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*
Institute of Physiology,
Albert-Ludwigs-Universität Freiburg, Freiburg,
Germany.
Department of Medicine, Division of Nephrology,
Albert-Ludwigs-Universität Freiburg, Freiburg,
Germany.
Correspondence to Prof. Dr. Hermann Pavenstädt, Department of Medicine, Division of Nephrology, Albert-Ludwigs-Universität Freiburg, Hugstetter-Strasse 55, D-79106 Freiburg, Germany. Phone: 0761-270-3270; Fax: 0761-270-3245; E-mail: nitschro{at}ruf.uni-freiburg.de
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of [Ca2+]i
The decapsulated glomerulus was held in the perfusion chamber with two
micromanipulator-controlled glass pipettes and was loaded with
fura-2/acetoxymethyl ester (AM) (5 µM) at room temperature for 20 to 25
min, as described previously
(11). Experiments were
performed using two different experimental configurations. One configuration,
for fluorescence digital video microscopy (FDIM), consisted of an inverted
fluorescence microscope (Axiovert 100 TV; Zeiss, Jena, Germany) equipped with
a x 100 objective (Fluar 40/1.3 oil; Zeiss), a fast-switching
monochromator (TILL; Photonics, Planegg, Germany) to change the excitation
wavelength, and a generation 3, intensified, charge-coupled device camera
(ICCD 350; Videoscope International, Sterling, VA) for fluorescence imaging
(13). Fluorescence images (all
emission signals above 505 nm) were acquired with an average of 16 frames, to
increase the signal/noise ratio. Control of the experiment, image acquisition,
and data analysis were performed with the software package MetaFluor/MetaMorph
3.5 (Universal Imaging, West Chester, PA). The number of the recorded
ratiometric images was varied during the experiment. Shortly before and during
hormone application, the ratiometric image acquisition rate was near 0.7 Hz;
during control periods, the rate was decreased to 0.2 to 0.05 Hz. Optical
filters and dichroic mirrors were obtained from Delta Light & Optics
(Lyngby, Denmark).
Confocal and Two-Photon Microscopy
The one-photon excitation measurements with fluo-3 were performed with
488-nm excitation and collection of the fluorescence emission above 505 nm
with a conventional confocal microscope (LSM 510; Zeiss). The confocal pinhole
was set to achieve an optical slice thickness of 1 µm. Time series of
fluorescence images (40/1.2 water objective, C-Apochromat, 60 to 150 images of
512 x 512 pixels, 2- to 4-µs pixel time) were recorded before and
during ACh stimulation. A prototype two-photon microscope system (LSM 560;
Zeiss) with a turnkey fiber laser (780 nm; pulse width, <180 fs; maximal
power, 7.4 mW) was used to image fluo-3-loaded glomeruli (loading for 20 min
with 5 µM fluo-3/AM at room temperature) with a x40 objective
(Plan-Neofluar 40x/1.3 oil; Zeiss). The mean laser power at the
objective plane was reduced to 1.2 mW using gray filters. The two-photon
excitation wavelength of 780 nm allowed sufficient excitation of fluo-3 for
the [Ca2+]i measurements. After the
[Ca2+]i measurements, the glomeruli were stained with
the general cell marker calcein/AM (5 µM for 20 min at room temperature).
Then, z-stacks (z-distance, 0.8 µm) of high-resolution
images (1024 x 1024 pixels) of the calcein-labeled glomeruli were
recorded. With both dyes, all fluorescence emission below 680 nm was collected
without using a pinhole in front of the fluorescence detector
(13).
Calibration of the FDIM [Ca2+]i measurements was performed with ionomycin at the end of the experiment, as described (11). No correction for background levels or autofluorescence was necessary in the FDIM or two-photon microscopy experiments, because the two parameters together exhibited a maximal pixel intensity of 5. The results of the successful calibrations were pooled and used for calculation of the [Ca2+]i values (mean ± SEM), according to the method of Grynkiewicz et al. (14). The averages of the pixel-by-pixel ratios (fura-2) or the raw fluorescence intensities (fluo-3) of user-selected areas, usually one or two for the FDIM experiments (emission at >470 nm with 345/380-nm excitation) and 15 to 20 for the two-photon images (emission at <680 nm with 780-nm excitation), were used for the data analyses. No Ca2+ calibration of the fluorescence signal could be performed for the confocal [Ca2+]i measurements. The glomerular structure is significantly altered by the application of ionomycin during the calibration procedure. These changes preclude reliable single-cell calibration, which would be necessary for single-wavelength dye calibration.
