From the Service de Biologie Cellulaire, Département de Biologie Cellulaire et Moléculaire, CEA Saclay, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A new type of G protein-coupled membrane receptor that is activated by increasing concentrations of extracellular ionized calcium ([Ca2+]e)1 and polyvalent cations has been cloned recently from bovine parathyroid cells (BoPCaR1, Ref. 1) and rat kidney (RaKCaR, Ref. 2). When expressed in Xenopus oocytes, these Ca2+-sensing receptors are coupled to phospholipase C stimulation (1, 2). Increasing [Ca2+]e also stimulates phospholipase C activity in parathyroid cells (3, 4) and inhibits hormone-dependent cAMP accumulation (5), but, so far, no interaction between these two transduction pathways has been established. The presence of BoPCaR1 in parathyroid cells explains the crucial role of [Ca2+]e to elicit a negative feedback on parathyroid hormone secretion (6, 7). In rat kidney, a predominant expression of RaKCaR mRNA has been localized in the cortical portion of the thick ascending limb (8, 9), a segment which ensures cAMP-stimulated paracellular Ca2+ reabsorption, from the lumen of the renal tubule to the extracellular fluid compartments (10). A functional Ca2+ receptor is expressed in the plasma membrane of the rat and mouse cortical thick ascending limb (CTAL) as evidenced by the properties of the dose-dependent increase in the concentration of intracellular calcium ([Ca2+]i) as a function of peritubular [Ca2+]e (11, 12). High [Ca2+]e (5 mM) in the mouse CTAL decreases hormone-dependent cAMP accumulation, an effect which has been ascribed to a direct inhibition of adenylyl cyclase (AC) activity (13).
Experiments using quantitative reverse transcription-polymerase
chain reaction (RT-PCR) have shown that the rat CTAL also expresses the
Ca2+-inhibitable type 6 AC mRNA (14). All the
Gs-coupled receptors studied so far in this segment
activate a single pool of AC catalytic units (15, 16), and in addition,
electron microscopy studies describe a single cell type in this
epithelium (17). These observations lead to the hypothesis that the
functional expression of the type 6 AC mRNA accounts for the
hormone-dependent cAMP synthesis in the rat CTAL.
The aim of the present study was therefore to investigate the
functional expression of the AC present in the rat CTAL and the
consequences of the possible co-localization in a same cell of a
Ca2+-inhibitable AC and of a Ca2+-sensing
receptor on the regulation of cAMP synthesis and/or hydrolysis. In
order to study the regulation of cAMP levels elicited by potentially similar or different mechanisms of action, we compared the effect of
extracellular Ca2+ to those of two agents also active in
this segment. The first agent, angiotensin II, induces
[Ca2+]i increases in the rat CTAL (11, 18). The
pattern of the responses observed demonstrates that a same
intracellular Ca2+ pool is released by angiotensin II and
extracellular Ca2+ (11). The second agent, prostaglandin
E2 (PGE2), inhibits
hormone-dependent cAMP synthesis (19) likely as a result of
the interaction of the PGE2 receptor with a
GTP-dependent, pertussis toxin-sensitive Gi
protein as demonstrated in the medullary portion of the rat thick
ascending limb (20).
The experiments were performed on rat CTAL isolated by microdissection,
and the results establish that PGE2 (coupled to pertussis toxin-sensitive Gi protein), angiotensin II, and
extracellular Ca2+ (both coupled to phospholipase C
pathway) are effective in a same cell to decrease arginine
vasopressin-dependent cAMP accumulation. [Ca2+]e, in the physiological range, decreases
hormone-dependent cAMP accumulation by more than 50%, an
effect which results from both an inhibition of cAMP synthesis and an
increase of cAMP hydrolysis. Angiotensin II also regulates both
mechanisms, but its ability to inhibit cAMP synthesis is much smaller.
The adenylyl cyclase present in this segment has the functional
properties previously described for the Ca2+-inhibitable
type 6 AC (21, 22); in particular, AC activity is inhibited by both
G
i and phospholipase C pathways by different mechanisms.
Accordingly, in situ hybridization shows a homogeneous distribution of type 6 AC mRNA in CTAL cells. Taken together, the
results establish that the co-expression of a Ca2+ sensing
receptor and of a Ca2+-inhibitable AC in the rat CTAL cell
allows a specific inhibition by physiological
[Ca2+]e of hormone-stimulated cAMP intracellular
content.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Unless otherwise specified, the compounds were from Merck (Damstardt, Germany), Sigma, and Calbiochem.
In Situ Hybridization--
A probe specific of type 6 AC was
chosen in the most divergent region of AC cDNA sequences. A 376-bp
fragment (PvuII-SphI, nucleotides 3766-4143) of
the type 6 AC cDNA, located in the 3'-untranslated region, was
subcloned in pGEM3Zf(+) in the corresponding sites (SmaI-SphI). This sequence was included in the
region previously used for RT-PCR experiments (14). A 1080-bp fragment
corresponding to nucleotides 1205 (EcoRI) to 2285 (PvuII) of the type 5 AC cDNA coding region was
subcloned in BSSK+. Sense and antisense cRNA probes were
transcribed in vitro with T3, T7, or SP6 RNA polymerase
(Promega Biotech, Madison, WI) according to the manufacturer's
instructions, in the presence of -35S-UTP (>3,000
Ci/mmol, Amersham Pharmacia Biotech, Les Ulis, France).
