Cellular localization of type 5 and type 6 ACs in collecting
duct and regulation of cAMP synthesis
Cécile
Héliès-Toussaint1,
Lotfi
Aarab1,
Jean-Marie
Gasc2,
Jean-Marc
Verbavatz1, and
Danielle
Chabardès1
1 Service de Biologie Cellulaire, Commissariat à
l'Énergie Atomique/Saclay, 91191 Gif-sur-Yvette, and
2 Laboratoire de Médecine Expérimentale,
Collège de France, 75005 Paris, France
 |
ABSTRACT |
The cellular distribution of
Ca2+-inhibitable adenylyl cyclase (AC) type 5 and type 6 mRNAs in rat outer medullary collecting duct (OMCD) was performed by in
situ hybridization. Kidney sections were also stained with specific
antibodies against either collecting duct intercalated cells or
principal cells. The localization of type 5 AC in
H+-ATPase-, but not aquaporin-3-, positive cells
demonstrated that type 5 AC mRNA is expressed only in intercalated
cells. In contrast, type 6 AC mRNA was observed in both intercalated
and principal cells. In microdissected OMCDs, the simultaneous
superfusion of carbachol and PGE2 elicited an additive
increase in the intracellular Ca2+ concentration,
suggesting that the Ca2+-dependent regulation of these
agents occurs in different cell types. Glucagon-dependent cAMP
synthesis was inhibited by both a pertussis toxin-sensitive
PGE2 pathway (63.7 ± 4.6% inhibition, n = 5) and a Ca2+-dependent carbachol
pathway (48.6 ± 3.3%, n = 5). The simultaneous addition of both agents induced a cumulative inhibition of
glucagon-dependent cAMP synthesis (78.2 ± 3.3%,
n = 5). The results demonstrate a distinct cellular
localization of type 5 and type 6 AC mRNAs in OMCD and the functional
expression of these Ca2+-inhibitable enzymes in
intercalated cells.
calcium-inhibitable andenylyl cylcase; in situ hybridization; microdissected collecting duct; intracellular calcium; glucagon; carbachol; prostaglandin E2; rat kidney
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INTRODUCTION |
IN MANY CELL
TYPES, the intracellular Ca2+ concentration
([Ca2+]i) regulates cAMP levels through
interactions of Ca2+ on cAMP synthesis and/or cAMP
hydrolysis. These effects of [Ca2+]i are
linked to the presence of Ca2+-sensitive adenylyl cyclases
(ACs) and/or Ca2+/calmodulin-dependent phosphodiesterases
(7, 13, 17). Among AC isoforms,
the enzymatic activity of type 5 and type 6 AC is directly inhibited by
submicromolar concentrations of Ca2+ (25,
31, 33). Ca2+-inhibitable AC
isoforms are sensitive to [Ca2+]i, typically
achieved in intact cells as a consequence of phospholipase C activation
and/or Ca2+ channel activation (4).
Two cell types have been described in kidney collecting duct: principal
cells, involved in water homeostasis (30), are the predominant cell type in the collecting duct. In rat kidney, type A
intercalated cells, involved in proton secretion and bicarbonate reabsorption, are found in cortical and outer medullary collecting ducts, whereas type B intercalated cells, mostly found in cortical collecting ducts, function in the other direction (5).
Type 5 and type 6 AC mRNAs are both expressed in the rat kidney outer medullary collecting duct (OMCD) (6), where principal and
type A intercalated cells are the two major cell types. In
microdissected OMCDs, AC activity is stimulated by arginine vasopressin
(AVP) and glucagon (8, 22). Physiological and
biochemical results support the conclusion that AVP stimulates AC
activity in principal cells, whereas glucagon is active in intercalated
cells ( 6, 8, 21).
An inhibitory effect of Ca2+ on the cAMP pathway in the rat
OMCD was first suggested by the negative regulation of a
Ca2+ ionophore on AVP-dependent cAMP accumulation
(19). PGE2 and carbachol, a muscarinic agonist
of the acetylcholine receptor, both induce a Ca2+-dependent
inhibition of hormone-stimulated intracellular cAMP accumulation
(1, 6), which is probably linked to the
[Ca2+]i increases elicited by
PGE2 and carbachol in rat OMCD (1, 20). However, the effect of these agents on cAMP
accumulation is cell type and agonist dependent. Indeed,
PGE2 induces only a small inhibition of AVP-stimulated cAMP
synthesis, and its Ca2+-dependent inhibitory effect on
AVP-dependent cAMP accumulation is due mainly to an increase in cAMP
hydrolysis (1, 9). In contrast, carbachol,
which has no effect on the response to AVP (2,
6), induces a marked Ca2+-dependent inhibition
of glucagon-stimulated AC activity (6). In addition,
glucagon-stimulated cAMP synthesis is inhibited by extracellular
Ca2+, whereas AVP-dependent cAMP synthesis is not
(6). These observations therefore show that the inhibitory
effect of either extracellular Ca2+ or agonist-mediated
[Ca2+]i changes on intracellular cAMP are
cell type dependent. These differences could be accounted for, at least
partly, by the cellular localization of type 5 and/or type 6 functional
AC proteins in the OMCD.
