Activation of Na+- and Ca2+-dependent Mg2+ extrusion by alpha 1- and beta -adrenergic agonists in rat liver cells

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 administration of selective alpha 1 (phenylephrine)-, beta  (isoproterenol)-, or mixed (epinephrine) adrenergic agonists induces a marked Mg2+ extrusion from perfused rat livers. In the absence of extracellular Ca2+, phenylephrine does not induce a detectable Mg2+ extrusion, isoproterenol-induced Mg2+ mobilization is unaffected, and epinephrine induces a net Mg2+ extrusion that is lower than in the presence of extracellular Ca2+ and quantitatively similar to that elicited by isoproterenol. In the absence of extracellular Na+, no Mg2+ is extruded from the liver irrespective of the agonist used. Similar results are observed in perfused livers stimulated by glucagon or 8-chloroadenosine 3',5'-cyclic monophosphate. In the absence of extracellular Na+ or Ca2+, adrenergic-induced glucose extrusion from the liver is also markedly decreased. Together, these results indicate that liver cells extrude Mg2+ primarily via a Na+-dependent mechanism. This extrusion pathway can be activated by the increase in cellular cAMP that follows the stimulation by glucagon or a specific beta -adrenergic receptor agonist or, alternatively, by the changes in cellular Ca2+ induced by the stimulation of the alpha 1-adrenoceptor. In addition, the stimulation of the alpha 1-adrenoceptor appears to activate an auxiliary Ca2+-dependent Mg2+ extrusion pathway. Finally, our data suggest that experimental conditions that affect Mg2+ mobilization also interfere with glucose extrusion from liver cells.

alpha 1-adrenoceptor; beta -adrenoceptor; hepatocyte; glucose


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IN THE LAST DECADE OUR UNDERSTANDING of cellular Mg2+ homeostasis and its role in regulating cytosolic or membrane-bound enzymes (6, 8, 35), ion channels (8, 35), and metabolic cycles (25, 42) has improved significantly. The concept that cellular Mg2+ content remains relatively constant under varying metabolic conditions has been largely reevaluated because several experimental reports indicate that major fluxes of Mg2+ can cross the plasma membrane of mammalian cells in either direction within minutes of the application of various hormonal or metabolic stimuli (see Refs. 10, 29, and 35 for review).

Despite increasing evidence for the operation of Mg2+ transporters in the cell membrane, the structure and nature of the transporters remain to be elucidated. The data available in the literature support the idea that two distinct Mg2+ transport mechanisms operate in the plasma membrane of mammalian cells. A Na+-dependent mechanism, tentatively identified as a Na+/Mg2+ exchanger (7, 13), likely represents the most abundant Mg2+ extrusion pathway in cardiac myocytes (30, 41), hepatocytes (18, 31), thymocytes (15), and other cell types as well. This exchanger specifically requires Na+ as counterion for Mg2+ extrusion (11, 13, 30, 31) and appears to operate in either direction based on the ion gradient across the cell membrane (11, 30, 31). Under conditions in which the operation of this exchanger is prevented by the absence of extracellular Na+ (30, 31) or by the presence of the Na+ transport inhibitors amiloride (18, 41) or imipramine (5), Mg2+ is extruded via an alternative pathway identified as a Na+-independent mechanism (14). The extrusion of Mg2+ via this second pathway appears to utilize divalent cations such as Ca2+ (30, 31) or Mn2+ (4, 17) or anions such as HCO3- or Cl- (12). Recently, this laboratory (1, 2) provided further evidence for the operation of distinct Na+- and Ca2+-dependent Mg2+ extrusion mechanisms, located in the basolateral domain and apical portion, respectively, of the hepatocyte plasma membrane.

