-Adrenergic agonists stimulate Mg2+ uptake in
mouse distal convoluted tubule cells
Hyung Sub
Kang,
Dirk
Kerstan,
Long-Jun
Dai,
Gordon
Ritchie, and
Gary A.
Quamme
Department of Medicine, University of British Columbia, Vancouver
Hospital and Health Sciences Centre, Vancouver, British Columbia,
Canada V6T 1Z3
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ABSTRACT |
-Adrenergic agonists
influence electrolyte reabsorption in the proximal tubule, loop of
Henle, and distal tubule. Although isoproterenol enhances magnesium
absorption in the thick ascending limb, it is unclear what effect, if
any,
-adrenergic agonists have on tubular magnesium handling. The
effects of isoproterenol were studied in immortalized mouse distal
convoluted tubule (MDCT) cells by measuring cellular cAMP formation
with radioimmunoassays and Mg2+ uptake with fluorescence
techniques. Intracellular free Mg2+ concentration
([Mg2+]i) was measured in single MDCT cells
by using microfluorescence with mag-fura-2. To assess Mg2+
uptake, MDCT cells were first Mg2+ depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and
then placed in 1.5 mM MgCl2, and the changes in
[Mg2+]i were determined.
[Mg2+]i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate,
d([Mg2+]i)/dt, of 168 ± 11 nM/s. Isoproterenol stimulated Mg2+ entry in a
concentration-dependent manner, with a maximal response of 252 ± 11 nM/s, at a concentration of 10
7 M, that
represented a 50 ± 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg2+ uptake was
completely inhibited with RpcAMPS, a protein kinase A inhibitor, and
U-73122, a phospholipase C inhibitor, and partially blocked by RO
31-822, a protein kinase C inhibitor. Accordingly, isoproterenol-mediated Mg2+ entry rates involve multiple
intracellular signaling pathways. Aldosterone potentiated
isoproterenol-stimulated Mg2+ uptake (326 ± 31 nM/s),
whereas elevation of extracellular Ca2+ inhibited
isoproterenol-mediated cAMP accumulation and Mg2+ uptake,
117 ± 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg2+ uptake in a cell line of mouse distal
convoluted tubules that is modulated by hormonal and extracellular influences.
isoproterenol; propranolol; intracellular magnesium; fluorescence; intracellular 3',5'-cyclic adenosine monophosphate; protein kinase A; phospholipase C; protein kinase C; phorbol ester; aldosterone; extracellular calcium
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INTRODUCTION |
RENAL NERVES INFLUENCE
ELECTROLYTE reabsorption in the proximal tubule, the loop of
Henle, and distal tubule (25). The proximal actions of
-adrenergic agonists have been extensively characterized; however,
they also directly influence electrolyte transport in the distal
tubule, but these effects have not been fully investigated. Among the
many direct tubular actions,
-adrenergic agonists, such as
isoproterenol, increase magnesium transport in the cortical segment of
the thick ascending limb (cTAL) (2). The cTAL reabsorbs a
significant portion of the filtered magnesium and as such plays an
important role in controlling renal magnesium balance
(35). Using RT-PCR, Elalouf et al. (14)
showed that the
1-receptor was principally the cTAL
receptor subtype but that some
2-receptor was also
present (14). Both receptor subtypes were shown to be associated with increases in intracellular cAMP accumulation, suggesting that receptor-mediated signaling pathways, involving protein
kinase A activity, may play a role in influencing electrolyte transport
(8). The distal tubule, comprising the distal convoluted tubule, connecting tubule, and collecting tubule collecting ducts, also
possess
-adrenergic-receptors (1, 7, 21, 29, 33, 36,
37). Isoproterenol influences distal tubular electrolyte handling by stimulating chloride absorption and K+,
H+, and bicarbonate secretion in the collecting tubule
(20, 23, 24, 28). Again, the receptor subtypes appear to
encompass both
1- and
2-receptors that
are associated with changes in intracellular cAMP and Ca2+
release (22, 36). Gesek and White (17)
demonstrated, with RT-PCR and receptor-binding studies, that the
immortalized mouse distal convoluted tubule cell line (MDCT) possesses
both
1- and
2-receptor subtypes. In
their study, isoproterenol stimulated cAMP formation and
22Na+ uptake but not
45Ca2+ entry into MDCT cells (17).