Isolation of Rat Kidney Cortical Thick Ascending Limbs--
The
experimental conditions used to perform microdissection of intact
segments have been detailed previously (20, 24). Experiments were
performed on male Sprague-Dawley rats (120-150 g of body weight,
Iffa-Credo, France) that were maintained on a standard diet with free
access to water. After anesthesia, the left kidney was perfused with
microdissection medium containing 0.16% collagenase (Serva,
Boehringer, Mannheim, Germany). After hydrolysis of the kidney (20 min
at 30 °C in 0.12% collagenase solution), single CTALs (0.3-1.5 mm
length) were microdissected at 4 °C. The standard microdissection
medium was composed of the following (in mM): NaCl, 137;
KCl, 5; MgSO4, 0.8; Na2HPO4, 0.33; KH2P04, 0.44; MgCl2, 1;
NaHCO3, 4; CH3COONa, 10; CaCl2,
0.5; glucose, 5; HEPES, 20, pH 7.4, and 0.1% (w/v) bovine serum
albumin (fraction V, protease-free, minimal fatty acid content, Pentex,
Miles Inc., Kankakee, IL). This medium was supplemented with 10 µM ibuprofen and 0.5 unit/ml adenosine deaminase
(Boehringer Mannheim) to prevent the synthesis of prostaglandins and
the release of adenosine, agents known to interact with the regulation
of cAMP content in the rat kidney (20, 24). A nominally
Ca2+-free medium used in some of our protocols had the same
composition except that calcium was omitted; Ca2+-chelating
agents were not added to this medium because they have been described
to modify the sensitivity to Ca2+ of the
Ca2+-inhibitable AC isoforms (25). The concentrations of
free Ca2+ present in the media were measured by using an
electrode sensitive to Ca2+ (Radiometer, Copenhagen,
Denmark). The relationship between the theoretical concentrations of
Ca2+ added (0.5-5.0 mM) and those measured in
the medium was as follows: y = 1.05x 0.07,
r = 1.0.
Measurement of Hormone-dependent cAMP Accumulation-- The experimental conditions used to measure hormone-dependent cAMP accumulation on an intact single segment (20, 24) will be recalled briefly. Microdissected pieces of CTAL were transferred in 2 µl of incubation medium onto glass slides (1 or 2 pieces per slide) and photographed in order to measure their length. Unless otherwise specified, each sample was preincubated for 10 min at 30 °C in 0.5 mM [Ca2+]e and, after addition of 2 µl of incubation medium containing the agonists to be tested, incubated for a further 4 min at 35 °C. Adenylyl cyclase activity was stimulated by arginine vasopressin (AVP) which binds to V2 receptor in the rat CTAL, the only AVP receptor expressed in this segment (26). Due to the small number of cells per tubular sample (from about 100 to 600 cells), hormone-dependent cAMP accumulation can be measured only in the presence of a phosphodiesterase inhibitor. Either 50 µM Ro 20-1724, a specific inhibitor of the low Km cyclic AMP phosphodiesterase (27, 28), or 1 mM IBMX, inhibitor of all phosphodiesterases in the rat kidney (28), was added to the incubation medium. The concentrations of the different agents given in the results are those present during the incubation step. For longer preincubation periods, in experiments performed with Bordetella pertussis toxin and bisindolylmaleimide I, all media were supplemented with essential and nonessential amino acids as well as vitamins (minimum Eagle's medium, Eurobio, Les Ulis, France).
The amounts of cAMP were measured on acetylated samples by radioimmunoassay (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). The micromethod used allows the determination of 2-80 fmol of cAMP per reaction tube. In our conditions, the basal level of cyclic AMP present in a single piece of tubule is close to, or below, the sensitivity threshold of the assay (20, 24). The results were calculated in femtomoles of cyclic AMP accumulated per mm of tubular length per 4 min incubation time (fmol·mmMeasurement of Inositol Phosphate Production-- Assays were performed by using the microtechnique developed for proximal tubule fragments (29) with slight modifications. Briefly, in each experiment, CTAL (150-200 mm of total length) were microdissected from a collagenase-treated kidney. The medium used was supplemented with vitamins and amino acids as described above but contained 2 mM [Ca2+]e. CTAL pieces were radiolabeled in 50 µl of this medium containing myo-[3H]inositol (1 mCi/ml, Amersham Pharmacia Biotech) for 2 h at 30 °C. After this labeling period, tubules were extensively rinsed in 0.5 mM [Ca2+]e medium, and tubule samples (4-7 mm length each) were incubated at 37 °C during 15 min in 0.5 mM [Ca2+]e medium supplemented with lithium. The reaction was stopped, and the radioactivity associated with phosphoinositides, free inositol, inositol phosphates (IP), and glycerophosphoinositol was separated and counted as described previously (29).
