Departamento de Biología, Bioquímica, y Farmacia, Universidad Nacional del Sur, 8000 Bahía Blanca, Argentina
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ABSTRACT |
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Direct effects of parathyroid hormone (PTH) on calcium uptake by
isolated rat duodenal cell preparations enriched in enterocytes were
investigated. PTH significantly stimulated enterocyte
45Ca2+
influx in a time-dependent (1-10 min) manner and at all doses tested (2 × 1013 to
10
7 M). The
Ca2+ channel antagonists verapamil
(10 µM) and nitrendipine (1 µM) completely blocked the stimulation
of Ca2+ influx by the hormone
(10
8 M). PTH markedly
increased cAMP levels in rat duodenal cells (88, 167, and 67%, after
1, 2, and 3 min, respectively). In agreement with these observations,
forskolin (adenylate cyclase activator), dibutyryl adenosine
3',5'-cyclic monophosphate (DBcAMP), and Sp-cAMPS (cAMP
analogs) mimicked, whereas Rp-cAMPS (cAMP antagonist) suppressed PTH
and DBcAMP activation of enterocyte calcium uptake. Furthermore, the
effects of DBcAMP were abolished by nitrendipine. These results show
direct rapid effects of PTH on duodenal cells'
Ca2+ influx, which involve the
activation of a dihydropyridine-sensitive Ca2+ influx pathway and the cAMP
second messenger system.
intestine; nongenomic effects; calcium channels; adenosine 3',5'-cyclic monophosphate signaling pathway
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INTRODUCTION |
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PARATHYROID HORMONE (PTH) is responsible for the regulation of calcium levels in blood and extracellular fluids in concert with other calciotropic hormones (12). PTH acts at its target tissues, kidney and skeleton, by enhancing calcium reabsorption at cortical sites within the distal portion of the nephron (1) or by stimulating bone resorption with the subsequent release of calcium and phosphate into the circulation.
Moreover, this peptide hormone exerts an indirect action on the
intestine. Through its effects on 1-hydroxylase, PTH stimulates the
formation of 1,25-dihydroxyvitamin
D3 (11), which in turn has a
direct biological effect on the gut, increasing the absorption of
dietary calcium.
Like other polypeptide hormones, PTH interacts with specific receptors on the cell plasma membrane in target tissues and modulates cellular responses mainly by activation of adenylate cyclase and the increase of adenosine 3',5'-cyclic monophosphate (cAMP) levels (17). Recent reports also suggest that PTH may act through other messenger systems that involve phosphoinositide breakdown, inositol trisphosphate accumulation, and protein kinase C (PKC) activation (16).
It is well known that 1,25-dihydroxyvitamin D3 is the principal regulator of duodenal calcium transport in mammalian intestine (23). However, early work also suggested certain direct actions of PTH on duodenum cells (21). Physiological concentrations of this peptide hormone increased lysosomal enzyme liberation and calcium uptake after 10-15 min of treatment. In the present work, using isolated rat enterocytes, we investigated the nongenomic action of PTH on calcium influx at very short treatment intervals (1-10 min) and tested the hypothesis that this event is related to the activation of intracellular second messenger pathways.
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MATERIALS AND METHODS |
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Materials. Synthetic rat PTH
(rPTH)-(134), verapamil,
N6,2'-O-dibutyryladenosine
3',5'-cyclic monophosphate (DBcAMP), and forskolin were
purchased from Sigma Chemical (St. Louis, MO). 45CaCl2
was provided by New England Nuclear (Chicago, IL). Nitrendipine was
provided by Bayer (Leverkusen, Germany). Rp and Sp isomers (Rp-cAMPS,
Sp-cAMPS) were provided by Biolog Life Science Institute (Bremen,
Germany). The cAMP
125I-radioimmunoassay kit was
obtained from DuPont (Boston, MA), and
[3H]cAMP assay kit was
obtained from Diagnostic Products (Los Angeles, CA). PTH antagonist,
PTH-(7
34), was obtained from Bachem California (Torrance, CA).