Although
-adrenergic agents have been shown to increase magnesium
absorption in the thick ascending limb, no experiments have been
directed at the distal convoluted tubule. The distal convoluted tubule
reabsorbs ~10% of the filtered magnesium and determines the final
urinary excretion as there is no transport beyond in the collecting
ducts (32).
In the present studies, we determined the effect of
-adrenergic
agonists on Mg2+ uptake into immortalized MDCT cells. The
MDCT cell line possesses many of the properties of the intact distal
convoluted tubule. MDCT cells exhibit amiloride-inhibitable
Na+ transport and chlorothiazide-sensitive NaCl cotransport
(16). Amiloride and chlorothiazide also stimulate
Mg2+ entry into these cells (11). Furthermore,
parathyroid hormone (PTH), glucagon, and arginine vasopressin (AVP)
increase Mg2+ entry in MDCT cells (9, 10, 12).
Accordingly, we used this cell line to investigate the actions of
isoproterenol on Mg2+ uptake in the distal convoluted
tubule. The distal convoluted tubule has not been extensively studied
because it is difficult to localize specific effects in intact
superficial tubules or to perform in vitro perfusion experiments. As
there is not an available isotope for magnesium to perform flux
measurements, we determined Mg2+ entry into
Mg2+-depleted MDCT cells. The cells were depleted of
intracellular Mg2+ by culturing in magnesium-free media for
16 h; then, the Mg2+-depleted MDCT cells were placed
in a medium containing 1.5 mM magnesium and the refill rate,
d([Mg2+]i)/dt, was measured with
microfluorescent studies by using mag-fura 2 (11).
Mg2+ uptake rate is concentration dependent and selective
for magnesium (11). Moreover, the influx rate is rapid and
reproducible so that it is possible to determine the effects of
extracellular influences on transport rates. In the present study, we
show that isoproterenol stimulates Mg2+ entry in MDCT
cells, in part, through cAMP-dependent mechanisms.
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METHODS |
Materials.
Basal DMEM and Ham's F-12 media were purchased from GIBCO
(Gaithersburg, MD). Customized magnesium-free media were purchased from
Stem Cell Technologies (Vancouver, BC). Fetal calf serum was from Flow
Laboratories (McLean, VA). Mag-fura-2-acetoxymethyl ester (AM) was
obtained from Molecular Probes (Eugene, OR). The protein kinase A
inhibitor RpcAMPS, the phospholipase C inhibitor U-73122, and protein
kinase C inhibitor RO 31-822 were purchased from Calbiochem, (San
Diego, CA). Isoproterenol, propranolol, the adenylate cyclase inhibitor
2'5'-dideoxyadenosine (DDA), and phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA), and other
materials were from Sigma (St. Louis, MO).
Cell culture.
Distal convoluted tubule cells were isolated from mice, immortalized by
Pizzonia (31), and functionally characterized as previously described by Friedman et al. (16). The MDCT
cell line was grown on 60-mm plastic culture dishes (Corning Glass Works, Corning Medical and Scientific, Corning, NY) in DMEM-Ham's F-12, 1:1, media supplemented with 10% fetal calf serum, 1 mM glucose,
5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml
streptomycin in a humidified environment of 5% CO2-95%
air at 37°C. For the fluorescent studies, confluent cells were washed
three times with PBS containing 5 mM EGTA, trypsinized, and seeded on
glass coverslips. Aliquots of harvested cells were allowed to settle
onto sterile glass coverslips in 100-mm Corning tissue culture dishes,
and the cells were grown to subconfluence over 1-2 days in
supplemented media as described above. The normal media contained 0.6 mM magnesium and 1.0 mM calcium. In the experiments indicated, MDCT
cells were cultured in nominally Mg2+-free media (<0.01
mM) where indicated for 16-24 h before study. Other constituents
of the Mg2+-free culture media were similar to the complete
media. These media contained 0.1% BSA rather than the fetal calf serum.
Determination of cAMP concentration.