In several experiments, it was checked that there was a good correlation between inositol phosphate formation expressed either per unit of tubule length or as a percentage of the total radioactivity counted (r = 0.93 ± 0.03, n = 4), an observation in agreement with that previously observed with proximal tubule fragments (29). Consequently, IP production was expressed as percentage of the total radioactivity counted. In each experiment, different experimental conditions (5 replicate samples each) were tested, and the mean of the IP values measured in each condition was taken as one single data point. The results are given as the mean values ± S.E. calculated from n different experiments. The statistical evaluation of the results was assessed by paired Student's t test.Measurement of Intracellular Ca2+ Concentration-- Intracellular Ca2+ concentration was measured in single CTAL samples microdissected from collagenase-treated kidneys by using the calcium-sensitive fluorescence probe fura-2 as described previously (11). Briefly, CTAL were loaded for 60 min with 10 µM fura-2 AM. Each CTAL was then transferred to a superfusion chamber fixed on the stage of an inverted fluorescence microscope (Zeiss IM 35, Oberkochen, Germany). The tubule was superfused at 37 °C at a rate of 10-12 ml/min, corresponding to about 10 exchanges per min. The composition of the superfusion medium was similar to that used in cAMP experiments, except that serum albumin, ibuprofen, and adenosine deaminase were not added since the superfusion medium was flushed continuously. After a 5- to 10-min equilibration period, agonists were added to either the 0.5 mM [Ca2+]e medium or to the Ca2+-free medium and superfused over tubule. Due to the dead space of the superfusion set up, the time necessary to achieve a full equilibration of agonist concentration in the chamber was of 15-20 s. The tubular portion selected for fluorescence measurement included about 30 cells. Double wavelength measurements of fura-2 fluorescence were recorded every 2 s.
Calculations of [Ca2+]i were performed as described previously (11). The results obtained from different tubules microdissected from several rats were expressed as mean values ± S.E. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Situ Hybridization
The localization of type 6 AC mRNA in the rat kidney was examined by in situ hybridization at the light microscope. The most intense labeling in the kidney cortex was found in thick ascending limb, including CTAL (Fig. 1), collecting duct, and glomeruli (not shown). Fig. 1 shows strong labeling of CTALs (a, c, and arrowheads), whereas proximal tubules (a, c, and *) are weakly labeled. In CTAL, labeling was equally intense in all cells. No labeling was observed when the sense cRNA probe was used (Fig. 1, b and d). In contrast to type 6 AC, no significant labeling for type 5 AC was found in CTAL (not shown). This is consistent with previous quantitative RT-PCR results showing that the type 6 AC mRNA was more abundant in CTAL than in proximal tubule and that type 5 AC mRNA was not detected in CTAL (14).
|
Effect of Extracellular Ca2+ on AVP-dependent cAMP Content and on the Activation of Phospholipase C
AVP was used at 10 nM, a concentration that induces a maximal stimulation of cAMP accumulation in intact cells (Ref. 19 and data not shown) and thus allows us to study the maximal amount of functional AC proteins present in the rat CTAL.
AVP-dependent cAMP accumulation was about 2-fold higher in the presence of 1 mM IBMX, which reflects AVP-stimulated cAMP synthesis only, than in the presence of 50 µM Ro 20-1724, which allows the measurement of cAMP accumulation that integrates both the synthesis and a partial catabolism of cAMP (Fig. 2, left panel).
|
In the presence of increasing [Ca2+]e, there was a dose-dependent inhibition of AVP-dependent cAMP levels with half-maximal inhibition of about 1.2 and 2.0 mM [Ca2+]e in the presence of Ro 20-1724 and IBMX, respectively (Fig. 2, right panel). With Ro 20-1724, a steep inverse relationship was observed, and thus a small variation of [Ca2+]e induced a high variation of hormone-dependent cAMP accumulation. The shape of the curve obtained with either IBMX or Ro 20-1724 shows that [Ca2+]e below 1.5 mM had a small effect on cAMP synthesis but increased cAMP hydrolysis; conversely, at higher [Ca2+]e the inhibition was mediated mainly by a decrease of cAMP synthesis.
Altogether in this study, the inhibition induced by 1.25 mM [Ca2+]e in the presence of Ro 20-1724 was of 63.3 ± 3.9% (p < 0.001 versus 10 nM AVP, n = 11) and that induced by 1.5 mM [Ca2+]e in the presence of IBMX was of 30.5 ± 3.5% (p < 0.01 versus 10 nM AVP, n = 8).
The implication of the Ca2+ receptor RaKCaR to explain the inhibitory effect of [Ca2+]e was supported by the results obtained with an agonist of this receptor, neomycin (1, 2). In 0.5 mM [Ca2+]e, 100 µM neomycin inhibited by 82.2 ± 5.8% (n = 3) and 43.5 ± 5.7% (n = 5) cAMP accumulated by 10 nM AVP in the presence of Ro 20-1724 and IBMX, respectively.