The purpose of the present experiments was to study the potential role
of Ca2+-inhibitable AC isoforms in the regulation of cAMP
synthesis in the rat OMCD cells.
The localization of type 5 and type 6 AC mRNAs at the cellular level
was performed by in situ hybridization. Only type 6 mRNA was detected
in rat kidney collecting duct principal cells, whereas both type 5 and
type 6 Ca2+-inhibitable AC isoforms were found in
intercalated cells. In superfused OMCDs, the effects of
PGE2 and carbachol on [Ca2+]i
were additive, suggesting that the Ca2+ increase elicited
by these agents occurs in different cell types. One fundamental
property of Ca2+-inhibitable AC isoforms is to be regulated
by independent Ca2+- and G
i-mediated
processes, leading to a cumulative inhibition of AC activity
(7, 14, 13, 29).
This property cannot be verified accurately in principal cells because
the Ca2+-dependent inhibition of PGE2 on
AVP-stimulated cAMP synthesis is of very low magnitude (1,
7, 9). In contrast, such a dual regulation
can be studied in OMCD intercalated cells, where about one-half of
glucagon-dependent AC activity is inhibited by either a
Ca2+-dependent carbachol pathway (6) or a
G
i-sensitive PGE2 pathway (3).
In our study, the simultaneous addition of carbachol and PGE2 induced a cumulative inhibition of glucagon-dependent
cAMP synthesis, demonstrating that these agents inhibit the same AC catalytic units. The results therefore localize
Ca2+-inhibitable AC mRNAs in OMCD and demonstrate the
functional expression of Ca2+-inhibitable AC isoforms in
intercalated cells.
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METHODS |
Unless otherwise specified, reagents used were purchased from
Merck (Damstardt, Germany), Sigma Chemical (St. Louis, MO), and
Calbiochem (San Diego, CA). Experiments were performed in male
Sprague-Dawley rats (140-180 g body wt, Iffa-Credo), maintained on
a standard diet with free access to water.
Northern blots.
Hybridization probes for type 5 and type 6 AC were prepared by random
primed labeling of the regions described for in situ hybridization
experiments using [
-32P]dCTP. A multiple rat tissue
Northern blot (Clontech Laboratories) was hybridized, as per the
manufacturer's instructions, in 5 ml of ExpressHyb solution at 68°C
for 30 min. The probe was added to 5 ml of fresh ExpressHyb solution
and incubated at 68°C for 1 h. Washes were performed as follows:
40 min (with 3 changes of the solution) in solution 1 (2×
SSC-0.05% SDS) at room temperature, followed by an incubation
of 40 min at 50°C in solution 2 (0.1× SSC-0.1% SDS). The
blot was then wrapped in plastic and exposed to X-ray film with an
intensifier screen at
80°C for 3 days.
In situ hybridization and immunostaining.
Probes specific for type 5 and 6 ACs were designed in the most
divergent regions of AC cDNA regions. A 376-bp fragment (Pvu II-Sph I, nucleotides 3965-4342) of type 6 AC cDNA,
located in the 3'-untranslated region, was subcloned in pGEM3Zf(+)
(Promega Biotech, Madison, WI). A 1,080-bp fragment corresponding to
nucleotides 1205-2285 (EcoR I, Pvu II) of
type 5 AC cDNA coding region was subcloned in BSSK+ (Stratagene). Sense
and antisense cRNA probes were in vitro transcribed with T3, T7, or SP6
RNA polymerases (Promega Biotech) according to the manufacturer's
instructions, in the presence of [35S]UTP
S
(>1,000 Ci/mmol, Amersham, Les Ulis, France).
Rat kidneys were fixed in 4% paraformaldehyde in PBS, washed in PBS,
and dehydrated with a graded series of ethanol and butanol. Tissues
were paraffin embedded, and 4- µm sections were collected on
silane-coated slides. In situ hybridization was performed as described
by Sibony et al. (26). Briefly, slides were deparaffinized in toluen and rehydrated by a graded series of ethanol (100-30%). After boiling in a solution of 0.01 M citric acid in a microwave oven,
slides were treated with 0.1% H2O2 in PBS.