Intracellular cAMP was determined in confluent MDCT cell monolayers
cultured in 24-well plates in DMEM-Ham's F-12 media without serum but
with 0.1% BSA. After addition of isoproterenol, MDCT cells were
incubated at 37°C for 5 min in the presence of 0.1 mM IBMX. cAMP was
extracted with 5% trichloroacetic acid, which was removed with ether,
and the extract was acidified with 0.1 N HCl. The aqueous phase was
dried, dissolved in Tris-EDTA buffer, and cAMP was measured with a
radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).
Cytoplasmic Mg2+ measurements.
The coverslips with MDCT cells were mounted into a perfusion chamber,
and intracellular free Mg2+ concentration
([Mg2+]i) was determined with the use of the
Mg2+-sensitive fluorescent dye mag-fura-2. The
cell-permeant AM form of the dye was dissolved in DMSO with pluronic
acid F-127 (0.125%, Molecular Probes) to a stock concentration of 5 mM
and then diluted to 5 µM mag-fura-2-AM in media for 20 min at 23°C.
Cells were subsequently washed three times with buffered salt solution
containing (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 20 HEPES/Tris, at pH 7.4. The MDCT
cells were incubated for a further 20 min to allow for complete
deesterification and washed once with this buffer solution before
measurement of fluorescence.
Epifluorescence microscopy was used to monitor changes in mag-fura-2
fluorescence within single subconfluent MDCT cells. The chamber (0.5 ml) was mounted on an inverted Nikon Diaphot-TMD microscope, with a
Fluor ×100 objective, and fluorescence was monitored under oil
immersion within a single cell over the course of study. Fluorescence
was recorded at 1-s intervals by using a dual-excitation wavelength
spectrofluorometer (Delta-scan, Photon Technologies, Princeton, NJ)
with excitation for mag-fura-2 at 335 and 385 nm (chopper speed set at
100 Hz), and emission at 505 nm. All experiments were performed at
23°C with continuous change of bathing solution (1 ml/min). Media
changes were made without interruption in recording.
[Mg2+]i was calculated from the ratio of the
fluorescence at the two excitation wavelengths as previously described
by using a dissociation constant of 1.4 mM for the
mag-fura-2-Mg2+ complex (9). The minimum and
maximum ratios were determined for the cells at the end of each
experiment using 20 µM digitonin. The maximum ratio for mag-fura-2
was found by the addition of 50 mM MgCl2, in the absence of
Ca2+, and the minimum ratio was obtained by removal of
Mg2+ and the addition of 100 mM EDTA, pH 7.2. The change in
[Mg2+]i with time
{d([Mg2+]i)/dt} was determined
by linear regression analysis of the fluorescence tracing over the
initial 500 s.
Statistical analysis.
Representative tracings of fluorescent intensities are given, and
significance was determined by Students' t-test or Tukey's analysis of variance as appropriate. A probability of P < 0.05 was taken to be statistically significant. All results are
means ± SE where indicated.
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RESULTS |
Isoproterenol stimulates Mg2+ uptake in MDCT cells.
To determine Mg2+ uptake, subconfluent MDCT monolayers were
cultured in magnesium-free medium for 16 h. These cells possessed a significantly lower [Mg2+]i, 0.22 ± 0.01 mM, than did cells cultured in normal media, 0.53 ± 0.02 mM.
When the Mg2+-depleted MDCT cells were placed in a bathing
solution containing 1.5 mM MgCl2, intracellular
Mg2+ concentration increased with time and leveled at a
[Mg2+]i of 0.54 ± 0.03 mM,
n = 9, which was similar to basal levels observed in
normal cells (Fig. 1). The average rate
of refill, d([Mg2+]i)/dt, measured
as the change in [Mg2+]i with time, was
169 ± 11 nM/s, n = 9 cells, as determined over the first 500 s after addition of 1.5 mM MgCl2 (Fig.
2). We used this approach to determine
the effects of
-adrenergic agonists on Mg2+ uptake into
MDCT cells.

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Fig. 1.
Isoproterenol stimulates Mg2+ uptake in
Mg2+-depleted mouse distal convoluted tubule (MDCT) cells.