In the presence of IBMX, 2.5 mM [Ca2+]e inhibited cAMP synthesis stimulated by either 10 µM forskolin or 10 nM AVP with a comparable efficiency (62.3 ± 7.2% of inhibition and 73.5 ± 2.5% in forskolin and AVP experimental groups, respectively, n = 3). These results suggest that [Ca2+]e inhibited AC activity at post-receptor sites.
In our experimental conditions, no detectable [Ca2+]i variations were obtained with 1.25 mM [Ca2+]e (Table I), and the response to 1.5 mM [Ca2+]e was characterized in most tubules by a low but sustained [Ca2+]i increase without a clear-cut peak phase (Fig. 3 and Table I). With 2.5 mM [Ca2+]e, the response was a transient peak followed by a lower sustained plateau (Fig. 3) that reflects Ca2+ entry (11). The simultaneous superfusion of 10 nM AVP and [Ca2+]e did not modify [Ca2+]i variations (data not shown). Unlike the peak, the plateau was blocked by nonspecific Ca2+ channel blockers such as La3+ and Ni2+, whereas addition of voltage-sensitive channel blockers, i.e. verapamil and nifedipine, did not modify [Ca2+]i (Ref. 11 and data not shown).
|
|
These [Ca2+]i variations were due, at least in part, to phospholipase C activation since increasing [Ca2+]e elicited a dose-dependent production of IPs (Table I). A significant stimulation was observed with 1.25 mM [Ca2+]e, although this concentration had no detectable effect on [Ca2+]i. This apparent discrepancy is probably linked to the following two methodological reasons: (i) discrete and local [Ca2+]i variations may not have been detected in fura-2-loaded CTAL; (ii) a small stimulation of IP production is amplified by the 15-min duration of the incubation performed in the presence of lithium, which results in IP accumulation, whereas [Ca2+]i changes are transient.
Comparison of the Effects of the Ca2+ Receptor and Others Agonists to Decrease AVP-dependent cAMP Accumulation
Angiotensin II and PGE2 were used at concentrations inducing maximal effects (18, 19), and in most experiments, their regulatory properties were studied in parallel to those of [Ca2+]e.
As expected from previous observations (11, 18), 0.1 µM angiotensin II increased IP production and [Ca2+]i (Fig. 3 and Table I). The pattern of [Ca2+]i variations was characterized by a single peak with no apparent plateau phase and thus was different from the response observed with [Ca2+]e. By contrast, 0.3 µM PGE2 did not increase [Ca2+]i or IP production (Fig. 3 and Table I).
Action on cAMP Synthesis and/or on cAMP Hydrolysis-- Fig. 4A shows the mean inhibitions obtained in the presence of either Ro 20-1724 or IBMX. In these two conditions, 0.3 µM PGE2 elicited an inhibition of about 60%, which suggests that PGE2-mediated inhibition was due only to a decrease of cAMP synthesis in the CTAL. In the presence of Ro 20-1724, the inhibition induced by angiotensin II was of smaller extent than that obtained with 2.5 mM [Ca2+]e (Fig. 4A). The use of IBMX instead of Ro 20-1724 decreased angiotensin II-mediated inhibition by more than 50% (p < 0.001 versus Ro 20-1724) and also reduced, but more slightly, the inhibitory effect of 2.5 mM [Ca2+]e (p < 0.005 versus Ro 20-1724). Therefore, although both 2.5 mM [Ca2+]e and angiotensin II induced high [Ca2+]i peak values (Fig. 3), the inhibition of AVP-dependent cAMP synthesis was much more pronounced with the addition of [Ca2+]e.
|
|
|
Interaction with the GTP-dependent Gi
Protein--
When expressed in Xenopus oocytes (1), the
Ca2+ receptor BoPCaR1 stimulates IP production by a process
sensitive to pertussis toxin (PTX). The possible role of a
GTP-dependent pertussis toxin-sensitive G protein in the
inhibitions observed was studied in CTAL preincubated with 500 ng/ml
PTX. When compared with the corresponding responses obtained with 10 nM AVP alone, PGE2-induced inhibition was of 63.4 ± 3.2% (p < 0.001) in control CTAL and was
decreased to 28.4 ± 3.4% (p < 0.05 when
compared with AVP) in PTX-treated CTAL (Fig. 5). A preincubation with PTX therefore
suppressed by more than 50% the inhibition elicited by
PGE2, a result identical to that observed in the medullary
portion of this segment and due very likely to the interaction of the
PGE2 receptor with G
i proteins (20). In
contrast, angiotensin II- and 2.5 mM
[Ca2+]e-mediated inhibition measured in the same
experiments were not modified by PTX (Fig. 5).