After fixation in 4% paraformaldehyde-PBS, and proteinase K treatment,
slides were covered with hybridization buffer [final concentrations: 50% formamide, 10% dextran sulfate, 1 mg/ml SS-DNA, 2× SSC, 70 mM
dithiothreitol (DTT)] and 0.5-1.106 counts · min
1 radiolabeled probe · tissue
section
1. Hybridization was performed overnight at 50°C
in a humidified chamber. Slides were rinsed in 5× SSC-10 mM DTT
followed by an incubation in 50% formamide-1× SSC-12.5 mM DTT. They
were further treated by RNAase A (20 µg/ml). For immunostaining, the
slides were then incubated for 5 min in PBS containing 1% BSA
(PBS/BSA), followed by overnight incubation of a 1:200 dilution of
anti-aquaporin-3 (AQP3) or anti-vacuolar H+-ATPase 56-kDa
subunit (kindly provided by Dr. D. Brown, Massachusetts General
Hospital, Charlestown, MA) antiserum in PBS/BSA. The sections were then
washed 3 × 10 min in PBS, followed by a 2-h incubation in
horseradish peroxidase-conjugated mouse anti-rabbit antibodies (6 µg/ml) in PBS/BSA. The sections were washed 2 × 10 min in PBS. Staining was revealed with diaminobenzidine, and slides were washed overnight in 50 mM Tris · HCl, pH = 8. Finally, the slides
were exposed for 3-5 wk to Kodak NTB2 liquid emulsion,
counterstained with toluidine blue and examined under the microscope
(Olympus Van OX).
Isolation of rat kidney OMCDs.
The experimental procedure used to microdissect intact segments from
collagenase-treated rat kidney has previously been detailed (8, 9). After the rats were anesthetized
(pentobarbital, 6 mg/100 g body wt), the left kidney was perfused with
microdissection medium containing 0.16% collagenase (Serva, Boehringer
Mannheim). After hydrolysis of the kidney (20 min at 30°C in 0.12%
collagenase solution), single pieces (0.3-1.5 mm length) of
collecting duct were microdissected at 4°C from the outer medulla.
The standard microdissection medium was composed of (in mM) 137 NaCl; 5 KCl; 0.8 MgSO4; 0.33 Na2HPO4; 0.44 KH2PO4; 1 MgCl2; 4 NaHCO3; 10 CH3COONa; 1.0 or 2.0 CaCl2 (see below); 5 glucose; and 20 HEPES, pH 7.4, and
0.1% (wt/vol) BSA (fraction V, Pentex, Miles, Kankakee, IL).
Measurement of [Ca2+]i.
[Ca2+]i was measured in single OMCD samples by using the
calcium-sensitive fluorescence probe acetoxymethyl ester of fura 2 (fura 2-AM, Molecular Probes, Eugene, OR) as previously described (1, 10, 27). Briefly, the
samples were loaded for 60 min with 10 µM fura 2-AM. Each tubule was
then transferred to a superfusion chamber fixed on an inverted
fluorescence microscope (Zeiss IM 35, Oberkochen, Germany). Tubules
were superfused at 37°C at a rate of 10-12 ml/min, corresponding
to ~10 exchanges/min. The superfusion medium (microdissection medium
without serum albumin) contained either 2 mM Ca2+ or no
Ca2+ (nominally Ca2+-free medium without
CaCl2 and containing 0.1 mM EGTA). After a 5- to 10-min
equilibration period, agonists were added to the medium and superfused
over tubules. Because of the dead space of the superfusion setup, the
time necessary to achieve a full equilibration was 15-20 s. A
circular area of 60-µM diameter was selected over the tubule (×400
magnification). The fluorescence intensity emitted from this area
(during brief excitation periods at 340 and 380 nm alternatively, at a
maximal rate of 30 cycles/min), was recorded every 2 s.
Tubule autofluorescence was subtracted from the fluorescence
intensities measured at 340 and 380 nm.
[Ca2+]i was calculated by using a
dissociation constant of fura 2 for calcium of 224 nM as previously
reported (10, 20, 27). Results obtained from different tubules (n) microdissected from
several rats were expressed as means ± SE. Statistical analysis
by one-way analysis of variance was followed by Fisher's least
significant difference test.
Measurement of glucagon-dependent cAMP synthesis.
Hormone-dependent cAMP synthesis in an intact single segment was
measured as previously reported (1, 9).