Fluorescence studies were performed in buffer solutions in absence of
external magnesium, and, where indicated, MgCl2 (1.5 mM
final concentration) was added to observe changes in intracellular
Mg2+ concentration ([Mg2+]i). The
buffer solutions contained (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 10 HEPES/Tris, pH 7.4, with and
without 1.5 mM MgCl2. Where indicated, 10 7 M
isoproterenol was added to the buffer solution from a stock solution.
Fluorescence was measured at 1 data point/s with 25-point signal
averaging, and the tracing was smoothed according to methods previously
described (11).
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Fig. 2.
Isoproterenol stimulates intracellular cAMP accumulation and
Mg2+ uptake in MDCT cells. Mg2+ uptake was
determined by techniques outlined in Fig. 1. The rate of
Mg2+ uptake
{d([Mg2+]i)/dt} was measured
over the first 500 s after addition of 10 7 M
isoproterenol and 1.5 mM MgCl2. Cellular cAMP
determinations were performed 5 min after the addition of
isoproterenol. In these studies, 10 7 M propranolol, the
-adrenergic antagonist, was added 5 min before 10 7 M
isoproterenol. Values are means ± SE for 3-6 cells.*, +:
P < 0.01 for Mg2+ entry rates and cAMP
concentrations, respectively, vs. respective control values.
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Isoproterenol added to the refill buffer solution increased the rate of
Mg2+ entry into Mg2+-depleted MDCT cells (Fig.
1). Isoproterenol (10
7 M) increased the mean
Mg2+ entry rate from 169 ± 11 to 252 ± 26 nM/s,
n = 6, measured at 23°C, which represented a
stimulation of 49 ± 7% above control values measured after
500 s (Fig. 2). Isoproterenol increased Mg2+ uptake so
that d([Mg2+]i)/dt was not linear,
in some cases, over the 500-s interval, but this time frame provided a
relative measurement for comparisons. In the cases when it was
measured, [Mg2+]i in isoproterenol-treated
cells returned to basal levels, 0.48 ± 0.12 mM, similar to that
in control observations. These experiments were repeated at 37°C:
control, 163 ± 13 nM/s, n = 4 and isoproterenol, 272 ± 38 nM/s, n = 3. As these values were not
significantly different from those measured at 23°C, the remaining
studies were performed at room temperature. The effect of isoproterenol
on Mg2+ uptake was concentration dependent, with the
maximal rate of stimulation at 10
6 M, 264 ± 28 nM/s, n = 4 and half-maximal stimulation at a
concentration of ~10
8 M (Fig.
3). We have previously reported that
dihydropyridines inhibit Mg2+ uptake into
Mg2+-depleted MDCT cells (11). To determine
whether isoproterenol-induced Mg2+ entry is mediated
through a dihydropyridine-sensitive pathway, we examined the effect on
the changes in [Mg2+]i of the channel blocker
nifedipine after placement in the refill buffer solution containing 1.5 mM MgCl2. The presence of 10
5 M nifedipine
inhibited isoproterenol-stimulated Mg2+ uptake, 25 ± 12 nM/s, which was similar to that observed in control cells
(11). These findings support the notion that
isoproterenol-stimulated Mg2+ uptake is the same as the
entry pathway observed in control cells.

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Fig. 3.
Concentration dependence of isoproterenol stimulation of cAMP
formation and Mg2+ entry in MDCT cells. The rate of
Mg2+ influx, as determined by
d([Mg2+]i)/dt, was measured with
the given isoproterenol concentrations by using fluorescence techniques
performed according to that in Fig. 1. Values of
d([Mg2+]i)/dt were determined over
the first 500 s of fluorescence measurements. Isoproterenol was
added, at the concentrations indicated, 5 min before the measurement of
cAMP in the presence of 3-isobutyl-1-methylxanthine. Values are
means ± SE for 3-6 cells. *, +:
P < 0.01 for Mg2+ entry rates and cAMP
concentrations, respectively, vs. respective control values.
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Next, we determined the effect of propranolol, a specific
-adrenergic receptor antagonist, on isoproterenol-stimulated
Mg2+ uptake (Fig. 2). Propranolol inhibited
isoproterenol-stimulated uptake, 140 ± 25 nM/s, n = 3, confirming that isoproterenol acts through
-adrenergic
receptor-mediated mechanisms.
Isoproterenol stimulates Mg2+ uptake by cAMP-dependent
protein kinase A- and phospholipase C-mediated intracellular signaling
pathways.