|
Experiments of Multiple Combined Inhibition--
The results
presented above indicate that only PGE2-mediated inhibition
was sensitive to pertussis toxin (Fig. 5), whereas both
PGE2 and 2.5 mM [Ca2+]e
inhibited AVP-dependent cAMP synthesis with the same efficiency (Fig. 4A). These results suggest that the two
agents inhibit cAMP synthesis by two different mechanisms. In order to confirm this hypothesis, and to examine whether these two effects occurred or not in the same cell, we performed experiments of multiple
combined inhibition in the presence of IBMX. Experimental conditions
and criteria previously defined (24) were used to test a possible
summation of inhibitions (if in different cells) or cumulative
inhibition (if different mechanisms in a same cell) of AC activity. The
residual cAMP content observed with the simultaneous addition of
PGE2 and [Ca2+]e was lower than the
value obtained with each inhibitor alone, but the response to AVP was
not fully abolished although inhibitions higher than 50% were obtained
with each agent alone (Table IV). This
result indicates that PGE2 and extracellular Ca2+ were active in a same cell. Analysis of the results
showed that the measured cAMP value (11.5 ± 3.3 fmol·mm1·4 min
1, Table IV) was not
different from the theoretical cAMP content calculated by assuming an
hypothesis of different mechanisms leading to a cumulative inhibition
(13.1 ± 4.0 fmol·mm
1·4 min
1).
These results and those obtained in pertussis toxin-treated tubules
(Fig. 5) demonstrate that PGE2 and
[Ca2+]e inhibited AVP-dependent AC
activity by different and independent mechanisms effective on a same
pool of catalytic units.
|
Role of Intracellular Ca2+ Release and/or Capacitive Ca2+ Influx to Inhibit cAMP Synthesis
The results presented above establish that the decrease of AVP-dependent cAMP accumulation induced by [Ca2+]e or angiotensin II results partly from an inhibition of cAMP synthesis. [Ca2+]e induced both cytosolic Ca2+ release from intracellular stores and extracellular Ca2+ influx (Ref. 11, Table I). Different approaches were used in the presence of IBMX to assess the relative importance of these respective [Ca2+]i variations in the inhibition of cAMP synthesis.
The potential inhibitory effect of the capacitive Ca2+ influx was investigated by the use of thapsigargin. This inhibitor of the endoplasmic reticular Ca2+-ATPase (31) depletes intracellular Ca2+ stores which was shown to produce capacitive Ca2+ entry (32). Thapsigargin added into the Ca2+-free medium evoked a transient [Ca2+]i rise which declined slowly to basal value reflecting Ca2+ release from intracellular stores and Ca2+ extrusion from the CTAL cell (Fig. 6A, lower part). On thapsigargin-treated CTAL, the addition of 200 µM neomycin did not evoke further [Ca2+]i increase indicating that 1 µM thapsigargin had actually emptied intracellular Ca2+ stores sensitive to Ca2+ receptor in the CTAL (data not shown). By contrast, the addition of 1.25 mM [Ca2+]e elicited high [Ca2+]i increases (Fig. 6A, lower part) which reflect a capacitive Ca2+ influx associated to the large emptying of intracellular Ca2+ pools induced by thapsigargin. Without thapsigargin, the addition of 1.25 mM [Ca2+]e to Ca2+-free medium induced only a small and progressive elevation of [Ca2+]i (Fig. 6A, upper part). This discrete increase likely reflects a slow Ca2+ entry consecutive to partial depletion of intracellular Ca2+ stores induced by superfusion in the Ca2+-free medium.
|
The consequences on cAMP synthesis of the changes in
[Ca2+]i elicited by the addition of 1.25 mM [Ca2+]e were studied in parallel
experiments on CTAL preincubated in the Ca2+-free medium
with or without thapsigargin. AVP-stimulated cAMP value was of the same
magnitude in both cases (139.6 ± 12.9 fmol·mm1·4 min
1 and 157.9 ± 13.3, n = 4, without and with thapsigargin, respectively), which indicates that thapsigargin-induced intracellular
Ca2+ release during the preincubation step had no
inhibitory effect on cAMP synthesized during the following incubation
step. AVP-dependent cAMP synthesis was significantly
inhibited by 1.25 mM [Ca2+]e in CTAL
treated with thapsigargin (mean value of 24.1 ± 1.9% of
inhibition, p < 0.05, n = 4), whereas
there was no inhibition in the absence of thapsigargin (Fig.
6B). In three of these experiments, the addition of 2.5 mM [Ca2+]e on CTAL treated with
thapsigargin inhibited cAMP synthesis by 69.1 ± 2.0%, a value
similar to that observed in control CTAL (see Figs. 2 and 4).
The potential inhibition of cAMP synthesis induced only by Ca2+ release from intracellular pools was studied in the Ca2+-free medium with the Ca2+-sensing receptor agonist neomycin to avoid Ca2+ influx associated with [Ca2+]e. Neomycin was added after 10 min superfusion or preincubation of CTAL samples in either the 0.5 mM [Ca2+]e control medium or the Ca2+-free medium. In control medium, neomycin induced [Ca2+]i responses similar to those observed with [Ca2+]e but with a low plateau phase (Fig. 7A compared with Fig. 3). The mean [Ca2+]i peak values were dose-dependent (Fig. 7B), an observation in agreement with the effect of neomycin on IP production; the mean stimulation factor of phospholipase C activity was 2.1 ± 0.3 (n = 4 experiments), 4.7 ± 0.3 (n = 3), and 10.0 ± 2.0 (n = 3) with 100, 200, and 400 µM neomycin, respectively. In Ca2+-free medium, neomycin-induced [Ca2+]i peak values were decreased at all the concentrations tested (Fig. 7B), and the plateau phase was not observed (Fig. 7A).
|
Fig. 7C shows the consequence on AVP-dependent cAMP synthesis of neomycin-evoked intracellular Ca2+ release. In the Ca2+-free medium, 100 µM neomycin did not impair cAMP synthesis in contrast to 200 and 400 µM. The mean data (Fig. 7C) indicate that the levels of inhibition obtained in the Ca2+-free medium (37.7 ± 3.4% of inhibition and 67.3 ± 4.7% with 200 and 400 µM neomycin, respectively) were decreased when compared with the corresponding values obtained in the 0.5 mM [Ca2+]e (64.9 ± 3.4% of inhibition and 79.5 ± 1.9% with 200 and 400 µM, respectively).