Microdissection medium (1 mM Ca2+) was supplemented with 5 µM indomethacin and 0.5 unit/ml adenosine deaminase (Boehringer
Mannheim) to prevent the endogenous synthesis of prostaglandins and the
release of adenosine, which interfere with the regulation of cAMP
levels in rat OMCD (9). The incubation medium similar to
the microdissection medium, included 0.1% (wt/vol) bacitracin (to
inhibit peptidase activity) and 1 mM IBMX, an inhibitor of all
phosphodiesterases in rat kidney (16). Microdissected tubules were transferred in 2 µl of incubation medium on glass slides
(1 or 2 pieces/slide) and photographed to measure their length. Each
sample was preincubated for 10 min at 30°C. After the addition of 2 µl incubation medium containing 1 µM glucagon (Neosystem
Laboratoire, Strasbourg, France), with or without other agonists,
samples were incubated for 4 min at 35°C. All agents were used at
concentrations inducing maximal effects (1,
9, 20). The reaction was stopped by rapidly
transferring the tubule together with 1 µl incubation medium into a
polypropylene tube containing a 20 µl mixture of formic acid in
absolute ethanol (5% vol/vol). Samples were evaporated to dryness
overnight at 40°C and then kept at
20°C until cAMP assay. The
amounts of cAMP were measured in acetylated samples by radioimmunoassay
(Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France, or NEN Life
Sciences Products, Le Blanc Mesnil, France). Under our conditions, the basal level of cAMP present in one single piece of tubule was similar
to, or below, the sensitivity threshold of the assay (1, 9). Thus only hormone-induced cAMP synthesis could be
measured. Results were expressed in femtomoles of cAMP accumulated per
millimeter of segment per 4-min incubation time (fmol · mm
1 · 4 min
1). In each experiment,
all experimental conditions were tested in six to nine tubule samples
from the same rat kidney. The mean cAMP value from each condition was
expressed in absolute value or in a percentage of inhibition calculated
from the mean value obtained with glucagon alone. Results are given as
means ± SE calculated from n experiments. Statistical
analysis by the one-way analysis of variance was followed by Fisher's
least significant difference test.
 |
RESULTS |
Northern blotting.
The specificity of probes used for in situ hybridization was checked by
Northern blotting of several rat tissues (Fig.
1). The probe for type 5 AC produced a
strong hybridization signal in heart and brain (Fig. 1A), a
weaker signal in kidney and lung and a very weak signal in spleen,
liver, skeletal muscle, and testis, consistent with previous reports
for type 5 AC (23). Similarly, type 6 AC was detected in
nearly all tissues (Fig. 1B), consistent with previous
localization (18, 23).

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Fig. 1.
Northern blotting of rat tissues. A: type 5 adenylyl
cyclase (AC) probe strongly stained heart and brain. A signal was also
observed in lung and kidney, whereas the staining of spleen, liver,
skeletal muscle, and testis was weak. B: type 6 AC probe
stained all tissues, including kidney. The strongest signal was
observed in heart and brain. Staining was distinct for type 5 and type
6 AC probes in all tissues, demonstrating the specificity of each
probe.
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In situ hybridization.
By in situ hybridization on 4-µm-thick rat kidney sections, both type
5 (Fig. 2a) and type 6 AC
(Fig. 2b) mRNAs were relatively abundant in glomeruli. The
labeling for type 5 AC mRNA was strongest in small arteries and blood
vessels (Fig. 2a, arrows), where little labeling was
observed for type 6 AC (not shown). Significant labeling for type 5 and
type 6 AC was also observed in the interstitium, between kidney
tubules. No significant labeling was observed in kidney with either
type 5 or type 6 sense probes (Fig. 2, c and d,
respectively). In kidney tubules, as previously reported
(14), type 6 AC mRNA was abundant in thick ascending limbs
and collecting ducts (Fig. 3,
c and d), but proximal tubules were weakly
labeled (Fig. 3c). Consistent with previous reports by
quantitative RT-PCR (6), type 5 AC was not detected in
proximal tubules or thick ascending limbs (Fig. 3, a and
b).

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Fig. 2.
In situ hybridization in kidney cortex. By in situ
hybridization, type 5 AC mRNA (a) was abundant in glomeruli
(G), small arteries, veins, and capillaries (arrows). Type 6 AC mRNA
was abundant in glomeruli (b) but was absent from arteries.
No labeling was observed with either type 5 (c) or type 6 (d) sense mRNA probes. Bars = 20 µm.
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Fig. 3.