However, whether the Na+-dependent and -independent Mg2+ extrusion mechanisms operate concomitantly or alternatively under different stimulatory conditions remains undefined. Activation of beta -adrenergic receptors by isoproterenol (Iso) (21, 41), epinephrine (Epi) (22), or norepinephrine (18, 30, 31, 34) in isolated cardiac (30, 33, 41) or liver (18, 31, 34) cells, perfused heart (33, 41) or liver (18, 31, 34), or anesthetized animals (15, 21) results in a marked mobilization of cellular Mg2+ into the extracellular compartment and ultimately into the bloodstream via a Na+/Mg2+ exchanger. The stimulatory effect of cell-permeant cAMP analogs or forskolin (18, 30, 33, 34) and the inhibitory effect of Rp-cAMP (43) strongly support the idea that the Na+/Mg2+ exchanger becomes active after phosphorylation by cAMP (15). In contrast, relatively little is known about hormonal activation of the Na+-independent mechanism. Our laboratory reported previously that a physiological extracellular Ca2+ concentration ([Ca2+]o) is required to observe a Mg2+ extrusion from cardiac (30) or liver cells (31) stimulated by norepinephrine, which supports the idea that the Na+-independent transport mechanism is involved, to some extent, in hormonal mobilization of cellular Mg2+. Furthermore, Jakob et al. (20) and, more recently, Keenan et al. (22) provided evidence for a role of the alpha 1-adrenergic receptor in mediating an extrusion of Mg2+ from liver cells after phenylephrine (Phe) administration through an uncharacterized transport mechanism.

Hence, in the present study we investigated the possibility that alpha 1-adrenoceptor-induced Mg2+ extrusion specifically occurs via the Na+-independent (Ca2+-dependent) mechanism. The results reported here indicate for the first time that the stimulation of the alpha 1-adrenoceptor results in an extrusion of Mg2+ from liver cells primarily via a Na+-dependent pathway and only marginally via a Ca2+-dependent mechanism. By contrast, the specific stimulation of the beta -adrenergic receptor results in the selective activation of a Na+-dependent Mg2+ extrusion mechanism. The administration of a mixed adrenergic agonist (e.g., Epi) results in a Mg2+ extrusion that is larger than that attained by the administration of a selective alpha 1- or beta -adrenergic agonist alone.


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Chemicals. Collagenase (CLS-2) was from Worthington (Lakewood, NJ). Enzymatic kits for glucose and lactate dehydrogenase (LDH) determinations in the perfusate and all other reagents were from Sigma (St. Louis, MO).

Perfused livers. Fed male Sprague-Dawley rats (250-300 g body wt) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The abdomen was opened, and the liver was perfused via the portal vein with a medium containing (mM) 120 NaCl, 3 KCl, 1.2 CaCl2, 12 NaHCO3, 1.2 KH2PO4, 15 glucose, and 10 HEPES, pH 7.2 at 37°C, equilibrated with an O2-CO2 (95:5 vol/vol) gas mixture (regular perfusion medium). The liver was quickly removed from the abdomen, placed on a platform for a 20-min washout period, and perfused at a flow rate of 3.5-4 ml · g-1 · min-1. Aliquots of the perfusate were collected at 30-s intervals, and Mg2+ content was measured by atomic absorbance spectroscopy (AAS) in a Perkin-Elmer 3100 atomic absorbance flame spectrophotometer. The first 10 min after the washout period provided a baseline for subsequent adrenergic agent addition. Phe (5 µM), Iso (10 µM), or Epi (5 µM) was dissolved directly into the perfusion medium. The concentration of Mg2+ present as contaminant in the buffer was measured by AAS and found to be <= 3 µM. For simplicity, this value was not subtracted from the curves of efflux reported in the figures.

Na+- or Ca2+-free medium. To determine the dependence of Mg2+ extrusion on the presence of extracellular Na+ and Ca2+, livers were perfused with a medium similar to the perfusion medium described in Perfused livers but devoid of Na+ (NaCl and NaHCO3 were replaced with equiosmolar concentrations of choline chloride and KHCO3, respectively, pH 7.4 with KOH) or Ca2+ (CaCl2 omitted from the buffer). To exclude that a reduced Mg2+ extrusion in the absence of extracellular Na+ or Ca2+ could be ascribed to an altered sensitivity of an alpha 1- or beta -adrenergic receptor, pharmacological doses of adrenergic agonists were used under all experimental conditions.

Estimation of total Mg2+ extrusion. To estimate the total amount of Mg2+ extruded in the perfusate, the Mg2+ concentrations of the last five points before the addition of adrenergic agonist were averaged and then subtracted from each of the subsequent time points under the curve of efflux. The net Mg2+ concentration (nmol/ml) was then expressed as micromoles, taking into account perfusion rate and collection intervals (37).