Gesek and White (17) reported that isoproterenol increased
cAMP formation in MDCT cells but not intracellular Ca2+
concentration, suggesting that
-adrenergic receptors mediate transport functions, in part, by activating protein kinase A
(17). As exogenous cAMP increases Mg2+ entry
into MDCT cells, we determined the effects of isoproterenol on cAMP
release in the MDCT cells used here (9). Isoproterenol (10
7 M) stimulated intracellular cAMP formation by about
sixfold in MDCT cells (Fig. 3). Propranolol inhibited
isoproterenol-mediated cAMP formation (Fig. 2). The
concentration-dependent manner of isoproterenol-stimulated
Mg2+ entry was associated with increases in intracellular
cAMP formation (Fig. 3). Accordingly, isoproterenol may stimulate
Mg2+ transport through cAMP-mediated signaling pathways.
To sort out some of the receptor-mediated signaling pathways, we used a
number of selective enzyme inhibitors to determine the effect on
isoproterenol-responsive Mg2+ uptake in MDCT cells. RpcAMPS
or DDA, two different protein kinase A inhibitors, were applied 5 min
before Mg2+ uptake measurements were performed
(9). RpcAMPS and DDA inhibited the effects of
isoproterenol on Mg2+ entry rates, 171 ± 25 nM/s,
n = 3 and 148 ± 12 nM/s, n = 3, respectively, suggesting that activation of protein kinase A is
involved in the actions of isoproterenol (Fig.
4). Pretreatment of MDCT cells with the
phospholipase C inhibitor U-73122 also diminished
isoproterenol-stimulated Mg2+ uptake, 143 ± 11 nM/s,
n = 3, whereas the protein kinase C inhibitor RO 31-822 diminished isoproterenol-stimulated uptake by 36%, 208 ± 14 nM/s, n = 3 (Fig. 4). These results suggest that
isoproterenol alters Mg2+ entry into MDCT cells through
cAMP-dependent protein kinase A- and phospholipase C-mediated signaling
pathways; the results with U-73122 were equivocal.

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Fig. 4.
Isoproterenol stimulates Mg2+ uptake though
cAMP-protein kinase A- and phospholipase C-mediated signaling pathways.
Inhibitors for protein kinase A (RpcAMPS, 0.5 µM) and
2'5'-dideoxyadenosine (DDA; 10 µM), phospholipase C (U-73122, 15 µM), and protein kinase C (RO 31-822, 0.1 µM) were added to
Mg2+-depleted MDCT cells 5 min before the addition of
10 7 M isoproterenol. Values are means ± SE for
3-5 cells.*P < 0.01 vs. control uptake rates.
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As isoproterenol increases Mg2+ uptake in MDCT cells, in
part, by RO 31-822-sensitive mechanisms, we tested whether phorbol ester-induced protein kinase C activation changes basal
Mg2+ entry or isoproterenol-stimulated uptake. The phorbol
ester TPA had no detectable effect on basal Mg2+ uptake,
144 ± 16 nM/s, n = 3, but potentiated the effects
of isoproterenol, 410 ± 38 nM/s, n = 3 (Fig.
5). Isoproterenol-stimulated intracellular cAMP accumulation was not altered with phorbol esters (Fig. 5). We infer from these studies that
-adrenergic agonists stimulated Mg2+ transport, in part, through protein kinase
A-mediated pathways and these effects may be modulated by other
intracellular signaling processes responsive to phorbol esters. In
support of this speculation, RpcAMPS, an inhibitor of protein kinase A,
abolished TPA-mediated potentiation of isoproterenol-stimulated
Mg2+ entry, indicating that phorbol esters interact with
the protein kinase A-signaling pathway beyond adenylate cyclase (Fig.
5).

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Fig. 5.
Phorbol ester potentiates isoproterenol-stimulated Mg2+
uptake. MDCT cells were incubated for 16 h in magnesium-free
buffer solution. 12-O-tetradecanoylphorbol-13-acetate (TPA;
0.01 µM), and RpcAMPS, 0.5 µM, were added with and without
10 7 M isoproterenol, where indicated. Values are
means ± SE for 3-6 cells. *,+: P < 0.01 for Mg2+ entry rates and cAMP concentrations,
respectively, compared with the respective control values.