Immunhistochemical Analyses
Fixation and Preparation of Tissue for Immunohistochemical Analyses.
The distribution of muscarinic receptor subtype M1, M3,
and M5 proteins in the kidney was determined by immunocytochemical
analyses, using respective polyclonal antisera (IgG fraction rabbit
anti-muscarinic M1, M3, and M5 receptor sera;
Biotrend, Köln, Germany). In addition, for
detection of the M3 receptor, two different polyclonal goat IgG
antibodies (N-18, epitope-mapping at the amino terminus of the M3
receptor; and C-20, epitope-mapping at the carboxyl terminus of the
M3 receptor; Santa Cruz Biochemicals, Santa Cruz, CA) were used.
M3 receptor staining was also tested in HT29 colon carcinoma cells,
which were cultured as described previously
(15).
Rats were anesthetized with Trapanal (5 to 7 mg/kg body weight; Byk Gulden, Konstanz, Germany). The kidneys were perfused at low pressure, through the artery, with 5 ml of cold (4°C) standard solution and then with approximately 5 ml of 4% paraformaldehyde solution. The kidneys were removed from the rats, decapsulated, and incubated for 24 h at 4°C in 4% paraformaldehyde solution.
Sectioning and Immunolabeling. Small pieces (4-mm thickness) of the kidneys were embedded in paraffin and cut into thin slices (5 or 7 µm). The slices were deparaffinized in xyloll for 45 min and gradually hydrated through graded alcohols (100 to 70%). The slices were rehydrated with phosphate-buffered saline (PBS), and antigen unmasking was performed by heating the slices in a sodium citrate buffer (1 mM) for 4 min, in a pressure cooker. To reduce nonspecific binding, sections were incubated for 20 min with blocking sera (10%) from the respective animals (Biotrend) and, throughout the labeling procedure, background-reducing buffer from DAKO (Copenhagen, Denmark), which also contained 1.5% of the respective blocking sera, was used. Sections were incubated for 24 h, in a humidified chamber at 4°C, with antibodies against M1, M3, or M5 receptors (M1, 1:500; M3, 1:400; M5, 1:500 or 1:800; Biotrend) and in addition, for double-labeling of podocytes, with a mouse monoclonal antibody against synaptopodin (1:2; Progen, Heidelberg, Germany). The slices were washed extensively with PBS and incubated for 45 min with the secondary antibody, Alexa Fluor 488 goat anti-rabbit Ig antibody (1:200 to 1:800; Mobitec, Göttingen, Germany). For double-immunostaining of synaptopodin, Cy3 goat anti-mouse Ig antibody (1:200 to 1:400; Dianova, Hamburg, Germany) was also used. Finally, nuclei were stained with HOE 33342 (10 µM; Mobitec) for 10 min. Slices were washed twice with PBS and twice with distilled water and were mounted with Glycergel (Dako). Sections were examined with conventional confocal microscopy (Zeiss LSM 510), using sequential image acquisition after excitation at 361 nm (HOE33342; emission, 385 to 470 nm), 488 nm (Alexa Fluor 488; emission, 505 to 550 nm), and 543 nm (Cy3; emission, >560 nm). Images of 1024 x 1024 or 2048 x 2048 pixels were recorded by setting the pinholes in the respective acquisition channels to obtain optical slices with a full-width half-maximum of 0.5 µm. The respective controls for nonspecific staining exhibited no fluorescence signals at the same instrument settings.
