Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
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ABSTRACT |
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Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+ channels, the PM Ca2+ pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+ (BKCa) channels were located in discrete peripheral sites, some with Cav. Immunoreactivites not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BKCa channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.
PM organization; cellular calcium compartments; caveolin association; neuronal nitric oxide synthase
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INTRODUCTION |
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EARLIER
(34, 35), we showed that canine lower esophageal sphincter
(LES) contains a plasma membrane (PM) constitutive nitric oxide (NO)
synthase (cNOS) that acts spontaneously to restrict tone development.
When it was inhibited by
N-nitro-L-arginine
(L-NNA), active tone persistently increased in tissues, and outward currents in isolated cells were diminished by
80%. Both tone development and outward currents were dependent on
continuing Ca2+ entry, inhibited by nifedipine. The same
outward currents inhibited by L-NNA were abolished by
iberiotoxin, a highly selective large-conductance Ca2+-activated K+ (BKCa) channel
blocker. However, the dependence of iberiotoxin-sensitive outward
currents on Ca2+ concentration in the pipette
([Ca2+]pipette) was inconsistent with
control of these channels primarily by the cytosolic
[Ca2+]; i.e., the EC50 for
activation of current by [Ca2+]pipette was
108 nM; NO donors did not increase outward currents further when the
[Ca2+]pipette was
200 nM but restored
iberiotoxin-sensitive outward currents fully when the
[Ca2+]pipette was less, even when it was 8 nM
(23, 35).
Moreover, in a Ca2+-free medium (with 100 µM EGTA), canine LES produced repeated sustained contractions in response to carbachol. These depended on ongoing Ca2+ entry because they were prevented or abolished by nifedipine or by high doses of EGTA (1 mM). They also depended on a functioning sarcoplasmic reticulum (SR) Ca2+ pump because cyclopiazonic acid inhibited them. Refilling of Ca2+ stores could be accomplished even when cyclopiazonic acid was present, provided L-Ca2+ channels were activated by BAY K 8644. This agent also enhanced tone recovery after depletion of Ca2+ stores (33). We postulated, based on these results and related ones involving canine tracheae and bronchi (2, 6, 7, 10, 28), that there was a close connection between the peripheral SR and a membrane site in which Ca2+ entry through L-Ca2+ channels occurred and that recycling of Ca2+ between these sites occurred, dependent on activities of the SR Ca2+ pump and on the L-Ca2+ channel.
When we discovered that canine LES had cNOS located in the PM, with its activity regulated by the L-Ca2+ channel and a local [Ca2+]i seemingly different from that of the general cytosol, we considered the probability that the site of colocalization of NOS and L-Ca2+ channels in the membrane might be the caveolae. These have been shown to have consensus sequences in both their cytoplasmic NH2 and COOH termini that contribute to oligomerization of Cav to form caveolae (36, 38) endothelial NOS (eNOS) or skeletal muscle neuronal NOS (nNOS) (13-15, 17-19, 22, 25, 26, 41, 43, 46).
The objectives of this study were to evaluate what Cav molecule(s) exists in canine LES and what Ca2+-handling molecules appear to be colocalized with it and associated with caveolae. We used light microscopy and ultrastructural immunohistochemistry to attain these objectives. Patch-clamp studies were done to evaluate if any of these proteins interact in a fashion that would be expected from their association with Cav.