#, : P < 0.01 for mean
Mg2+ entry rate of TPA plus isoproterenol vs.
isoproterenol alone and TPA+RpcAMPS vs. TPA alone, respectively.
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Aldosterone potentiates isoproterenol-stimulated Mg2+
uptake in MDCT cells.
We have previously shown that aldosterone, applied 16 h before
experimentation, increases PTH-, glucagon- and AVP-mediated cAMP
generation that, in turn, potentiates hormone-mediated Mg2+
uptake (12, 13). Short-term (10-15 min) aldosterone
administration changes neither Mg2+ uptake
(13) nor intracellular Ca2+ signaling:
control, 104 ± 5 nM and aldosterone, 104 ± 6 nM
(n = 4). Although the cellular mechanisms are not
known, it has been speculated that aldosterone-induced proteins
modulate receptor signaling in epithelial cells (30). In
the present study, we determined whether pretreatment of MDCT cells
with aldosterone for 16 h potentiates the acute actions of
isoproterenol. Treatment of cells with aldosterone, for 16 h
before study, did not significantly affect basal Mg2+
uptake but potentiated isoproterenol-stimulated Mg2+ entry.
(Fig. 6). Aldosterone did not change the
level of isoproterenol-mediated cAMP formation, suggesting that the
actions are downstream of the generation of this second message
(Fig. 6).

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Fig. 6.
Aldosterone potentiates isoproterenol-mediated cAMP generation and
Mg2+ uptake. MDCT cells were incubated for 16 h in
magnesium-free buffer solution containing 10 7 M
aldosterone. Isoproterenol (10 7 M) was added where
indicated, and cAMP was measured after 5 min in the presence of IBMX or
Mg2+ uptake was determined after 500 s in 1.5 mM
MgCl2. Values are mean ± SE for 3-5
observations. *, +: P < 0.01 for mean
Mg2+ entry rates and cAMP determinations, respectively,
compared with the respective control values.
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Elevation of extracellular Ca2+ inhibits
isoproterenol-stimulated cAMP generation and Mg2+ uptake.
MDCT cells possess an extracellular
Ca2+/Mg2+-sensing receptor, Casr,
that, on activation with polyvalent cations such as, Ca2+,
Mg2+ or neomycin, inhibits PTH-, glucagon-, and
AVP-mediated cAMP generation and glucagon- and AVP-stimulated
Mg2+ uptake (3, 4). To determine whether
activation of Casr alters isoproterenol actions, we
pretreated cells for 5 min with 5.0 mM CaCl2 before the
addition of isoproterenol. Elevation of extracellular Ca2+
did not have any effects on basal Mg2+ entry but abolished
isoproterenol stimulation of cAMP generation and
Mg2+ uptake (Fig. 7).
Elevation of extracellular Ca2+ also inhibits
aldosterone-potentiated, isoproterenol-stimulated cAMP formation and
Mg2+ uptake (Fig. 7). The mechanisms by which
Casr inhibits isoproterenol actions remain unclear, but the
receptor is coupled to G
i proteins, which is consistent
with the conclusion that isoproterenol responses in MDCT cells are
dependent, in part, on cAMP-mediated signaling pathways.

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Fig. 7.
Summary of the effects of extracellular Ca2+ on
isoproterenol-stimulated cAMP formation and Mg2+ uptake.
cAMP was measured by radioimmunoassay and
d([Mg2+]i)/dt was determined with
1.5 mM extracellular Mg2+ in the absence and presence of
5.0 mM CaCl2 as indicated. CaCl2 was added 5 min before the addition of 10 7 M isoproterenol. In those
studies indicated, MDCT cells were treated with 10 7 M
aldosterone 16 h before experimentation. Mg2+ uptake
rate was determined over the initial 500 s after addition of
isoproterenol. Values are mean ± SE for 3-5 cells. +,*:
P < 0.001 for cAMP determinations and Mg2+
uptake, respectively, vs. respective control values. Ald, aldosterone;
Ca, CaCl2. #, :
P < 0.001 for cAMP determinations and Mg2+
uptake of Ald +5 Ca vs. Ald alone, respectively.