Animals. Wistar rats (3-5 mo old) were fed with standard rat food (1.2% Ca:1.0% phosphorus), given water ad libitum, and maintained on a 12:12-h light-dark cycle. Animals were killed by cervical dislocation.
Duodenal cell isolation. Duodenal cells were isolated essentially as previously described (20). The method employed yields preparations that contain only highly absorptive epithelial cells and that are devoid of cells from the upper villus or crypt (26, 27). The duodenum was excised, washed with 0.9% NaCl, and trimmed of adhering tissue. The intestine was slit lengthwise and cut into small segments (2 cm length) and placed into solution A containing (in mM) 96 NaCl, 1.5 KCl, 8 KH2PO4, 5.6 Na2HPO4, 27 sodium citrate, pH 7.3, for 10 min at 37°C. The solution was discarded and replaced with solution B (isolation medium) containing (in mM) 154 NaCl, 10 NaH2PO4, 1.5 EDTA, 0.5 dithiothreitol (DTT), 5.6 glucose, pH 7.3, for 15 min at 37°C with shaking (87 oscillations/min). The cells were sedimented by centrifugation at 750 g for 10 min; washed twice with 154 mM NaCl, 10 mM NaH2PO4, and 5.6 mM glucose at pH 7.4; and then resuspended in solution D (incubation medium) containing (in mM) 154 NaCl, 5 KCl, 1 Na2HPO4, 1 MgCl2, 10 3-(N-morpholino)propanesulfonic acid sodium salt, pH 7.4, 5.6 glucose, 0.5% bovine serum albumin, 1 CaCl2, and 2.5 glutamine. Duodenal cells were preequilibrated in the incubation medium before hormone treatment and measurement of calcium uptake for 20 min. All the above-mentioned steps were performed under an atmosphere of 95% O2-5% CO2 and using oxygenated solutions. Cell viability was assessed by Trypan blue exclusion in well-dispersed cell preparations. Exclusion of the dye in >90% of the cells was observed for at least 90 min after isolation. Morphological characterization was performed by phase-contrast microscopy. Although enterocytes isolated by this procedure have been shown to possess functional characteristics of intestinal cells (26, 27), the possibility that these isolated cell preparations contain nonepithelial cells should not be ruled out.
Calcium uptake. After the
preequilibration period, duodenal cells were incubated in
solution
D with rPTH-(134),
Sp-cAMPS [protein kinase A (PKA) activator], forskolin, or
DBcAMP in the presence of
45CaCl2
(0.2 µCi/ml; 1 mM). To the corresponding control cell suspensions, distilled water was added except in the case of forskolin, when ethanol
(<0.1%) was employed. When calcium channel blockers or cAMP
antagonists (Rp-cAMPS) were used, they were added during the
preequilibration period before hormone addition. Immediately after
treatment, aliquots of cell suspension were diluted 25-fold in ice-cold
unlabeled medium [in mM: 140 NaCl, 10 tris(hydroxymethyl)aminomethane (Tris) · HCl, pH
7.4, 1 LaCl3, and 1 CaCl2] and quickly
centrifuged for 45 s at 1,500 g, and then the pellet was
solubilized in 1 N NaOH-0.1% sodium dodecyl sulfate. Under these
conditions, extracellularly bound
45Ca2+
is completely removed. Aliquots were taken for measurement of radioactivity and for protein determination by the method of Lowry et
al. (19) using bovine serum albumin as standard.
Measurement of cAMP levels. Immediately after hormone treatment, aliquots of cell suspension were quickly transferred to ice-cold 6% tricloroacetic acid and centrifuged at 1,200 g for 15 min (4°C), and the supernatant was washed six times with four volumes of water-saturated diethyl ether so that the final pH value was 5.5-6.0. The extract was used for cAMP measurements by a radioimmunoassay technique using a commercially available kit (14). The sensitivity of the method was 0.025 pmol/ml, and the variation between assays was characterized by a coefficient of variation of 7.83%. The results obtained represent the means ± SD of four experiments performed separately.