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METHODS |
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Animals and Tissues
Mongrel dogs, chosen irrespective of gender, were euthanized with an overdose of pentobarbital sodium (100 mg/kg) in accordance with a protocol approved by the McMaster Animal Ethics Committee and the guidelines of the Canadian Council for Animal Care. The gastroesophageal region was then carefully removed from the dog and placed in a cold (4°C) Krebs-Ringer solution composed of (in mM) 115.0 NaCl, 4.6 KCl, 22.0 NaH2PO4, 2.5 CaCl2, and 11.0 glucose. The Krebs-Ringer solution was also equilibrated with 5% CO2- 95% O2. The gastroesophageal junction was then opened on the gastric greater curvature side, and the mucosa was removed by sharp dissection. This revealed the LES as a thickened ring of muscle composed of clasp fibers with oblique gastric sling fibers on either side and skeletal muscle in the longitudinal layer above. The LES used for experimentation was taken only from the clasp region of the LES.Fixation and Preparation of Tissues for Light Microscopic Immunohistochemistry
These methods have been described in detail (11, 12, 34, 35, 49). In brief, fixation was usually in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7. When cryostat sections were to be prepared, tissues were cryoprotected with 20% sucrose, frozen and sectioned 10-µm thick, and studied with indirect immunofluorescence with the use of a Leitz LaborLux fluorescence microscope with an I2 filter for Cy3 and an N2 filter for FITC. Laminar preparations were prepared by dissection of the relevant layers after they were stretched by being mounted on Sylgard and cleared with DMSO. Cryostat sections were immunostained by incubation overnight with the appropriate antibody and washed before being treated with a secondary antibody labeled with a fluorescent molecule.Staining for immunocytochemistry used antibodies to Cav-1 and -3, nNOS,
connexins 43, 45, and 40, vinculin, calreticulin, calsequestrin, the SR
Ca2+ pumps, and the PM. Table
1 summarizes the primary and secondary (fluophore labeled) antibodies used. Usually, smooth muscle cells were
cut in cross sections.
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Specificity of staining was determined by a series of tests: omission of primary antibody (to rule out nonspecific staining by secondary antibody), omission of secondary antibody (to rule out autofluoresence), and saturation of primary antibody by antigen when available (to ensure that the antibody stained only specific antigen sites). Additional procedures were carried out to reduce background staining, e.g., pretreating sections with an antibody made against an immunoglobin from a species different from that used to raise either the primary or the secondary antibody.
For normal colocalization studies, primary antibodies from different species were used with previously detailed procedures (12, 13, 49), e.g., when colocalization of proteins appeared to be possible based on staining with antibodies against individual proteins. We first carried out colocalization studies at the level of light microscopy. Because orientation or section thickness problems limit resolution in studies of colocalization of proteins with Cav-1, we used both normal immunocytochemistry and dual-laser confocal microscopy (Carl Zeiss, LSM 510) on 1-µm sections. Additional studies were conducted at the ultrastructural level.
Methods for Fixation and Preparation of Tissues for Ultrastructural Study
Tissues were fixed after dissection by immersion in 2% glutaraldehyde and 4% paraformaldehyde with 4.5% sucrose in 0.75% cacodylate buffer at pH 7.4. The methods have been described in detail (3, 4). After fixation, the tissues were washed, stained en bloc with uranyl acetate, dehydrated in graded ethanol and propylene glycol, and embedded in Spurr resin. After ultrathin sections were cut on a Reichert Ultracut E microtome, they were stained in grids with lead citrate and studied.Fixation for ultrastructural immunohistochemistry was
carried out by immersion in 0.1% glutaraldehyde with 4%
paraformaldehyde and 3% sucrose in 0.1 M phosphate buffer (pH 7.4)
followed by washing, dehydration in ethanol, infiltration with LR
White acrylic resin, polymerization at 20°C, and sectioning.
Labeling was with protein A gold or related methods. Colocalization was
done with protein A gold of different sizes. Silver enhancement was
carried out with a silver-enhancing kit (British BioCell, Cardiff, UK). Ultrastructural studies were carried out on a JOEL-1200 EX Biosystem electron microscope at 80 kV.
Patch-Clamp Techniques
Cell isolation.
The LES was dissected as described in Methods for Fixation
and Preparation of Tissues for Ultrastructural Study, and
strips were cut into 1- to 2-mm2 pieces and placed
in the dissociation solution. Cells were dissociated in (mM) 0.25 EDTA,
125 NaCl, 4.8 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose for 30 min. An enzyme solution containing papain (130 mg/ml), ()-1,4 dithio-L-threitol (L-DTT, 15.4 mg/ml), BSA (100 mg/ml), and Sigma collagenase blend H (occasionally F)
was added to the tissue pieces for 30-60 min. After incubation,
the enzyme solution was decanted off, and the tissue pieces were rinsed in enzyme-free dissociation solution. Single cells were gently agitated
mechanically with siliconized Pasteur pipettes to disperse and isolate
single smooth muscle cells. Cells used in this study were patch clamped
at room temperature (22-24°C), usually within 8 h of isolation.