Glucose and LDH determination. Aliquots of perfusate were collected every minute, and glucose content was determined with an enzymatic kit (Sigma) monitoring the variations in NADH+ content at 340 nm. The absence of cell damage was assessed by measuring LDH release in aliquots of the perfusate with an NADH+-coupled enzymatic kit (Sigma).

Collagenase-dispersed cells. Collagenase-dispersed rat hepatocytes were isolated according to the procedure of Seglen (36). After isolation, hepatocytes were resuspended (final concentration 1 × 106 cells/ml) in a medium containing (mM) 120 NaCl, 3 KCl, 1.2 KH2PO4, 12 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH 7.2, at 37°C under O2-CO2 (95:5 vol/vol) flow and kept at room temperature until used. Cell viability was 88 ± 3% (n = 5) as assessed by trypan blue exclusion test and did not significantly change over the course of 3-4 h (85 ± 2%, n = 5).

Mg2+ determination in cell suspensions. To determine Mg2+ extrusion, 1 ml of cell suspension was transferred in a microfuge tube, and the cells were rapidly sedimented at 600 g for 30 s. After the supernatant was removed, the cells were washed with 1 ml of a medium having a composition similar to that mentioned in Collagenase-dispersed cells but devoid of Mg2+ (incubation medium). The concentration of Mg2+ present as contaminant was measured by AAS and found to be <= 3 µM. After the washing, the cells were transferred to 10 ml of incubation medium, prewarmed at 37°C, and incubated under continuous stirring and O2-CO2 flow. After 3 min of equilibration, the reported doses of adrenergic agonist were added to the incubation system. Ca2+ channel blockers or Na+ transport or glucose transport inhibitors were added to the incubation system together with the cells. At the time points indicated in Figs. 5A and 6A, 700-µl aliquots of the incubation mixture were withdrawn in duplicate and the cells were sedimented in microfuge tubes (3,500 g × 30 s). The supernatants were removed, and the Mg2+ content was measured by AAS. For the experiments in the absence of Na+ or Ca2+, isolated hepatocytes were washed and incubated in the Na+- or Ca2+-free buffers. The net Mg2+ extrusion was estimated as follows. The Mg2+ content in the supernatant at the two time points before adrenergic agonist administration was averaged and then subtracted from each of the subsequent time points. The net Mg2+ content in the supernatant (nmol/mg protein) at the latest time point of incubation (time = 6 min after agonist addition) was then plotted in Figs. 5B, 6B, and 7.

Other procedures. Protein content was measured using the procedure of Lowry et al. (24).

Statistical analysis. Data are 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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Rat livers perfused with regular medium in the absence of any stimulatory agent maintained a stable Mg2+ baseline in the perfusate over 35 min of perfusion (Fig. 1A). The administration of the selective beta -adrenergic agonist Iso (10 µM), the alpha 1-adrenergic agonist Phe (5 µM), or Epi (5 µM), an endogenous catecholamine that stimulates both alpha - and beta -adrenergic receptors, resulted in a significant extrusion of cellular Mg2+ into the perfusate (Fig. 1A). The Mg2+ extrusion was detectable within 1-2 min of the agonist addition and became maximal by 8 min before returning toward baseline despite the persistence of the agonist in the perfusion medium. The Mg2+ extrusion was not associated with LDH release into the perfusate (not shown), thus excluding that the process depended on a nonspecific alteration of cell membrane integrity. The total amount of Mg2+ released into the perfusate, estimated as described in MATERIALS AND METHODS, accounted for ~1.09, 1.42, and 2.64 µmol of Mg2+ after Iso, Phe, or Epi administration, respectively (Fig. 1B). These values correspond to 2.8, 3.6, and 6.8% of total liver Mg2+ content, respectively.