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DISCUSSION |
Isoproterenol stimulates Mg2+ reabsorption in the
distal tubule.
In the present studies, we show that isoproterenol stimulates
Mg2+ uptake in MDCT cells. The response is concentration
dependent and involves selective
-adrenergic receptors as
propranolol inhibited isoproterenol actions. As the MDCT cell line
demonstrates the properties of the intact distal convoluted tubule, we
infer that
-adrenergic innervations and neurally released
catecholamines regulate distal magnesium conservation. Bailly et
al. (2) have also reported that isoproterenol increases
magnesium absorption in mouse cTAL. Accordingly,
-adrenergic
agonists control magnesium reabsoption by its actions within both the
thick ascending limb and the distal convoluted tubule.
Barajas et al. (5) demonstrated that the thick ascending
limb and distal convoluted tubule have the greatest adrenergic innervation. The receptor subtypes within the distal tubule, including thick ascending limb segments, distal convoluted tubule, and collecting tubule, have been reported to be
1- and
2-adrenoreceptors (7, 14, 27, 29, 37).
Using RT-PCR, Gesek and White (17) reported that MDCT
cells possess
1- and
2-receptor mRNA, the DNA of which was identical to the reported mouse receptor sequences (17). Receptor-binding studies confirmed the membrane
expression of these subtypes, and receptor-mediated cAMP accumulation
demonstrated functional presence in the immortalized mouse cell line.
These studies with MDCT cells are consistent with those in the intact kidney (6, 7, 14). Gesek and White (17)
showed that both
1- and
2-receptor
subtypes enhanced Na+ uptake into MDCT cells. Bailley et
al. (2) and Levine et al. (26) reported that
-adrenergic agonists stimulated Na+, Cl
,
and passive Ca2+ and Mg2+ absorption in the
cTAL, and other investigators have demonstrated that isoproterenol
increases K+, H+, and bicarbonate secretion in
the collecting tubule (19, 20, 23, 24, 28). Levine et al.
(26) reported that isoproterenol diminishes bicarbonate
reabsorption in the distal convoluted tubule (26). Gesek
and White (17) demonstrated that isoproterenol stimulated
22Na+ entry but had no effect on
45Ca2+ uptake in MDCT cells. These reports
indicate that both
1-receptors, principally activated by
neurally released norepinephrine, and
2-receptors,
stimulated by circulating epinephrine, have direct effects on
electrolyte transport in the distal tubule including the thick
ascending limb, distal convoluted tubule, and collecting tubule.
The cellular mechanisms by which
-adrenergic agents influence distal
electrolyte transport are not fully understood. In most tissues, the
major route for
-agonist effects is the stimulation of adenylate
cyclase and increases in intracellular cAMP accumulation (6,
18). The evidence is that
-adrenergic receptors directly influence epithelial transport, in part, through G
-coupled proteins (8, 18, 19, 22, 27, 34). Again, Gesek and White
(17) have carefully characterized some of the
receptor-mediated signaling pathways in the immortalized MDCT cell
line. They showed that
1- and
2-receptor
subtypes increase intracellular cAMP accumulation but not intracellular
transient Ca2+ signaling in MDCT cells. This response is
unlike those observed in rat collecting ducts, where calcium signals
are associated with
-agonist stimulation of cAMP (27).
However, in support of the observations of Gesek and White
(17), we have shown that isoproterenol does not increase
intracellular Ca2+ in MDCT cells so that transient
Ca2+ signaling is not likely involved with
isoproterenol-mediated Mg2+ uptake (17, data not shown).
The notion that adrenergic agonists act through cAMP-dependent
mechanisms is persuasive; first, isoproterenol stimulates cAMP
accumulation and cAMP enhances Mg2+ entry (9).
Second, inhibition of cAMP-dependent protein kinase A abolishes
isoproterenol-stimulated Mg2+ uptake (Fig. 4). Third,
aldosterone potentiates isoproterenol-mediated cAMP concentrations that
are associated with greater Mg2+ uptake (Fig. 6). Finally,
elevation of extracellular Ca2+ diminishes
isoproterenol-induced cAMP and Mg2+ transport (Fig. 7).