In situ hybridization in kidney outer medulla. In kidney
tubules, type 5 AC mRNA was only detected in collecting ducts ( ),
where principal cells were stained by anti-aquaporin-3 (AQP3)
antibodies (a) and intercalated cells were stained by anti
H+-ATPase antibodies (b). In these tubules,
labeling for type 5 AC was localized in intercalated cells (arrowheads)
but absent from principal cells (arrows). Type 6 AC mRNA (c,
d) was abundant in both collecting duct ( ) and thick
ascending limbs (T). Weak labeling was also detected in proximal
tubules (P). In the collecting duct, both principal cells (arrows)
stained with anti-AQP3 antibodies (c) and intercalated cells
(arrowheads) stained with anti-H+-ATPase antibodies
(d) were labeled by the type 6 AC mRNA probe. Both type 5 and type 6 AC mRNA were detected in the interstitium. Bars = 20 µm.
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The characterization of cell types in which type 5 and type 6 AC mRNAs
were expressed in collecting tubules was achieved by immunoperoxidase
staining with anti-AQP3 or anti-vacuolar H+-ATPase
56-kDa-subunit rabbit polyclonal antibodies after the in situ
hybridization. As previously reported (see Ref. 30), anti-AQP3
antibodies specifically stained basolateral plasma membranes of
collecting duct principal cells (Fig. 3, a and
c), whereas staining for the proton pump was localized to
intercalated cells in collecting ducts (Fig. 3, b and
d). In these tubules, labeling for type 6 AC was abundant in
both AQP3-positive principal cells (Fig. 3c) and proton
pump-positive intercalated cells (Fig. 3d). In contrast,
labeling for type 5 AC was primarily observed in collecting duct
intercalated cells, negative for AQP3 (Fig. 3a) but positive
for the vacuolar proton ATPase (Fig. 3b). Accordingly, no
labeling for type 5 AC mRNA was observed in inner medullary collecting
ducts, devoid of intercalated cells (Fig.
4a), whereas these tubules
were still labeled for type 6 AC (Fig. 4b). Although there
was no evidence of intercalated cells negative for either type 5 or
type 6 AC in the OMCD, labeling with the type 5 AC probe in cortical
collecting duct intercalated cells was usually weaker than in the outer
medulla, often undetectable (not shown). Altogether, these results in
rat kidney OMCD therefore suggest that type 6 AC is expressed in both
principal and intercalated cells, whereas type 5 AC is only found in
intercalated cells. In the OMCD, most intercalated cells were labeled
with both type 5 and type 6 AC, suggesting that both mRNAs could be
expressed in type A intercalated cell. Type 5 AC-negative intercalated
cells were only observed in cortical collecting ducts, where type B
intercalated cells are more abundant than in the OMCD. This result may
suggest a greater expression of type 5 AC in type A intercalated cells.

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Fig. 4.
In
situ hybridization in kidney inner medulla. No labeling was observed in
inner medullary collecting ducts ( ) with the type 5 AC mRNA probe
(a). In contrast, type 6 AC was still detected in inner
medullary collecting duct cells (b). Sections were stained
with anti-AQP3 antibodies. Bars = 20 µm.
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Quantification of in situ hybridization labeling.
The in situ observations above were confirmed by quantification of
labeling in cells unambiguously detected as either principal or
intercalated cells by antibody (anti-AQP3 or anti-proton pump) staining. Results are reported in Table 1
and show no statistically significant differences of labeling for type
6 AC in OMCD principal and intercalated cells. In contrast, labeling
for type 5 AC was much greater in intercalated cells and significantly
different from principal cells, where labeling was not significant.
Effect of carbachol and PGE2 on
[Ca2+]i increases in rat
OMCD.
As underlined at the beginning of this study,
[Ca2+]i increases induced by PGE2
and carbachol in rat OMCD (1, 20) appear a
prerequisite condition to observe the inhibition elicited by PGE2 on AVP-dependent cAMP accumulation (1)
and the inhibition elicited by carbachol on glucagon-dependent cAMP
synthesis (6). These observations led to the hypothesis
that a PGE2-mediated [Ca2+]i
increase might be mainly effective in the vasopressin-sensitive cells,
whereas a carbachol-mediated [Ca2+]i increase
might be located in the glucagon-sensitive cells. This hypothesis was
tested by comparing [Ca2+]i variations
induced by the addition of both agents to the responses obtained with
each agent added alone to the superfusion medium.
Carbachol and PGE2 were used at concentrations inducing
maximal [Ca2+]i increases and did not elicit
homologous desensitization (20 and data not shown). In a same tubule,
the superfusion of 0.3 µM PGE2 followed by the
superfusion of 100 µM carbachol, or conversely, did not give evidence
of a heterologous desensitization in 2 mM Ca2+ medium (Fig.
5). Both agents induced a peak of
Ca2+ of a comparable magnitude, and carbachol elicited a
pronounced plateau phase (Fig. 5).

