, A, and Scarpa A. Regulation of cell magnesium. Arch Biochem Biophys 298: 1-12, 1992[ISI][Medline].

39.   Scarpa, A, and Brinley F. In situ measurements of free cytosolic magnesium ions. Fed Proc 40: 2646-2652, 1981[ISI][Medline].

40.   Schatzmann, HJ. Asymmetry of the magnesium sodium exchange across the human red cell membrane. Biochim Biophys Acta 1148: 15-18, 1993[ISI][Medline].

41.   Seglen, PO. Preparation of isolated rat liver cells. Methods Cell Biol 13: 29-83, 1976[Medline].

42.   Smoake, JA, Moy G-MM, Fang B, and Solomon SS. Calmodulin-dependent cyclic AMP phosphodiesterase in liver plasma membranes: stimulated by insulin. Arch Biochem Biophys 323: 223-232, 1995[ISI][Medline].

43.   Streb, H, Irvine RF, Berridge MJ, and Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 306: 67-69, 1983[ISI][Medline].

44.   Tashiro, M, and Konishi M. Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum. Biophys J 73: 3371-3384, 1997[Abstract].

45.   Tessman, PA, and Romani A. Acute effect of EtOH on Mg2+ homeostasis in liver cells: evidence for the activation of an Na+/Mg2+ exchanger. Am J Physiol Gastrointest Liver Physiol 275: G1106-G1116, 1998[Abstract/Free Full Text].

46.   Vormann, J, and Gunther T. Amiloride-sensitive net Mg2+ efflux from isolated perfused rat hearts. Magnesium 6: 220-224, 1987[ISI][Medline].

47.   Wolf, FI, Di Francesco A, Covacci V, and Cittadini A. Regulation of magnesium efflux from rat spleen lymphocytes. Arch Biochem Biophys 344: 397-403, 1997[ISI][Medline].

48.   Zhang, GH, and Melvin JE. Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini. J Biol Chem 267: 20721-20727, 1992[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 280(6):G1145-G1156
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