However, receptor-mediated cAMP-protein kinase A activity is not the
full explanation as phospholipase C and protein kinase C inhibition
also mitigate hormone-stimulated Mg2+ uptake (Fig. 4).
Furthermore, phorbol esters potentiate isoproterenol-mediated Mg2+ uptake without changing cAMP concentrations (Fig. 5).
The intracellular mechanisms involved with isoproterenol responses are
yet to be fully explored.
Aldosterone potentiates isoproterenol-stimulated Mg2+
entry in MDCT cells.
Mineralocorticoid hormones stimulate NaCl cotransport, Na+
conductance, and Na+ pump activity in the distal convoluted
tubule (27). Aldosterone also modulates hormone-responsive
Mg2+ transport into MDCT cells (13).
Incubation of aldosterone, for 16 h before determination of
Mg2+ uptake, failed to have any effect on basal magnesium
transport; however, pretreatment of MDCT cells with aldosterone
potentiated isoproterenol-stimulated Mg2+ entry (Fig. 6).
This was not associated with potentiation of hormone-mediated cAMP
release in the aldosterone-treated MDCT cells, suggesting that the
aldosterone actions were either downstream from the generation of this
second messenger or involve the induction of other signaling pathways
(Fig. 6). The prominent mechanism of steroids, which operate through
nuclear receptors, is to control transcriptional regulation,
expression, and posttranslational targeting of heterotrimeric G
proteins (30). Regulation of G protein subunits by steroid
hormones has been studied in a variety of systems. Changes in the
levels of expression of G protein subunits in adrenalectomized animals
reflect changes in subunit mRNA, suggesting that adrenocorticoids
activate genes encoding G
s , G
i, G
,
G
, and phospholipase C (30). Other aldosterone-induced
proteins may also be involved (38). These studies indicate
that distal magnesium transport is regulated at two levels: first by
membrane-receptor signaling and, second, by nuclear transcriptional
expression of receptor subunits.
Extracellular Ca2+ affects isoproterenol-stimulated
Mg2+ uptake in MDCT cells.
Casr within the distal tubule is important in the control
of renal electrolyte handling (3). We have reported that
elevation of extracellular Ca2+, Mg2+, or the
addition of the polyvalent cation neomycin, completely inhibits peptide
hormone-stimulated cAMP formation and glucagon- and AVP-stimulated
increases in Mg2+ uptake in MDCT cells (3, 4).
Activation of Casr inhibits isoproterenol-mediated cAMP and
isoproterenol stimulation of Mg2+ uptake in MDCT cells
(Fig. 7). The extracellular Ca2+- and
Mg2+-sensing mechanisms provide a negative feedback loop to
diminish the renal conserving actions of hormones such as
-agonists.
Role of
-adrenergic agonists on renal magnesium reabsorption.
The loop of Henle reabsorbs ~70% of the filtered magnesium.
Isoproterenol increases magnesium absorption within the thick ascending
limb (2). However, the distal convoluted tubule reabsorbs significant amounts of magnesium and plays an important role in determining the final urinary excretion rate (32). In
contrast to more proximal segments of the nephron, distal magnesium
transport processes are postulated to be active and transcellular in
nature (35). Hormonal control of magnesium transport in
this segment provides the fine-tuning of renal conservation,
contributing to whole body magnesium balance. In the present study, we
show that isoproterenol stimulates Mg2+ uptake in MDCT
cells, in part, through increases in cellular cAMP levels. We infer
from these results that
-adrenergic agonists may modulate distal
tubule magnesium transport and, together with its actions within the
loop, regulate renal magnesium conservation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter A. Friedman for providing the MDCT cell line.
 |
FOOTNOTES |
Dr. Hyung Sub Kang is a Postdoctoral Fellow of the Korean Science and
Engineering Foundation. This work was supported by research grants from
the Medical Research Council of Canada (MT-5793) and from the Kidney
Foundation of Canada.
Address for reprint requests and other correspondence:
G. A. Quamme, Dept. of Medicine, Univ. of British
Columbia, Vancouver Hospital and Health Sciences Centre, Keorner
Pavilion, 2211 Westbrook Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail:
quamme{at}interchange.ubc.ca).
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 10 April 2000; accepted in final form 11 August 2000.
 |
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