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Fig. 5.
PGE2- and carbachol-mediated intracellular
Ca2+ concentration ([Ca2+]i)
increases. A: representative traces of [Ca2+]i
variations elicited by repeated superfusion of the same tubule with 100 µM carbachol, then 0.3 µM PGE2, and finally carbachol.
Experiment was performed in 2 mM Ca2+ medium. Solid
horizontal bars, superfusion period of each agonist. B: mean
values of [Ca2+]i variations at the peak are
expressed as maximal increases over basal value. Values are means ± SE obtained in tubules superfused successively with the 2 agonists:
either a first superfusion with 0.3 µM PGE2 and then 100 µM carbachol (n = 7 tubules) or conversely
(n = 5 tubules). For a given agonist,
[Ca2+]i increases are similar whatever the
order of superfusion.
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The simultaneous superfusion of PGE2 and carbachol induced
a higher increase in [Ca2+]i than the
response observed with the superfusion of only one agonist (Fig.
6A). The same observation was
made whatever the order of superfusion of the agonists. Intracellular
Ca2+ concentrations were calculated from the maximal peak
values that allowed accurate determinations of
[Ca2+]i increases (Fig. 6A). As
shown by the mean data, the [Ca2+]i increase
obtained with the addition of both PGE2 and carbachol was
statistically higher than the response observed with each agonist
(Table 2). In addition, the experimental
value obtained with the superfusion of both PGE2 and
carbachol was not different from the theoretical value calculated by
assuming a full additivity of the individual responses (Table 2).

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Fig. 6.
Additive effect of PGE2 and carbachol on
[Ca2+]i increases in rat outer medullary collecting duct
(OMCD). A: representative traces of [Ca2+]i
variations elicited by different agonists added in 2 mM
Ca2+ medium. The tubule was superfused with 100 µM
carbachol, then the combination of both agonists, and finally 0.3 µM
PGE2. Horizontal bars, superfusion period of each solution.
The magnitude of the responses was independent from the order of the
superfusion (see Table 2). B: representative traces of
[Ca2+]i variations elicited by different
agonists added in Ca2+-free medium. The tubule was
superfused with 2 mM Ca2+ and then with
Ca2+-free medium. Rapid superfusion of PGE2 (3 µM) and then carbachol (100 µM) elicited successive
[Ca2+]i increases (left). The
magnitude of carbachol- or PGE2-mediated
[Ca2+]i increase was independent from the
order of superfusion [carbachol = 48 ± 8 nM
(n = 8) and 38 ± 5 (n = 5) with
the 1st and 2nd superfusion, respectively; PGE2 = 121 ± 15 nM (n = 5) and 111 ± 11 (n = 8) with the 1st and 2nd superfusion,
respectively]. Note that, in contrast to successive superfusions of
different agonists, a second superfusion with the same agonist did not
elicit a second response (data not shown). The combination of both
agonists added in Ca2+-free medium induced a response
higher than that elicited by either carbachol or PGE2
(right). Note that, after the treatment with
PGE2 followed by the superfusion of carbachol, it was
necessary to superfuse the tubule with 2 mM Ca2+ medium to
refill the intracellular Ca2+ stores. The magnitude of the
different responses was independent from the order of superfusion (see
Table 2).
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Additional experiments were conducted in a Ca2+-free
medium. As usually observed on a same tubule superfused with a
Ca2+-free medium, the [Ca2+]i
increase elicited by one given agonist was no longer observed with a
second superfusion of the same agonist (data not shown). In contrast,
the superfusion of PGE2 followed by that of carbachol, or
conversely, led to successive [Ca2+]i
increases (Fig. 6B). The simultaneous superfusion of
PGE2 and carbachol induced a response statistically higher
than the individual responses, and this response was not different from
the theoretical value calculated by assuming a full additivity of the
increases of [Ca2+]i elicited by
PGE2 and carbachol (Fig. 6B and Table 2).
Altogether, these data establish that PGE2 and carbachol
release Ca2+ from different Ca2+ pools located
in either the same cell or different cells of rat OMCD.
Multiple combined inhibition of glucagon-dependent AC activity.