The lower inhibitions of cAMP obtained in the Ca2+-free medium could be due to the absence of Ca2+ influx and/or to the decrease of intracellular Ca2+ release (Fig. 7). In the absence of extracellular Ca2+, this last effect may be secondary to partial emptying of intracellular Ca2+ stores or to an alteration of the binding of neomycin to the Ca2+ receptor. Additional experiments were performed in CTAL superfused or preincubated in a Ca2+-free medium for only 2 min to prevent the emptying of intracellular Ca2+ stores. In such conditions, 200 µM neomycin evoked intracellular [Ca2+]i peaks of magnitude (418 ± 52 nM, Fig. 8) similar to that observed in 0.5 mM [Ca2+]e medium (436 ± 47 nM, Fig. 7B). With a same experimental timing, the inhibition of cAMP production was 59.9 ± 3.7% (Fig. 8), a value close to that obtained in control medium (64.9 ± 3.4%, Fig. 7C). The similarity of the responses observed in Ca2+-free medium and control medium indicates that the binding of neomycin is not affected by the absence of extracellular Ca2+. Moreover, these results establish that neomycin-evoked intracellular Ca2+ release alone can induce an inhibition of cAMP synthesis comparable to that obtained in 0.5 mM [Ca2+]e medium, a condition in which neomycin induces only a small Ca2+ entry (Fig. 7A). By comparison, angiotensin II elicited high [Ca2+]i peak values in CTAL superfused for 2 min in the Ca2+-free medium (603 ± 104 nM, Fig. 8), and its inhibitory effect on cAMP synthesis was in the range of that obtained with angiotensin II in 0.5 mM [Ca2+]e medium (Fig. 4) with a mean value of only 14.4 ± 2.8% (Fig. 8). Altogether these results therefore support the hypothesis of a specific role played by the activation of the Ca2+ receptor in the inhibition of AVP-dependent cAMP synthesis.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study demonstrates that in a same epithelial cell of the rat
renal tubule PGE2, angiotensin II and the activation of the
Ca2+ receptor inhibit the same pool of
AVP-dependent intracellular cAMP content by different
mechanisms, i.e. Gi-mediated or
Ca2+-mediated inhibition of AC activity and PDE-mediated
hydrolysis of cAMP. In particular, the strong and specific inhibition
of hormone-dependent cAMP synthesis by
[Ca2+]e in the CTAL likely results from the
co-expression of the Ca2+ receptor RaKCaR (8, 9, 11) and of
the Ca2+-inhibitable type 6 AC (Ref. 14 and this
study).
Transduction Pathways Involved in PGE2-, Angiotensin
II-, and [Ca2+]e-mediated Inhibition of cAMP
Content--
Previous studies have established that PGE2
decreases AVP-mediated cAMP synthesis in the rat thick ascending limb
(19, 20). The preincubation of isolated segments with PTX reverses, at
least partly, this inhibition in medullary (20) and cortical (Fig. 5)
segments. The PGE2 receptor is therefore coupled to
Gi proteins in the thick ascending limb, in agreement
with the presence of the PGE2 receptor EP3
subtype (33) usually associated with the inhibition of AC activity
(34). Multiple isoforms of the EP3 subtype have been
described, some of which coupled to both G
i-mediated inhibition of AC activity and activation of phospholipase C (35). In
our experiments, PGE2 did not increase IP production or
[Ca2+]i (Table I). In addition, the magnitude of
PGE2-mediated inhibition did not depend on the type of
phosphodiesterase inhibitor added to the incubation medium. Thus, very
likely, the coupling of PGE2 receptor to
G
i-mediated inhibition of AC activity alone accounts for
the effect of PGE2 observed on cAMP synthesis in the rat
CTAL.
Mechanisms of Inhibition Induced by Angiotensin II and [Ca2+]e on AVP-dependent cAMP Accumulation-- Different mechanisms account for the inhibition of AVP-dependent cAMP accumulation induced by [Ca2+]e, depending on the Ca2+ concentration studied. Low [Ca2+]e, below 1.5 mM, and angiotensin II used at maximal concentration elicit a modest inhibition of cAMP synthesis (Figs. 2 and 4). These inhibitions are in the range of those previously observed with agonist-evoked phospholipase C activation in cell lines that express type 6 AC (41, 42). Angiotensin II and low [Ca2+]e also induce an hydrolysis of cAMP likely mediated by Ca2+/calmodulin-dependent PDEs. The data from Fig. 2 suggest that regulation of cAMP hydrolysis has a higher sensitivity to Ca2+ than regulation of cAMP synthesis, and thus the hydrolysis of cAMP appears to be the major process that accounts for the decrease of AVP-dependent cAMP accumulation (Figs. 2 and 4 and Table II). Therefore, the small inhibitory effect on cAMP synthesis induced by 1.25 mM [Ca2+]e or angiotensin II is amplified by the cAMP hydrolysis leading to a total inhibition of AVP-dependent cAMP accumulation of about 50% (Figs. 2 and 4 and Table II).