Western Blotting
Whole-brain tissue and glomeruli were homogenized in ice-cold Tris-buffered
saline containing 2 mM ethylenediaminetetraacetate, 100 mM NaCl, 20 mM Tris, 2
mM ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 2 mM phenylmethylsulfonyl fluoride, and a proteinase inhibitor cocktail
(Roche Diagnostics, Mannheim, Germany) and were centrifuged (14,000 x
g at 4°C). The cell membranes were sonicated in buffer containing
1% Nonidet P-40 (Boehringer Mannheim, Mannheim, Germany). The samples were
resuspended in Laemmli sample buffer, boiled (5 min), and subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transfer
electrophoresis. The transblots were probed with the primary antibodies
(anti-muscarinic M5 receptor; Biotrend), followed by
peroxidase-labeled secondary antibodies (donkey anti-rabbit; Amersham
Pharmacia Biotech, Piscataway, NJ), and were detected with chemiluminescence
detection reagents (Amersham Pharmacia Biotech).
Solutions and Chemicals
The standard solution contained 145 mM NaCl, 1.6 mM
K2HPO4, 0.4 mM KH2PO4, 1.3 mM
calcium gluconate, 1 mM MgCl2, and 5 mM D-glucose, pH 7.4. The
specific compositions of the experimental solutions were recently described
(11). Fura-2/AM, fluo-3/AM,
and calcein/AM were obtained from Mobitec. All other chemicals were of the
highest purity grade available and were obtained from Sigma (Deisenhofen,
Germany) or Merck (Darmstadt, Germany). All experiments were performed at
37°C.
Statistical Analyses
The data are presented as mean ± SEM values; n refers to
the number of experiments. Paired or unpaired t tests were used to
compare mean values within or between independent experimental series. A
P value of 
0.05 was considered to indicate statistical
significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence that the ACh-Induced [Ca2+]i Increase
Is Attributable to Intracellular Ca2+ Release and Ca2+
Influx
Figure 2A shows the effects
of ACh on [Ca2+]i in the presence of a reduced
extracellular Ca2+ concentration. Reduction of the external
Ca2+ concentration significantly reduced resting
[Ca2+]i from 92 ± 14 nM to 45 ± 7 nM
(n = 9). The ACh (10 µM)-induced [Ca2+]i
plateau was abolished (52 ± 8 nM), and the peak
[Ca2+]i increase was reduced. One-half of the
experimental series of the type presented in
Figure 2A were performed in the
reverse order, to avoid effects of receptor desensitization. In seven of nine
experiments with 1 µM Ca2+, a mean reduction of the peak
[Ca2+]i increase of 47 ± 6% was measured. In the
two other experiments, the peak was increased (130 and 157%). We have no
explanation for the peak increase under theses conditions, because the removal
of Ca2+ had reduced resting [Ca2+]i in these
two glomeruli to a similar extent, compared with the other glomeruli.
Figure 2B demonstrates that the reduction of the extracellular Ca2+ concentration from 1.3 mM to 1 µM completely abolished the maximal [Ca2+]i plateau and reduced [Ca2+]i below the resting value measured before the reduction of the external Ca2+ concentration. The mean resting [Ca2+]i in these experiments was 136 ± 31 nM before ACh stimulation, compared with a value of 80 ± 34 nM for ACh stimulation with 1 µM Ca2+ (n = 8).
To examine whether ACh might activate L-type Ca2+ channels in podocytes, experiments were performed with the L-type Ca2+ channel blocker nicardipine. Nicardipine (1 µM) did not change the ACh-induced [Ca2+]i increase (n = 6), as shown in a typical experiment in Figure 3A. However, lanthanum (La3+) and gadolinium (Gd3+), which are nonspecific blockers of Ca2+-influx pathways activated after inositol trisphosphate-induced Ca2+ store release, efficiently blocked the [Ca2+]i plateau at 1 µM (Figure 3, B and C). In paired experiments (n = 11), La3+ was more effective than Gd3+. The mean reduction of the plateau obtained with 1 µM Gd3+ was 29 ± 11%, compared with - 14 ± 8% obtained with 1 µM La3+, which is below the resting [Ca2+]i before ACh stimulation. The effects of La3+ and Gd3+ were not always fully reversible, even after a washout period of 15 min. Possible effects of La3+ and Gd3+ on the [Ca2+]i peak were measured in another experimental series, in which La3+ or Gd3+ (both at 1 µM) was added 2 min before stimulation with ACh. The ACh-induced peak [Ca2+]i values obtained during the application of La3+ or Gd3+ were not significantly different from the ACh-induced [Ca2+]i peaks measured before and after the application of La3+ (98 ± 4%, n = 4, data not shown) or Gd3+ (109 ± 6%, n = 5, data not shown).