Glucagon-dependent cAMP synthesis is inhibited by both carbachol
through a Ca2+-dependent process (6) and
PGE2 through a Ca2+-independent,
G
i-mediated process (3). If present in a
same cell, these regulations suggest that different mechanisms may inhibit the same AC enzymatic activity. This hypothesis was tested in
multiple, combined inhibition experiments by using criteria previously
defined (1). PGE2 or carbachol inhibited to a
comparable extent, close to 50-60%, the response to glucagon
(Table 3). The simultaneous addition of
both agents led to a residual cAMP value lower than that obtained with
each agent alone, but the response to glucagon was not fully abolished
(Table 3). This result establishes that PGE2 and carbachol
were active in the same glucagon-sensitive cells. The results were
further analyzed by comparing the values measured to those that could
be expected if a different mechanism of inhibition on AC activity
accounted for carbachol- and PGE2-mediated regulation,
i.e., if these two agents elicited a cumulative inhibition of cAMP
synthesis. The measured value (8.7 ± 0.9 fmol · mm
1 · 4 min
1, Table 3) was not
different from the theoretical value calculated assuming an hypothesis
of cumulative inhibition (7.5 ± 0.6 fmol · mm
1 · 4 min
1). These results
therefore demonstrate that PGE2 and carbachol inhibit the
same pool of glucagon-sensitive AC catalytic units in rat OMCD by
different and independent mechanisms.
 |
DISCUSSION |
In this study, Ca2+-inhibitable AC isoforms were
localized by in situ hybridization at the cellular level in the rat
OMCD to further define the regulation of cAMP content in this segment. Hormone-dependent cAMP accumulation is inhibited by Ca2+ in
both cell types of the OMCD, but the agonist involved and the mechanism
of Ca2+ inhibition are cell specific. Indeed, the
muscarinic agonist carbachol induces a Ca2+-dependent
inhibition of glucagon-mediated cAMP synthesis but has no effect on
AVP-stimulated cAMP synthesis (6) or cAMP accumulation
(2). On the other hand, PGE2 inhibits
AVP-dependent cAMP accumulation, mainly by an increase in cAMP
hydrolysis, through a Ca2+-mediated process that is
insensitive to pertussis toxin (1, 3). In
addition, PGE2 inhibits glucagon-dependent cAMP synthesis through a Ca2+-independent, G
i-mediated
process (3). These mechanisms are summarized in Fig.
7.

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Fig. 7.
Schematic summary of intracellular cAMP regulations in rat kidney
OMCD. PLC, phospholipase C; PDE, phosphodiesterase; ag, agonist; R,
receptor.
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In the OMCD, carbachol and PGE2 increase
[Ca2+]i through an interaction with the m1
subtype of the muscarinic receptor (20) and, very likely,
with the EP1 subtype of the PGE2 receptor (3), respectively. These receptor subtypes are usually coupled to the phospholipase C pathway (12, 15). In our
experiments, simultaneous superfusion of PGE2 and
carbachol, in either 2 mM Ca2+ or Ca2+-free
medium, produced [Ca2+]i peaks corresponding
to a full additivity of the effects of both agonists. This result and
the observation of a Ca2+-dependent inhibition of cAMP
content elicited by either PGE2 in vasopressin-sensitive
cells (1) or carbachol in glucagon-sensitive cells
(6) strongly suggest a cell-specific
[Ca2+]i increase induced by PGE2
and carbachol in principal and intercalated cells, respectively (Fig.
7).
Type 5 and type 6 AC mRNAs have been detected by quantitative RT-PCR in
the OMCD, and functional data have suggested that this localization
corresponds to the expression of functional proteins (6).
Consistently with previous RT-PCR (6), type 6 AC mRNA was
highly expressed in thick ascending limbs and collecting ducts.
Colocalization of type 6 AC mRNA by in situ hybridization and cells
positive for AQP3 as well as cells positive for vacuolar H+-ATPase by immunocytochemistry demonstrated that type 6 AC mRNA is present in both collecting duct principal and intercalated cells. In contrast, type 5 AC mRNA labeling was mostly detected in
intercalated cells, where the proton pump was found, but where no
staining for AQP3 could be detected. Because mRNA for type 5 and type 6 AC was detected in every type A (proton secreting) intercalated cell,
these observations suggest that principal cells express only type 6 AC,
whereas both type 5 and type 6 AC mRNAs are expressed in type A
intercalated cells.
A property of Ca2+-inhibitable ACs is their sensitivity to
both submicromolar concentrations of Ca2+ and to a
G
i-mediated pathway (7, 13,
29). In the presence of an inhibitor of
phosphodiesterases, the simultaneous addition of carbachol and
PGE2 elicited a cumulative inhibition of glucagon-mediated cAMP synthesis. This dual negative regulation demonstrates the functional expression of Ca2+-inhibitable AC in
intercalated cells. In addition, although two distinct
Ca2+-inhibitable AC isoforms were detected by in situ
hybridization in intercalated cells, the cumulative inhibition
demonstrates that carbachol and PGE2 regulate a single pool
of AC catalytic subunits in this cell type, either type 5 AC, type 6 AC, or both. The enzymatic properties of type 5 AC and type 6 AC
isoforms and the regulation of their activities are closely similar
(7, 13). Thus at the present time it appears
difficult to further define the functional isoform(s) involved in cAMP
synthesis in intercalated cells. Figure 7 summarizes previous and
present data on the regulation of cAMP accumulation in the OMCD.