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Fig. 1.   Time course (A) and total amount (B) of Mg2+ extruded from perfused rat livers stimulated by different adrenergic agonists under varying experimental conditions. Rat livers were perfused with a regular medium. After a 25-min washout period, aliquots of the perfusion medium were collected every 30 s to establish baseline. After 10 min, 5 µM phenylephrine (Phe), 5 µM epinephrine (Epi), or 10 µM isoproterenol (Iso) was infused for 10 min (A). Data were collected every 30 s but are reported at 150-s intervals for clarity. In B, the net amount of Mg2+ extruded from the livers into the perfusate under varying experimental conditions is shown. The net Mg2+ extrusion was calculated as reported in MATERIALS AND METHODS. The data are means ± SE of 9 different experiments for all experimental conditions. *Statistically significant vs. control (Ctl), Iso, Phe (P < 0.05). #Statistically significant vs. control only. &Statistically significant vs. Iso and control.

Previous reports from this laboratory (30, 31) and other laboratories (18, 41) indicate that the stimulation of beta -adrenergic receptor by catecholamine or Iso via increase in the cytosolic cAMP level induces an extrusion of Mg2+ from cardiac and liver cells through the operation of an Na+/Mg2+ exchanger (7, 13, 18, 30, 31, 41). Under conditions in which the operation of this transport pathway is inhibited by amiloride (41) or imipramine (5) or by the removal of extracellular Na+ (30, 31), Mg2+ is extruded through a Na+-independent (Ca2+-dependent) mechanism (14, 30, 31). However, the modality of activation of this alternative mechanism is still undefined.

To investigate the possibility that the Mg2+ extrusion induced by Phe occurs via activation of the Na+-independent (Ca2+-mediated) pathway, livers were perfused with a Ca2+-free medium. The results, reported in Fig. 2A, indicate that, in the absence of a physiological [Ca2+]o, Phe was ineffective at inducing a Mg2+ extrusion from perfused livers (inhibition ~90%). In contrast, under the same experimental conditions, the Mg2+ extrusion induced by Iso was unaffected (Fig. 2A), whereas that prompted by Epi was inhibited ~60%. The estimation of the net amount of Mg2+ extruded by the liver after Iso or Epi administration in the absence of extracellular Ca2+ accounted for ~1 and 0.9 µmol Mg2+, respectively.


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Fig. 2.   Mg2+ extrusion from perfused rat liver stimulated by different adrenergic agonists in the absence of extracellular Ca2+ (A) or Na+ (B). Rat livers were perfused with a medium devoid of extracellular Ca2+ or Na+ (see MATERIALS AND METHODS). After a 25-min washout period, aliquots of the perfusion medium were collected every 30 s to establish baseline. After 10 min, 5 µM Phe, 5 µM Epi, or 10 µM Iso was infused for 10 min. Data were collected every 30 s but are reported at 150-s intervals for clarity. The data are means ± SE of 9 different experiments for all experimental conditions. *Statistically significant vs. control.

When similar perfusion experiments were carried out with a medium devoid of extracellular Na+, no Mg2+ extrusion from liver cells was observed (Fig. 2B) regardless of the adrenergic agonist and the dose used. Qualitatively similar results were also obtained when 0.1 mM imipramine was used as an inhibitor of Na+ transport. The administration of imipramine to livers perfused in the presence of physiological extracellular Na+ concentration ([Na+]o) and [Ca2+]o 5 min before the addition of 5 µM Phe resulted in a 60% inhibition of Phe-induced Mg2+ extrusion (net Mg2+ extrusion was ~0.60 vs. 1.88 µmol). A similar inhibitory effect was also observed in livers pretreated with imipramine and stimulated by 10 µM Iso or 5 µM Epi (not shown). Under these experimental conditions, doses of Phe or other adrenergic agonists larger than those reported here were ineffective at eliciting a Mg2+ extrusion (not shown). A similar inhibitory effect was also exerted by 500 µM amiloride (not shown), another agent able to significantly reduce Mg2+ extrusion from liver cells (37).

It is well documented that the stimulation of both alpha 1- and beta -adrenergic receptors results in activation of glycogenolysis (26) and glucose extrusion from liver cells (39). We recently reported (32) that insulin-stimulated Mg2+ accumulation in cardiac cells appears to be associated with and dependent on glucose transport. To investigate whether a similar association also exists for glucose and Mg2+ extrusion from liver cells and to determine whether changes in extracellular ion composition affect glucose extrusion in addition to Mg2+ mobilization, the amount of glucose extruded from the liver into the perfusate after adrenergic agonist administration was measured enzymatically. Figure 3 shows that, in the absence of physiological [Na+]o or [Ca2+]o in the perfusion medium, the administration of Phe, Iso, or Epi resulted in the extrusion of a negligible amount of hepatic glucose into the perfusate compared with the amount mobilized from stimulated livers perfused with a medium containing physiological [Na+]o or [Ca2+]o. Glucose output was also reduced by ~50% in livers pretreated with imipramine and stimulated by Phe (13.9 ± 1.8 vs. 27 ± 2 µmol glucose/ml, n = 4; P < 0.05) or other adrenergic agonists (not shown).