Effects of Muscarinic Antagonists on the ACh-Induced
[Ca2+]i Increase
Figure 4 presents original
recordings of the effects of different concentrations of the muscarinic
M3/M5 receptor antagonist
4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP)
(Figure 4A) and the muscarinic
M1 receptor antagonist pirenzepine
(Figure 4B)
(16). The summary of these
types of experiments (Figure
5), as well as the original recording
(Figure 4B), shows that low
concentrations (10 and 100 nM) of pirenzepine augmented, whereas higher
concentrations inhibited, the [Ca2+]i response to ACh
(IC50 of approximately 1 µM). Similar results were obtained with
4-DAMP (Figure 5A); however,
the concentration-response curve was shifted to the left by approximately 2.5
decades, compared with pirenzepine (IC50 of approximately 5 nM,
compared with 1 µM).
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Measurement of [Ca2+]i with Two-Photon Laser
Scanning Microscopy
To investigate whether the ACh-mediated [Ca2+]i
increase was attributable to a [Ca2+]i increase in
podocytes, [Ca2+]i measurements were performed using
two-photon laser scanning microscopy (n = 10).
[Ca2+]i was measured confocally on the single-cell level
with fluo-3, in a confocal slice near the lower surface of the decapsulated
glomerulus. Typical fluo-3 fluorescence images (recorded at 780-nm excitation)
are presented in Figure 7, A through
C. Cells that responded to ACh (10 µM) with a
[Ca2+]i increase are shown in
Figure 7B. Only approximately
one-third to one-half of the cells in the image responded. The fluo-3
fluorescence traces from the marked responding cells are presented in
Figure 7D. Using stepwise
changes in the focus, it was possible to identify podocytes because of their
typical structure and location in the glomerulus. To facilitate
identification, in some experiments the glomeruli were incubated with
calcein/AM, a general marker of living cells. Calcein yielded much brighter
and less noisy images, compared with fluo-3, and allowed clear identification
of podocytes on the basis of their characteristic morphologic features and
location in relation to the capillary network.
Figure 8 presents four images
from a z-stack of calcein fluorescence images through a glomerulus.
Podocyte foot processes located on the basal membrane around the capillaries
could be clearly observed. From the confocal two-photon images, it can be
concluded that podocytes respond to ACh stimulation with a
[Ca2+]i increase.
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Fluorescence Immunohistochemical Staining Demonstrating M5
Receptor Expression in Podocytes
M3 receptors could not be detected in glomerular cells but were
found in colon carcinoma cells, which are known to express M3
receptors (17) (data not
shown). Figure 9A demonstrates
the positive immunoreactivity for synaptopodin, a protein that, within the
glomerulus, is specifically expressed in podocyte foot processes
(18). In comparison with
synaptopodin, M5 receptors demonstrated similar immunoreactivity
localization but were also detected in cell bodies of podocytes
(Figure 9B). The specific
localization of M5 receptors in podocytes was also demonstrated by
double-immunostaining of the glomeruli with antibodies against synaptopodin
and the M5 receptor (Figure
9C). Figure 9D
shows that staining of M1 receptors could be detected only in the
parietal sheet of Bowman's capsule and not in podocytes. Negative control
slides incubated only with secondary antibodies demonstrated no appreciable
staining (data not shown).
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Figure 10 shows that the expression of muscarinic M5 receptors in rat glomeruli could be confirmed by Western blot analysis (n = 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The [Ca2+]i response to ACh was biphasic, with a peak and a plateau. The [Ca2+]i peak was reduced by approximately 50% in a solution with a reduced extracellular Ca2+ concentration, indicating that the peak was partly attributable to a release of Ca2+ from intracellular stores. The subsequent [Ca2+]i plateau was mainly the result of Ca2+ influx from the extracellular space, as supported by the finding that low extracellular Ca2+ concentrations completely abrogated the plateau. Nicardipine, a blocker of L-type Ca2+ channels, did not inhibit the [Ca2+]i plateau, which is in line with our recent results showing that depolarization by high extracellular K+ concentrations did not affect [Ca2+]i (11). The data suggest that the effect of ACh is not mediated by L-type Ca2+ channels. However, the strong reduction of the [Ca2+]i plateau by low concentrations of Gd3+ or La3+ indicates that other Ca2+ influx pathways, perhaps of the ICRAC type, are activated in response to ACh stimulation.