Determination of adenylate cyclase
activity. The enzyme activity was indirectly determined
in vitro by a binding protein assay kit (9) measuring the amount of
cAMP present in a sample after a timed incubation, using enterocyte
microsomal membranes as an experimental model. Microsomes were obtained
by centrifugation at 100,000 g during
60 min (4°C) of the postmitochondrial supernatant (12,000 g) of cells homogenized in 50 mM
Tris · HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM
ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 0.5 µM phenylmethylsulfonyl fluoride, 1 mM DTT, 20 µg/ml aprotin, and 20 µg/ml leupeptin. Membranes were incubated for 3 min
at 30°C with vehicle (distilled water), rPTH-(1
34), or PTH-(7
34) in the assay buffer (50 µM ATP, 10 mM
MgCl2, 10 mM creatine phosphate,
50 U/ml creatine phosphokinase, 100 µM 3-isobutyl-1-methylxanthine, 1 mM DTT, 10 mM Tris · HCl, pH 7.4). The reaction was
stopped with HClO4, neutralized
with KHCO3 (so that the final pH
was 5.5-6.0), and the solution was quickly centrifuged at 10,600 g for 15 min (4°C).
Aliquots of the supernatant were taken for cAMP measurements with the
[3H]cAMP assay kit.
The results obtained represent the means ± SD of five experiments
performed separately.
Statistical evaluation. Data are presented as means ± SD and derived from at least two independently valid assays giving statistically homogeneous results. The significance of the results was evaluated by Student's t-test, and P < 0.050 was considered significant. In addition, for multiple comparisons, Bonferroni test and analysis of variance were employed (25).
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RESULTS |
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Although PTH is a single-chain 84-amino acid peptide in several species, the structural requirements necessary for full biological activity are satisfied by the NH2-terminal 34-amino acid fragment, which was used in our study (10, 12).
Figure 1 shows the dose-response profile of
the rapid effects of rPTH-(134) on rat enterocyte calcium uptake.
Results are expressed as percent of control values after a 5-min
treatment interval. The hormone significantly increased
45Ca2+
influx at all doses tested
(10
12 to
10
7 M). At concentrations
of 7 × 10
13 and 2 × 10
13 M,
the stimulatory action of PTH on
Ca2+ uptake was still evident,
whereas at 10
13 M the
hormone was without effects (control: 3.40 ± 0.58; 7 × 10
13 M: 4.42 ± 0.53, P < 0.025; 2 × 10
13 M: 3.98 ± 0.30, P < 0.025;
10
13 M: 3.51 ± 0.21, not significant; values given in nmol Ca2+/mg protein,
n = 8, for each of 2 independent
experiments). The fall observed at
10
9 M PTH was not
statistically different from the responses induced by the other PTH
doses tested (P > 0.05; the
possibility that this decline is due to a type II,
, error was ruled
out). A hormone concentration of
10
8 M was chosen for the
following experiments because it has been widely used for studies of
PTH actions in vitro (2, 17).
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The stimulation of calcium uptake was time dependent (Fig.
2). Thus, 1 min after its addition,
108 M rPTH-(1
34) elicited
a significant increase (53% above control values) in calcium uptake,
whereas no significant changes were observed at shorter treatment
intervals (30 and 45 s; data not given). This elevation reached a
maximum of 96% at 5 min after exposure of the cells to the hormone and
decreased up to 10 min to 31%.