From about 1.5 mM, [Ca2+]e induces a higher inhibition of AVP-dependent cAMP accumulation due to an additional decrease of cAMP synthesis, an effect becoming dominant with high [Ca2+]e (Fig. 2). Previous experiments in the mouse medullary thick ascending limb have established that the inhibitory effect of 5.0 mM [Ca2+]e on hormone-dependent cAMP synthesis is not due to metabolic or cellular damages (43). These observations support the hypothesis that, even at high concentrations, the effect of [Ca2+]e is actually a regulating process. Although both angiotensin II and [Ca2+]e stimulate phospholipase C pathway in the CTAL, the relative effects of these agents on cAMP synthesis suggest that [Ca2+]e elicits an additional and specific mechanism of action. As will be discussed in the following sections, this specific action of [Ca2+]e might be linked to the co-expression of a functional Ca2+-inhibitable AC and of a Ca2+-sensing receptor in the CTAL cell.Functional Characteristics of the Adenylyl Cyclase Expressed in the Cortical Thick Ascending Limb-- Among the different isoforms of adenylyl cyclase known up to date, types 4-6 sequences have been cloned from rat tissues and are expressed in the kidney (21). The type 9 AC cloned from mouse cell lines or tissues (44, 45) appears also expressed in the rat kidney (46). Experiments of quantitative RT-PCR have demonstrated the expression of the Ca2+-inhibitable type 6 AC mRNA in the CTAL but not types 4 or 5 AC mRNA (14). Our in situ hybridization results establish that type 6 AC mRNA was expressed in all cells along CTAL (Fig. 1), whereas type 5 AC mRNA was not detected. If present in epithelial cells, the localization of type 9 AC mRNA along the rat renal tubule is not known.
Several properties of regulation of AVP-dependent cAMP accumulation were observed in this study as follows. (i) [Ca2+]e has an inhibitory effect on either hormone- or forskolin-stimulated cAMP production. (ii) cAMP synthesis is inhibited by agents coupled either to an activation of phospholipase C or to a GSpecific Inhibition of cAMP Synthesis by Extracellular Ca2+-- As already underlined, both [Ca2+]e and angiotensin II activate the phospholipase C pathway in the same cell but have different consequences on the parameters studied. Indeed, for a comparable magnitude of inhibition of cAMP synthesis (15-20%, Fig. 2 and 4), a high [Ca2+]i peak was observed with angiotensin II, whereas no detectable [Ca2+]i variations were obtained with 1.25 mM [Ca2+]e in our experimental conditions (Table I). Conversely, for comparable [Ca2+]i peak values, activation of the Ca2+ receptor, but not angiotensin II, induced about 60% of inhibition of cAMP synthesis (Fig. 8). We also observed in the rat CTAL that a maximal concentration of bradykinin has properties similar to those of angiotensin II on [Ca2+]i variations and inhibition of cAMP accumulation (data not shown). These results support the hypothesis of a specific role of Ca2+ receptor activation in the inhibition of cAMP synthesis.