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Fig. 3.   Total glucose extrusion from perfused rat livers stimulated by different adrenergic agonists under varying experimental conditions. The net amount of glucose extruded from the livers was calculated as described in MATERIALS AND METHODS. Briefly, the last 5 time points before adrenergic agonist addition were averaged and subtracted from the time points during which the adrenergic agonist was present in the perfusion system. The amount of glucose extruded from the organ into the perfusate was then calculated, taking into account the perfusion rate and the sampling time. The data are means ± SE of 9 different experiments for all experimental conditions. All values obtained in the absence of extracellular Ca2+ or Na+ were significant vs. those obtained with regular perfusion medium; symbols of statistical significance are omitted for simplicity.

To exclude that changes in extracellular ion composition could affect the responsiveness of the beta -adrenergic receptor, rat livers were perfused with a Na+- or a Ca2+-free medium and stimulated by the addition of glucagon (30 nM) or 8-chloroadenosine 3',5'-cyclic monophosphate (8-chloro-cAMP; 250 µM). We reported previously (31, 34) that this cell-permeant cAMP analog induces an extrusion of Mg2+ from liver cells comparable to that elicited by beta -adrenergic agonists. The administration of either of these two agents resulted in an extrusion of Mg2+ that accounted for >= 60% of that induced by Iso (Fig. 4A). This extrusion was unaffected by the removal of Ca2+ from the perfusion medium but was completely prevented in the absence of extracellular Na+ (Fig. 4A). The amplitude of glucose extrusion was also affected by Na+ but not by Ca2+ removal (Fig. 4B).


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Fig. 4.   Total amount of Mg2+ (A) and glucose (B) extruded from perfused rat livers stimulated by glucagon and cAMP under varying experimental conditions. Rat livers were perfused with the media described in Figs. 1 and 2 and stimulated by addition of 30 nM glucagon or 250 µM 8-chloroadenosine 3',5'-cyclic monophosphate (8-chloro-cAMP). The results obtained with 10 µM Iso, already shown in Figs. 1B and 3, are reported for comparison. Net Mg2+ and glucose extrusions are reported as previously described. Data are means ± SE of 9 experiments for isoproterenol and 5 experiments for glucagon or 8-chloro-cAMP under the different experimental conditions. All values obtained in the absence of extracellular Ca2+ or Na+ were significant vs. those obtained with regular perfusion medium; symbols of statistical significance are omitted for simplicity.

Qualitatively similar results were also obtained in collagenase-dispersed hepatocytes. When incubated in the presence of extracellular Na+ or Ca2+, nonstimulated hepatocytes retained cellular Mg2+ for several minutes (Fig. 5A). The addition of 10 µM Iso, 5 µM Epi, or 5 µM Phe resulted in a time-dependent extrusion of Mg2+ from the cells into the extracellular compartment, which reached the maximum (net Mg2+ extrusion approx  1.5 nmol/mg protein) within 6 min of the agonist addition (time = 8 min in Fig. 5A). Therefore, for simplicity, the following data are expressed as the net amount of Mg2+ released from the hepatocytes at 6 min after agonist addition. In the absence of extracellular Ca2+ (Fig. 5B), the Mg2+ extrusion elicited by Iso, Epi, or Phe was inhibited by ~60%, regardless of the agonist used. When similar experiments were performed in hepatocytes incubated in Na+-free medium, the Mg2+ extrusion elicited by Iso was completely abolished and that induced by Phe or Epi was reduced by ~80% (Fig. 5B).