Patch-clamp methodology.
Cells from the suspension were placed in a glass-bottomed dish.
Within 30 min, cells adhered to the dish. The cells were then washed by
perfusion with Ca2+-containing external solution containing
(in mM) 140 NaCl, 4.5 KCl, 2.5 CaCl2, 1 MgCl2,
10 HEPES, and 5.5 glucose (pH adjusted to 7.35 with NaOH). Patch
electrodes were made from borosilicate glass capillary tubes with a
Flaming/Brown micropipette puller (Sutter Instruments). After being
polished with a microforge (Narishige MF-83) and being filled, the
pipettes had resistances of 2-5 M. High-Ca2+ pipette solution contained (in mM) 2.5 CaCl2, 140 KCl, 1 MgCl2, 10 HEPES, 4 Na-ATP,
and 0.3 EGTA to obtain free [Ca2+] of 8 µM.
CaCl2, KCl, and EGTA levels were adjusted to obtain 50 or
200 nM free Ca2+ levels as calculated with MAX Chelator
software (version 6.72) by Bers et al. (5).
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RESULTS |
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Nature and Distribution of Caveolins in Canine LES
Cav-1 is present in endothelial cells but Cav-3 is considered to be the Cav present in muscle cells (13, 41, 43, 46, 50), but Segal et al. (40) found Cav-1 in vascular smooth muscle, accompanied by Cav-3 in arterial smooth muscle. Only Cav-3 was found in the skeletal muscle overlying the LES. In our tissue, a sparse distribution of immunoreactivity against an antibody to Cav-3 was found in canine LES when evaluated both at the light and ultrastructural levels (Figs. 1D and 2c). Positive controls, skeletal muscle that overlies the LES, showed heavy staining for Cav-3 (Figs. 1C and 2b). In contrast, an antibody to Cav-1 found dense immunoreactivity at the cell periphery of LES cells and little in skeletal muscle (Fig. 1, A and B, and Fig. 2a). The ultrastructural studies showed this immunoreactivity to be located in or near caveolae. As expected from the difficulty in maintaining proteins without excessive denaturation during fixation, embedding, sectioning, and staining, labeling did not occur at all caveolae. Additional studies found that Cav-1 was the primary Cav in other canine gastrointestinal, airway, and vascular smooth muscle (Ref. 12 and C. Y. Kwan, E. E. Daniel, and Y. F. Wang, unpublished observations).
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Localization of nNOS in Canine LES
Intestinal muscle cells contain several isoforms of nNOS (20, 37), one likely targeted to the membrane by an NH2-terminal extension (31). A previous study (40) reported that nNOS was present in vascular smooth muscle. Canine LES cells show immunoreactivity to three nNOS antibodies, one of which was raised in guinea pig (See Table 1), was directed against an epitope to the COOH-terminal end of nNOS. Figure 3, A and C, shows that this immunoreactivity, like that to Cav-1, was punctate and located at the cell periphery of LES cells. Immunoreactivity was also present in many myenteric neurons in the myenteric plexus (Fig. 3B). Occasional sites of immunoreactivity were present on skeletal muscle. One antibody, directed against an NH2-terminal epitope in rabbit, recognized a site in the periphery of skeletal muscle as well as Z lines (Fig. 3, F and G), where nNOS may be located. It stained LES muscle diffusely (Fig. 3H). An antibody raised against a COOH-terminal peptide also recognized immunoreactivity in nerve cells of the myenteric plexus (Fig. 3D) and stained the periphery of LES muscle cells (Fig. 3E) but with less fluorescence intensity than the antibody raised in guinea pig.