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Voltage-sensitive calcium channels are modulated by intracellular second-messenger signaling systems (5). First, the possibility that the activation of a dihydropyridine- and phenylalkylamine-sensitive pathway was involved in the early stimulation induced by the hormone of rat duodenal calcium influx and, second, the potential involvement of the cAMP-dependent pathway were investigated. As shown in Table 1, nitrendipine (1 µM) and verapamil (10 µM) completely abolished the increment in calcium uptake produced by the addition of the hormone after 3 min of treatment. These calcium channel antagonist concentrations have been previously shown to block calcium influx in similar rat enterocyte preparations (20).
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Changes in enterocyte cAMP content induced by the peptide hormone and
the effects of known activators and inhibitors of this messenger system
on rat duodenal Ca2+ uptake were
studied. Incubation of the cell suspension with
108 M rPTH-(1
34) rapidly
increased cAMP levels. As shown in Fig. 3,
already after 1 min of hormone addition a significant elevation (+88%)
in enterocyte cAMP content was observed. Maximum response (+167%) was
elicited by 2 min of treatment with the peptide. Although less
markedly, cAMP levels remained higher (+67%) than basal values after
3-5 min of incubation of enterocytes with rPTH-(1
34). Treatment with 10
12 M rPTH-(1
34)
for 2 min also markedly increased cAMP production (1.20 ± 0.12 vs.
4.41 ± 0.66 pmol/mg protein for control and PTH-treated enterocytes, respectively; P < 0.005; n = 3 for each of 2 independent experiments). Preincubation of cells with 10 µM verapamil did not
abolish the fast increase in cAMP induced by PTH in enterocytes after 2 min of treatment [1.20 ± 0.12 vs. 3.00 ± 0.24 vs. 1.26 ± 0.20 vs. 3.26 ± 0.33 pmol/mg protein for control,
10
8 M rPTH-(1
34), 10 µM
verapamil, and 10
8 M
rPTH-(1
34) + 10 µM verapamil, respectively]. Moreover, the elevation of cAMP caused by 10 µM forskolin was not blocked
by 10 µM verapamil either (data not shown).
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Furthermore, calcium uptake was also measured in the presence of
forskolin, an adenylate cyclase activator, and the cAMP analogs DBcAMP
and Sp-cAMPS. Three minutes of treatment of rat duodenal cells with
these agents, similar to rPTH-(134), resulted in a stimulation of
45Ca2+
uptake (Table 2). Rp-cAMPS is a specific
PKA-competitive inhibitor that blocks first messenger-stimulated
phosphorylation by cAMP-dependent protein kinase (8). The presence of
the analog at a concentration of 200 µM completely suppressed the
increment in calcium uptake induced by 5 min of treatment with the
hormone (Fig. 4). The inhibitory effect at
lower concentrations of the cAMP antagonist was less pronounced (data
not shown). Similar to the hormone, 200 µM Rp-cAMPS completely
blocked the stimulatory action of 100 µM DBcAMP on 45Ca2+
influx whereas 100 µM Rp-cAMPS only inhibits by 61% the effect of
the cAMP analog (3.1 ± 0.5, 4.3 ± 0.5, and 3.8 ± 0.4 nmol Ca2+/mg protein for control, DBcAMP,
and DBcAMP + 100 µM Rp-cAMPS, respectively). Previous studies have
shown that the optimal Rp-cAMPS concentration to block cAMP-dependent
processes varies between 50 and 500 µM according to the cell type
employed (8). In addition, the stimulation of
45Ca2+
influx in enterocytes by DBcAMP could be blocked by 1 µM
nitrendipine (Table 1).
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Finally, we measured adenylate cyclase activity using isolated
enterocyte microsomes as an experimental model. As can be seen in Fig.
5, treatment of the membranes with
108 M rPTH-(1
34) for 2 min significantly increased the enzyme activity (188% above the
control value). However, the
NH2-terminal-shortened fragment of
the hormone, PTH-(7
34), was not able to activate the enzyme.