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Fig. 5.   Time course (A) and net Mg2+ extrusion (B) from collagenase-dispersed hepatocytes stimulated by different adrenergic agonists in vitro under varying experimental conditions. Collagenase-dispersed hepatocytes were incubated in Mg2+-free medium, and the amount of Mg2+ extruded into the extracellular compartment was determined as described in MATERIALS AND METHODS. After a few minutes of equilibration, 10 µM isoproterenol, 5 µM phenylephrine, or 5 µM epinephrine was added to the incubation mixture (A). The total amount of Mg2+ extruded from the cells after 6-min stimulation with different agonists under varying experimental conditions (time = 8 min in A) was then plotted as the net amount of Mg2+ (B). The data are means ± SE of 6 different cell preparations, each performed in quadruplicate, for all experimental conditions. *Statistically significant vs. control sample.

Isolated hepatocytes were also used to evaluate the efficacy of the glucose transport inhibitor phloretin at inhibiting adrenergic-induced Mg2+ extrusion. When 15 µM phloretin was used to block glucose transport, the amplitude of Iso-induced Mg2+ extrusion was reduced by ~50% (Fig. 6; P < 0.05). The presence of 15 µM phloretin in the incubation medium also reduced the amplitude of Phe- or Epi-induced Mg2+ extrusion to a comparable extent (Fig. 6B). A similar percent inhibition of Mg2+ and glucose extrusion was observed in perfused livers stimulated by adrenergic agonist in the presence of 15 µM phloretin in the perfusion medium (not shown).


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Fig. 6.   Time course (A) and net Mg2+ extrusion (B) from collagenase-dispersed hepatocytes stimulated by different adrenergic agonists in vitro in the presence of phloretin. Collagenase-dispersed hepatocytes were incubated in Mg2+-free medium in the presence or absence of 15 µM phloretin (Phlo) as a glucose transport inhibitor. Phloretin was added together with the hepatocytes. The amount of Mg2+ extruded into the extracellular compartment was determined as described in MATERIALS AND METHODS. After a few minutes of equilibration, 10 µM isoproterenol, 5 µM phenylephrine, or 5 µM epinephrine was added to the incubation mixture (A). The total amount of Mg2+ extruded from the cells after 6-min stimulation with the different agonists in the presence of the glucose transport inhibitor was then plotted as the net amount of Mg2+ (B). The data are means ± SE of 4 different cell preparations, each performed in triplicate, for all experimental conditions. *Statistically significant vs. control and phloretin alone. #Statistically significant vs. all other experimental samples.

Consistent with the results reported in Fig. 4, the addition of 250 µM 8-chloro-cAMP to suspensions of isolated hepatocytes elicited a Mg2+ extrusion comparable to that induced by isoproterenol (compare Fig. 7 with Fig. 5). Also in this experimental model, the cAMP-induced Mg2+ extrusion was not affected by the removal of extracellular Ca2+ but was significantly reduced in the absence of extracellular Na+ or in the presence of 15 µM phloretin (Fig. 7).


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Fig. 7.   Net Mg2+ extrusion from collagenase-dispersed hepatocytes stimulated by 8-chloro-cAMP in vitro under varying experimental conditions. Collagenase-dispersed hepatocytes were incubated in Mg2+-free medium in the presence or absence of 15 µM phloretin or in a medium devoid of extracellular Ca2+ or Na+. When used, phloretin was added together with the hepatocytes. The amount of Mg2+ extruded into the extracellular compartment was determined as described in MATERIALS AND METHODS. After a few minutes of equilibration, 250 µM 8-chloro-cAMP was added to the incubation mixture. The total amount of Mg2+ extruded from the cells after 6-min stimulation under the varying experimental conditions was then plotted as the net amount of Mg2+. The data are means ± SE of 4 different cell preparations, each performed in triplicate, for all experimental conditions. *Statistically significant vs. cells incubated in regular medium.