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Figure 4a shows that,
ultrastructurally, nNOS immunoreactivity was, like Cav-1, often located
in or near caveolae. Not all caveolae were immunostained, possibly
because of technical limitations in preserving antigenic sites. Cav-1
immunoreactivity was similarly located and appeared, like nNOS, to be
associated with caveolae as expected (Fig. 4, b and
c). The absence of the colocalization of immunoreactivity to
Cav-1 and to nNOS at a molecular level is considered later.
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Localization of Ca2+-Handling Proteins in LES
Figure 5, A-G, shows the localization of calreticulin (Ca-R), the L-Ca2+ channel, the SR Ca2+ pump, the PM Ca2+ pump, calsequestrin, the
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Colocalizations of Cav-1 and nNOS with Ca2+ Handling Proteins
In light microscopy studies with confocal microscopy, Cav-1 and nNOS were clearly colocalized in LES, as expected from their common presence in caveolae (Fig. 6, A-C). Similarly, nNOS and calreticulin appeared to be colocalized (Fig. 6, D-F). Moreover, the PM Ca2+ pump appeared to colocalize closely with both Cav-1 (Fig. 6, J-L) and with nNOS (Fig. 6, G-I), in agreement with previous biochemical studies (39).
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Proteins Not Colocalized with nNOS or Cav-1
We compared the localization of PM proteins, vinculin, and connexins 43, 40, and 45 as well as the SR Ca2+ pump to nNOS and Cav-1. Figure 7, A-C, illustrates for connexin 43 that these proteins, none of which is expected to be present in caveolae, were usually not colocalized with Cav-1 or nNOS (data not shown). When they were, it is possible that this was a result of inadequate resolution from the technique. Vinculin, expected to be largely located at sites distinct from Cav (30), was expressed at the cell periphery in a nonpunctate fashion and with some overlap to that of Cav (Fig. 7, D-F) or nNOS (data not shown).
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Colocalization of SR Ca2+ Pump and Calreticulin or Cav-1
Calreticulin rather than calsequestrin is reported to be the primary Ca2+-binding site of low affinity and high capacity in SR of smooth muscle (1, 21, 27, 32, 47) but is also distributed outside the SR (24, 44, 45). Figure 7, J-L, shows the colocalization of calreticulin and the SR Ca2+ pump. As noted earlier, immunoreactivity to calreticulin and to the SR Ca2+ pump is punctate at the periphery of cells but also present in the central portion of some cells, either as a circle or a sphere. Figure 7, J-L, illustrates that both sets of structures have the Ca2+ pump and calreticulin colocalized. However, Fig. 7, G-I, shows that Cav-1 is colocalized only with the peripheral, punctate location for the SR Ca2+ pump not with locations in the cell centers.Patch-Clamp Data
When the anti-Cav-1 antibody was placed in a patch pipette with 200 nM Ca2+ during whole cell patch-clamp studies, it decreased the outward currents. After 20 min, currents induced by depolarization to +10 mV or more were significantly decreased, from P < 0.05 to P < 0.01. These decreases were reversed by addition to the bath of 10
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DISCUSSION |
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These studies show that nNOS and a number of Ca2+-handling proteins appear colocalized with Cav-1 in punctate sites around the cell periphery of cross-sectioned LES cells. Because caveolae, formed by the presence of Cav, are arranged in rows in the longitudinal axis of smooth muscle cells, punctate sites around the periphery of cross-sectioned cells are the expected arrangement. However, sections for immunohistochemistry were 10-µm-thick, and out-of-register membrane domains can appear colocalized with caveolae. Even in 1-µm sections evaluated with dual laser confocal microscopy, such spurious colocalization can occur. Thus studies done with either light or confocal microscopy cannot establish molecular colocalization but do support or negate such a possibility. Even ultrastructural studies require careful interpretation because many antigenic sites are lost during preparation by fixation, embedding, and processing. Also, false positive sites will inevitably occur.