Repetitive addition of high concentrations of ACh did not inhibit the [Ca2+]i increase induced by ACh, indicating that repetitive ACh application did not cause receptor desensitization. A lack of desensitization of the ACh-induced glomerular [Ca2+]i response was also shown by Lebrun et al. (8). We recently demonstrated that, in contrast to ACh, angiotensin II produced strong desensitization of the AT1 receptor; after a second or third application of angiotensin II, no [Ca2+]i response or only a small response was observed (11). Therefore, ACh- and angiotensin II-induced signaling in podocytes might affect their respective receptors in different ways.
To date, five subtypes of muscarinic receptors (M1 to M5), with unique distributions, have been identified by molecular cloning (19). M2 and M4 receptors are preferentially coupled to Gi proteins and inhibit adenylate cyclase, whereas M1, M3, and M5 receptors are preferentially coupled to phospholipase C activation (19). On the basis of pharmacologic criteria, only four subtypes of muscarinic receptors can be distinguished using selective antagonists, because clear discrimination between M3 and M5 receptors is currently not possible (16,20).
To determine which muscarinic receptor subtype is coupled to calcium mobilization in glomeruli, the muscarinic receptor antagonists 4-DAMP (an M3/M5 receptor antagonist) and pirenzepine (an M1 receptor antagonist) were used (16). Both compounds inhibited the [Ca2+]i response to ACh, but 4-DAMP was approximately 2.5 orders of magnitude more potent than pirenzepine, suggesting that the ACh-mediated [Ca2+]i response is primarily dependent on M3/M5 receptor activation.
An unexpected result was that both 4-DAMP and pirenzepine, at concentrations lower than required for inhibition, significantly stimulated the ACh-induced [Ca2+]i response. We currently have no explanation for this observation.
To test for the existence of M1, M3, and M5 receptor protein expression in glomeruli, fluorescence immunohistochemical studies were performed with specific antibodies against the respective receptors. These studies indicated that podocytes express M5 receptors. These receptors could be detected on the cell surface of podocytes (Figure 9B) and were also colocalized with synaptopodin, a protein that, within the glomerulus, is expressed only in podocyte foot processes (17) (Figure 9, A and C). By using Western blot analyses, appropriate bands for the muscarinic M5 receptor (approximately 65 kD) were identified in isolated rat glomeruli. Brain tissue served as a positive control sample. Two glycosylation sites have been identified in this receptor (21). The lower band observed for glomeruli might thus be explained by the presence of nonglycosylated M5 receptors.
M1 receptors were present only in the parietal sheet of Bowman's capsule (Figure 9D), suggesting that ACh-induced [Ca2+]i increases in Bowman's parietal cells, which have been reported by Lebrun et al. (8), are mediated via M1 receptors. Using three different antibodies against the M3 receptor, we could not detect this receptor in glomerular cells, indicating that glomerular cells do not express M3 receptor protein. In positive control experiments, the antibodies against the M3 receptor stained colon carcinoma cells, which are known to express M3 receptors (18).
This study demonstrates that podocytes express functionally active M5 receptors in vivo. M5 receptors are known to be predominantly expressed in the brain (striatum, hippocampus, midbrain, pons, medulla, and cerebellum), where they might play a role in dopaminergic transmission. There are few data concerning the expression and function of M5 receptors in peripheral tissues. M5 receptor expression in rat salivary gland tissue and in a melanoma cell line has been suggested (for review, see reference 20). In the melanoma cell line, activation of M5 receptors inhibited cloning capacity, indicating that M5 receptors might have antioncogenic functions in this cell type (22).
This study indicates that M5 receptors in podocytes might play a role in the regulation of podocyte function and thus might participate in the regulation of the renal glomerular filtration process. Further studies must demonstrate the ultrastructural localization of M5 receptors in podocytes and clarify their precise role in podocyte function. Because podocyte foot processes contain contractile machinery, it might be speculated that the effect of ACh on [Ca2+]i in podocytes contributes to ACh-induced glomerular contraction.
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Acknowledgments |
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