    DISCUSSION
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In recent years, our laboratory (30, 31) and other laboratories (15, 18, 41) have provided compelling evidence for the operation of a Na+-dependent Mg2+ extrusion mechanism in several tissues, including liver (18, 31), after beta -adrenergic stimulation. Although the transport mechanism has not been structurally characterized, experimental results suggest that this Mg2+ is extruded primarily through an Na+/Mg2+ exchanger, most likely activated via phosphorylation (15) by the increase in cellular cAMP level that follows the stimulation of beta -adrenergic receptor (28, 31, 34) or the administration of cell-permeant cAMP analogs or forskolin (28, 31, 34). Under conditions in which the operation of the exchanger is prevented by the absence of extracellular Na+ or by agents that block Na+ transport, namely amiloride (41) or imipramine (5), Mg2+ extrusion can still occur via a Na+-independent transport mechanism. The nature of this alternative transporter is not fully characterized, because either cations such as Ca2+ (30, 31) or Mn2+ (4, 17) or anions such as HCO3- or Cl- (12) have been reported to favor Mg2+ extrusion. Recently, our laboratory reported (1) that two distinct Mg2+ transport mechanisms operate in purified rat liver plasma membranes. On the basis of their characteristics, these transporters have tentatively been identified as a Na+/Mg2+ and a Ca2+/Mg2+ exchanger, respectively (1). These transporters appear to be specifically localized in the basolateral (the Na+/Mg2+ exchanger) and apical (the Ca2+/Mg2+ exchanger) domains of the hepatocyte plasma membrane (2).

The present study was aimed at elucidating the possibility that the stimulation of the alpha 1-adrenergic receptor, which also induces Mg2+ extrusion from liver cells (20, 22), specifically activates the Na+-independent (Ca2+-dependent) transport mechanism. The obtained results indicate that this is not the case, in that alpha 1-adrenergic stimulation mobilizes cellular Mg2+ primarily via the Na+-dependent mechanism and only marginally via the Ca2+-dependent pathway.

Role of extracellular Na+ and Ca2+ on Mg2+ extrusion. Perfused livers respond to the stimulation of alpha 1- and/or beta -adrenergic receptors by extruding a sizable amount of cellular Mg2+ into the perfusate, provided that a physiological concentration of Na+ or Ca2+ is present extracellularly. Some significant differences are observed. The Mg2+ extrusion elicited via alpha 1-adrenoceptor by Phe requires both extracellular Na+ and Ca2+ to occur. In the absence of either of these two cations, Mg2+ extrusion is reduced to a mere 10% in perfused organs and to <50% in isolated hepatocytes. By contrast, Iso-induced Mg2+ extrusion via beta -adrenoceptor only requires extracellular Na+, the absence of external Ca2+ being ineffective at preventing Mg2+ mobilization. The mixed adrenergic agonist Epi also requires both extracellular Na+ and Ca2+ to elicit maximal Mg2+ extrusion. However, Epi can still induce a sizable Mg2+ extrusion in the absence of extracellular Ca2+, the amplitude of which is comparable to that elicited by Iso. These results would suggest that the stimulation of alpha 1- or beta -adrenergic receptors ultimately results in the activation of a Na+-dependent Mg2+ extrusion mechanism (most likely the Na+/Mg2+ exchanger) via changes in cellular Ca2+ signaling (3) or increase in cellular cAMP (15, 18, 30, 31), respectively. Although the effect of cAMP is likely mediated via phosphorylation (15), whether the effect of Ca2+ is mediated via calmodulin is not yet defined.

Presently, we do not have a clear explanation for the discrepant percent inhibition of Mg2+ extrusion after Phe or Iso administration in perfused livers vs. isolated hepatocytes incubated in the absence of physiological [Ca2+]o or [Na+]o. (Fig. 1B vs. Fig. 5B). Most likely, this discrepancy depends on a drastic change in cell-cell connection after collagenase digestion. The apical domain, which appears to specifically possess the Ca2+-dependent Mg2+ extrusion mechanism (2), accounts for ~10% of the total hepatocyte plasma membrane in situ (19) compared with ~90% of the basolateral domain, where the Na+/Mg2+ exchanger is localized (2). The percent expression of these transporters would be consistent with the percent inhibition of Mg2+ extrusion observed in perfused livers in the absence of extracellular Na+ or Ca2+. In contrast, the percent distribution of these transporters drastically changes in collagenase-dispersed hepatocytes, which now entirely expose their plasma membrane surface. Furthermore, the discovery of the Ca2+/Mg2+ exchanger is too recent for us to propose a physiological role for this mechanism. The nonreversibility of this transporter and its localization in the apical domain of the hepatocyte cell membrane (2), facing the bile space, may suggest that the Ca2+/Mg2+ exchanger is important to maintain physiological concentrations of Ca2+ and Mg2+ in the bile.

