Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The stimulation of the
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
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+.
1-adrenergic receptor; magnesium homeostasis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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
-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 1-adrenoceptor induces
Mg2+ extrusion via a cAMP-independent process.
The stimulation of 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
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
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
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
-adrenergic receptor.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Collagenase type Cls-2 (235-280 U/mg) was from Worthington (Freehold, NJ). SKF-96365, (+)BAY-K8644 and (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.
|
|
|
|
|
|
|
|
|
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 -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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 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
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 cells1 · 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
1-agonist but rather decreased
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
cells1 · 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
-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).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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
-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
- and
1-adrenergic-mediated signaling pathways, respectively
(23). The ability of phenylephrine to induce a
Mg2+ extrusion from liver cells via
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
1-adrenoceptor.
Activation of Mg2+ Extrusion Pathway by Phenylephrine
The stimulation of theA 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
1-adrenoceptor by phenylephrine has to be reconciled
with the observation that involvement of protein kinase C, which also
occurs after activation of the
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
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
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
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
|
In conclusion, the data reported here indicate that the
increase in cytosolic Ca2+ elicited via stimulation of the
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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
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
4.
Exton, JH.
Molecular mechanisms involved in -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 -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.
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
22.
Keenan, D,
Romani A,
and
Scarpa A.
Differential regulation of circulating Mg2+ in the rat by 1- and
2-adrenergic receptor stimulation.
Circ Res
77:
973-983,
1995
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
26.
Lowry, OH,
Rosenbrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275,
1951
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
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
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
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
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