Extracellular Ca2+, or carbachol-mediated
[Ca2+]i increases, inhibit glucagon-dependent
AC activity by ~50% (6). By comparison, in OMCD
principal cells or in the cortical thick ascending limb (cTAL), where
the Ca2+-inhibitable type 6 AC is also expressed,
PGE2 or angiotensin II induces a Ca2+-mediated
inhibition of cAMP synthesis of only 10-20% (7,
9, 14). Ca2+-inhibitable AC can
be inhibited by either [Ca2+]i peaks or
Ca2+ entry (11, 14). The
following two major hypotheses can be discussed to explain the high
sensitivity of OMCD glucagon-sensitive cells AC activity to
Ca2+.
Role of Ca2+ in
intercalated cell AC activity.
The activation of the Ca2+-sensing receptor RaKCaR can
inhibit cAMP synthesis by up to 90% in the cTAL, in contrast to the
small inhibitory effect of angiotensin II in this segment. This high inhibition involves both a [Ca2+]i peak and a
capacitive Ca2+ entry (14). Although there is
no evidence for the expression of RaKCaR in OMCD basolateral plasma
membranes (10, 24, 32), the
presence of a yet unknown Ca2+-sensing receptor in
intercalated cells could account for the great sensitivity of
intercalated cell AC to extracellular Ca2+. The inhibition
of glucagon-dependent, but not AVP-dependent, AC activity
(6) by extracellular Ca2+ supports also the
hypothesis that Ca2+ channels are specifically expressed in
intercalated cells. Accordingly, the presence of non-voltage-gated
Ca2+ channels has been demonstrated in rat OMCD
(10). In addition, the [Ca2+]i
increase elicited by carbachol in glucagon-sensitive cells is
characterized by a plateau phase of markedly larger amplitude than that
observed with PGE2 in AVP-sensitive cells (Refs. 1 and 20
and this study) or with angiotensin II in cTAL (14). This
plateau reflects Ca2+ entry triggered by a
[Ca2+]i release (1,
20) and could also result from the activation of
Ca2+ channels. It can be noted that in some cell types,
carbachol was described to induce a direct activation of
Ca2+ channels (15, 28). A
carbachol-induced Ca2+ entry could therefore account for
the high inhibition of glucagon-dependent AC activity by
Ca2+.
Role of AC isoforms in intercalated cell sensitivity to
Ca2+.
Type 5 AC is expressed only in intercalated cells. The rabbit type 5 AC
isoform was previously reported to be more sensitive to
Ca2+ than type 6 AC (31). However, recent
results with the canine type 5 AC isoform did not confirm this property
(25). Additional experiments are therefore necessary to
demonstrate a different sensitivity to Ca2+ of type 5 and
type 6 AC that might account for the high Ca2+-dependent
inhibition of AC activity observed in intercalated cells.
In conclusion, our results in rat kidney demonstrate the localization
of type 6 AC mRNA in both OMCD principal and intercalated cells. In
contrast, type 5 AC was only detected in intercalated cells, where both
AC mRNA isoforms are therefore expressed. Functional data establish the
expression of Ca2+-inhibitable AC proteins, which allow the
cumulative inhibition of glucagon-dependent AC synthesis by both
PGE2, through a G
i-mediated process
(3), and carbachol, through an increase of
Ca2+ (6, 20). The simultaneous
action of these two inhibitory pathways therefore can deeply decrease
the physiological functions achieved by glucagon in intercalated cells
of the rat collecting duct, i.e., proton secretion and/or bicarbonate
reabsorption (21).
 |
ACKNOWLEDGEMENTS |
This work was supported by the Commissariat à
l'Énergie Atomique and the Centre National de la Recherche
Scientifique (URA 1859). C. Héliès-Toussaint was supported
by a postdoctoral fellowship from the Commissariat à l'Energie
Atomique, and L. Aarab was supported in part by a grant from the
Commissariat à l'Energie Atomique.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
J-M Verbavatz, DBCM/SBCe, Bât. 532, CEA/Saclay, F-91191
Gif-sur-Yvette, Cedex France (E-mail: jmverbavatz{at}cea.fr).
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. §1734 solely to indicate this fact.
Received 20 September 1999; accepted in final form 29 February 2000.
 |
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