From our observations, two main conclusions can be drawn. The first conclusion is that a redundancy of adrenergic-activated Mg2+ transport pathways is present in liver cells. Not only can these Mg2+ transport mechanisms be activated by specific alpha 1- or beta -adrenergic agonists but they also appear to be concomitantly stimulated by the mixed adrenergic agonist Epi, at least in perfused livers. Figure 1B shows that the net amount of Mg2+ mobilized from the liver by Epi is equivalent to the sum of Mg2+ amounts mobilized by Iso and Phe. The second conclusion is that in perfused livers the transmembrane Na+ gradient is the main driving force for Mg2+ extrusion to occur, irrespective of the adrenergic signaling pathway activated, whereas Ca2+ is primarily used to activate the Na+-dependent Mg2+ extrusion pathway and only partially to support a subsidiary Ca2+-dependent Mg2+ transport mechanism.

Concomitance of Mg2+ and glucose extrusion. Despite the large number of reports indicating that adrenergic agonist administration to anesthetized animals (16, 21), perfused organs (18, 30, 31, 41), or isolated cells (30, 31) results in an extrusion of Mg2+ into the extracellular compartment or in the circulation, the physiological significance of this phenomenon remains elusive.

We recently reported (32) that the administration of insulin to perfused rat hearts or isolated cardiac myocytes results in a parallel accumulation of glucose and Mg2+ in the cells. The stimulation of alpha 1- and beta -adrenergic receptors in liver cells results in the activation of glycogenolysis and in glucose extrusion (26, 39) through the glucose transporter GLUT-2 (9) and a subsidiary undefined mechanism (9). Thus we decided to evaluate whether an association between Mg2+ and glucose also existed for the extrusion. The data reported here indicate that in the absence of extracellular Na+ or Ca2+, i.e., conditions under which adrenergic-induced Mg2+ extrusion from the liver is decreased to a varying extent, glucose extrusion is also decreased, although not in a consistent and corresponding manner. In fact, a marked inhibition of both glucose and Mg2+ extrusion after administration of different adrenergic agonists, glucagon, and cAMP is observed under conditions in which no extracellular Na+ is present (Figs. 1B, 3, and 4). In the absence of extracellular Ca2+, however, a major difference is observable in liver cells stimulated by adrenergic agonists (Figs. 1B and 3) or by glucagon or cAMP (Fig. 4). Glucagon and cAMP, in fact, elicit a comparable extrusion of glucose and Mg2+ both in the presence and in the absence of extracellular Ca2+. In contrast, Iso and Epi elicit a negligible glucose extrusion from liver cells despite their ability to mobilize cellular Mg2+ even in a Ca2+-free medium. The reason for this discrepancy is not apparent. Whether it can be attributed to the activation of a beta 2-adrenergic-mediated signaling pathway other than cAMP, as suggested by several experimental reports (27, 44), is a topic for future investigations. An inhibitory effect on both adrenergic-induced Mg2+ and glucose extrusion is also exerted by the glucose transport inhibitor phloretin. The inhibitory effect of phloretin on glucose extrusion can be explained by the binding of the agent to the extracellular site of the glucose transporter (40) such as GLUT-2 in liver cells. However, because phloretin also affects several other cellular functions and transporters (23, 38), whether this agent blocks the Mg2+ extrusion pathway directly or indirectly remains undetermined.

In conclusion, our data indicate that the stimulation of alpha 1- and beta -adrenergic receptors by selective or mixed agonists results in an extrusion of Mg2+ from liver cells. The Mg2+ extrusion predominantly occurs via a Na+-dependent mechanism, activated by changes in cytosolic cAMP (beta -adrenoceptor signaling) and Ca2+ (alpha 1-adrenoceptor stimulation), and marginally via a Ca2+-dependent pathway. Finally, changes in extracellular cation composition appear to affect both Mg2+ and glucose extrusion from livers stimulated by adrenergic agonists.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-18708, National Institute of Alcohol Abuse and Alcoholism Grant AA-R9AA11593A1, 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, OH 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 3 December 1999; accepted in final form 1 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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