By contrast to angiotensin II, [Ca2+]e and the Ca2+ receptor agonist, neomycin, elicit a sustained increase of [Ca2+]i which reflects an entry of Ca2+ (Ref. 11 and Figs. 3 and 7). An activation of cation channels by neomycin has been observed in Ca2+ receptor transfected cells (51), and such an effect in CTAL cell could account, at least in part, for this Ca2+ influx. The entry of Ca2+ might partly explain the properties of [Ca2+]e in the inhibition of cAMP synthesis since (i) previous experiments have demonstrated that capacitive Ca2+ influx inhibits type 6 AC activity in a glioma cell line (42), and (ii) in the present study, a high inhibition of cAMP synthesis can be obtained in thapsigargin-treated CTAL. However, the experiments with neomycin in Ca2+-free medium (Figs. 7 and 8) establish that a release of Ca2+ from intracellular stores can also induce a high inhibition of cAMP synthesis. Therefore, the presence of a Ca2+ influx, on its own, does not totally explain the specific inhibition of cAMP synthesis induced by the activation of the Ca2+ receptor. The differential inhibitory effect observed with angiotensin II and [Ca2+]e might be linked to the activation of another transduction pathway by one of these agents, and we have no formal argument to support or exclude such an hypothesis. The specific properties observed with the activation of the Ca2+ receptor recalled above lead, however, to another hypothesis supported by observations made in other polarized epithelial cells (52, 53). Indeed in the renal line of Madin-Darby canine kidney cells, inositol 1,4,5-triphosphate receptors have multiple cellular localization including sites close to the basolateral plasma membrane and can thus generate localized [Ca2+]i increases (52). In nasal epithelial cell preparations, phospholipase C activation results in [Ca2+]i release and Ca2+ influx in close proximity to the stimulated receptor (53). These data might explain the high efficiency of cAMP synthesis inhibition by [Ca2+]e if the Ca2+ receptor was more closely associated to type 6 AC than the angiotensin II receptor in the CTAL plasma membrane. An intimate co-localization in a subdomain of the basal membrane of the Ca2+ receptor and of the Ca2+-inhibitable AC might also explain the similar inhibition of cAMP synthesis observed with either a local [Ca2+]i increase (Ca2+ influx and/or Ca2+ release) elicited by low [Ca2+]e or a high intracellular Ca2+ release induced by angiotensin II. In addition, the cAMP hydrolysis observed with low [Ca2+]e or angiotensin II was similar (Table II), suggesting that a local [Ca2+]i increase must be effective to activate Ca2+/calmodulin-dependent PDEs. These hypotheses cannot be tested on isolated tubule samples at the present time. Whatever the precise mechanism involved, the co-expression of the Ca2+-inhibitable type 6 AC and of the Ca2+-sensing receptor RaKCaR in the plasma membrane very likely allows the specific inhibition of AC activity observed with [Ca2+]e. This conclusion is supported by previous results obtained in the vasopressin-sensitive cell of the rat outer medullary collecting tubule (14). In this cell type, different data support the functional expression of the type 6 AC, but [Ca2+]e increases do not inhibit cAMP synthesis (14). In contrast to the CTAL, there is no evidence for the presence of the Ca2+ receptor RaKCaR in the plasma membrane of the medullary collecting tubule (8, 9). The co-expression (CTAL) or the absence of co-expression (vasopressin-sensitive cell of the outer medullary collecting tubule) of the type 6 AC and of the Ca2+ receptor RaKCaR may explain, therefore, the different effect of [Ca2+]e on AC activity observed in these segments. From a physiological point of view, the inhibition of AC activity in the CTAL cell, together with an effect on cAMP hydrolysis, confers a high sensitivity to [Ca2+]e on the inhibition of hormone-stimulated cAMP accumulation. The steep slope (Fig. 2) obtained shows that a large variation of cAMP content is elicited by a small change in [Ca2+]e. It is noticeable that a same slope has been observed in parathyroid cells, and 1.0-1.2 mM [Ca2+]e defined as the "set point" of parathyroid hormone secretion, i.e. the concentration at which hormonal secretion is inhibited by 50% (6). Similarly in the kidney, the present data show that inhibition of hormone-stimulated cAMP accumulation is exquisitely sensitive to variations in [Ca2+]e that are physiologically relevant thus allowing the modulation of cAMP-mediated luminal Ca2+ reabsorption usually present in the CTAL (10). At higher [Ca2+]e, like in hypercalcemia (6, 7), the strong inhibition of cAMP synthesis may be a potent factor to decrease sharply hormone-dependent cAMP content and thus to block Ca2+ reabsorption. Our functional data support that the co-expression of a Ca2+-inhibitable AC and of a Ca2+-sensing receptor in the plasma membrane of the CTAL cell contributes to the regulation of one of its main physiological functions. ![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to N. Griffiths and A. Doucet for critical advice and careful review of this manuscript; to J. M. Gasc for helpful suggestions to develop the in situ hybridization technique; to R. Rajerison and M. Bloch-Faure for their valuable advice in the development of phosphoinositide assay; and to H. Moysan and S. David for helpful technical assistance.
![]() |
Note Added in Proof |
---|
While this manuscript was being typeset, results describing an inhibitory effect of [Ca2+]e increases on chloride reabsorption in rat CTAL have been accepted for publication (M. C. de Jesus Ferreira and C. Bailly, Am. J. Physiol., in press).
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant URA 1859 from the Centre National de la Recherche Scientifique and from the CEA (DBCM, SBCe).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.
Supported by a doctoral fellowship from the Ministère de
l'Education Nationale, de la Recherche et de la Technologie.
§ Supported by a postdoctoral fellowship from the Commissariat à l'Energie Atomique.
¶ To whom correspondence should be addressed: DBCM, SBCe, Bât. 520, CEA Saclay, 91191 Gif sur Yvette, France. Tel.: 33 (0)1 69 08 97 61; Fax: 33 (0)1 69 08 35 70; E-mail: chabardes{at}dsvidf.cea.fr.
1 The abbreviations used are: [Ca2+]e, extracellular free concentration of Ca2+; AC, adenylyl cyclase; AVP, arginine vasopressin; [Ca2+]i, intracellular free concentration of Ca2+; CTAL, cortical portion of the thick ascending limb; IBMX, 3-isobutyl-1-methylxanthine; IP, inositol phosphates; PDE, phosphodiesterase; PGE2, prostaglandin E2; PTX, Bordetella pertussis toxin; RT-PCR, reverse transcription-polymerase chain reaction; Ro 20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|