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DISCUSSION |
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Despite the fact that the classical target tissues for PTH action on
calcium fluxes are bone and kidney, the results reported in the present
study provide evidence of a rapid (1-10 min), direct action of the
peptide hormone on intestinal calcium uptake of mammals in a wide
concentration range (2 × 1013 to
10
7 M). The mode of action
elicited by the hormone involves the participation of the adenylate
cyclase messenger system and calcium channel activation.
Fast alterations in Ca2+ fluxes induced by PTH in hepatocytes (13), connecting tubules (18), bone cells (10), and exocytosis of lysosomal enzymes (21) or cell Ca2+ by forskolin in the HT-29 intestinal cell line (7) have been reported. More important, it has also been shown that PTH rapidly stimulates Ca2+ transport in perfused duodena from normal chicks (22).
The mechanism of action of PTH in the classical bone and kidney target cells involves the interaction with hormone-specific receptors on the plasma membrane, which results in the activation of the adenylate cyclase-cAMP-PKA and/or the phospholipase C-diacylglycerol-PKC pathways (6, 10, 12).
However, observations of hormone receptor interaction and activation of adenylate cyclase have been expanded greatly to other cellular systems (13, 24). In the present study, we demonstrate that direct treatment with PTH rapidly induced a marked cAMP elevation in rat duodenal cells and an elevation of adenylate cyclase activity in isolated microsomal membranes derived therefrom. Future studies should also investigate whether the phospholipase C-PKC messenger system also mediates PTH effects in this cell system. In addition, coupling of these signaling pathways to a PTH receptor should be also experimentally addressed.
The increase in cAMP elicited by the hormone paralleled the stimulation
of Ca2+ influx. Several lines of
evidence in our study support the hypothesis that the production of
cAMP by PTH activates calcium channels via cAMP-dependent
phosphorylation and/or by a direct action on cyclic
nucleotide-gated Ca2+ channels
(28). First, the adenylate cyclase activator forskolin increased
calcium uptake in rat duodenal cells. Second, cAMP analogs, DBcAMP and
Sp-cAMPS, like PTH stimulation, induced similar effects on
45Ca2+
influx. Third, the competitive inhibitor of cAMP binding to the R
subunit of PKA, Rp-cAMPS, completely blocked PTH increment of calcium
uptake at a concentration similar to that required for inhibition of
the effects of DBcAMP on this process. Finally, the calcium channel
blockers, nitrendipine and verapamil, fully suppressed PTH stimulatory
action. Nitrendipine also inhibits DBcAMP-induced calcium uptake.
However, electrophysiological data is required to conclusively
establish the participation of
Ca2+ channels in PTH stimulation
of duodenal Ca2+ influx. The
possibility that the increase in
Ca2+ uptake is responsible for the
elevation of cell cAMP levels is not supported by the fact that
verapamil did not abolish the changes in cAMP induced by
rPTH(134).
It is widely known that the principal regulator of intestinal calcium transport is the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (4). With regard to the physiological significance of the results obtained in this study, it should be considered that, because it was carried out with nonpolarized cells, it is not possible to distinguish apical versus basolateral Ca2+ transport. However, there is evidence indicating that voltage-dependent L-type Ca2+ channels are located at the basolateral membranes of mammalian duodenal cells (15). In addition, according to the currently accepted mechanism of transcaltachia, e.g., rapid modulation of intestinal Ca2+ transport by vitamin D (3), Ca2+ influx through duodenal basolateral membranes triggers calcium transfer to the circulation by exocytosis.
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ACKNOWLEDGEMENTS |
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Assistance with statistical analysis by Susana Iturmendi (Dept. Matemáticas, Universidad Nacional del Sur) is gratefully acknowledged.
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FOOTNOTES |
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Laboratorios Rontag SA (Buenos Aires, Argentina) generously provided a research fellowship to G. Picotto. Experimental activities were supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Universidad Nacional del Sur, Argentina, and from the World Academy of Sciences, Trieste, Italy.
Address for reprint requests: R. Boland, Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina.
Received 6 September 1996; accepted in final form 19 May 1997.
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