Loss of normal relations of antigenic sites is likely the case with Cav-1 or -3 in relation to eNOS or nNOS. The association between these molecules is based on the NOS protein binding reversibly to sites on both the NH2- and COOH-terminal cytoplasmic arms of the hairpinlike Cav molecule and becoming inactive (8, 13-15, 25, 26, 46). This binding is thought to be reversed by elevation of Ca2+ levels near the membrane. Because membrane permeabilization occurs during fixation of tissues, Ca2+ levels likely rise and dissociate nNOS from Cav (Fig. 4). However, nNOS appears to remain associated with caveolae under our conditions for ultrastructural immunocytochemistry, as eNOS and nNOS appear to do functionally, allowing rebinding to Cav when Ca2+ levels fall in cells (8, 13-15, 25, 26, 46).
Despite these caveats, the apparent colocalizations we observed may explain earlier studies. In them, L-Ca2+ channel activity was required to support nNOS activity when [Ca2+]pipette was 1,000 nM, and nifedipine reduced the activation of BKCa channels by SNP when [Ca2+]pipette was 8 nM (35). These studies implied that [Ca2+]i near the nNOS, the L-Ca2+ channel, and the BKCa channel was controlled differently than that in the general cytosolic [Ca2+]i. Subsequently we found that in the absence of extracellular Ca2+, achieved by eliminating it from the medium and adding 100 µM EGTA, the LES could contract tonically and repeatedly to carbachol. However, these contractions were abolished by nifedipine and by high levels of external EGTA and reduced by blockade of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump with cyclopiazonic acid (33). These findings, along with similar ones made in canine tracheae and bronchi (2, 6, 7, 28) suggested that Ca2+ was bound weakly at an extracellular site near the L-Ca2+ channel from which it was made available by carbachol and recycled by the activity of the SERCA pump activity.
The apparent colocalization of some of these proteins in caveolae may result from their possession of sequences that recognize scaffolding peptides in the cytoplasmic COOH and NH2 termini of Cav-1, as eNOS and nNOS do (8, 9, 13-15, 17-19, 22, 25, 26, 41-43, 46). Whether this or some other mechanism accounts for the apparent colocalization of Ca2+ proteins with Cav, their presence in close proximity helps explain the control of active tone in this sphincter.
Earlier, we showed that there was a spontaneously active cNOS, requiring Ca2+ entry through L-Ca2+ channels and operating to activate iberiotoxin-sensitive BKCa and other K channels (34, 35). As described above, there was also recycling of Ca2+ between an extracellular site, which retained Ca2+ even when the extracellular medium had no added Ca2+ and contained 100 µM EGTA, and peripheral SR.
These properties are difficult to explain unless there are close
spatial relationships between the site of Ca2+ entry, the
nNOS, the BKCa channels, and the peripheral SR. Also, there
must be a region between the peripheral SR and the site of
extracellular Ca2+ entry in which the [Ca2+]
differs from the general cytoplasmic [Ca2+].
This study provides evidence that caveolae, composed primarily of Cav-1
molecules, may provide a biochemical, organizational basis to localize
Ca2+-handling proteins close together and near peripheral
SR. It provides evidence that an nNOS, possibly nNOS-, is associated
with Cav-1 as it is with Cav-3 in skeletal muscle (40, 46,
50). There is already biochemical evidence that both Cav-1 and
-3 can interchangeably bind this nNOS (46). Thus our
findings are consistent with expectations from the clear evidence that
canine LES has membrane cNOS that is spontaneously active based on
Ca2+ entry through L-Ca2+ channels (34,
35). As expected, these channels too were associated with
caveolae and nNOS immunoreactivity based on our immunochemical findings. Our data do not exclude that they were present elsewhere in
the PMs, as suggested by studies with isolated Cav-rich membranes compared with other membrane fractions in canine trachea
(12).
In addition to the L-Ca2+ channel, the PM Ca2+ pump was present associated with caveolae and nNOS. Its presence may be associated with transport of Ca2+ from intracellular sites to the extracellular low-affinity binding sites suggested by our studies showing that repeated contractions to carbachol were possible in Ca2+-free medium provided the L-Ca2+ channels and the SR Ca2+ pump were working.
As demonstrated in this study, the BKCa channel was colocalized in part with Cav-1. When present, its immunoreactivity was close to that of Cav, but there were regions where Cav reactivity, but not that for BKCa channels, was present. If this is generally the case, it implies that some caveolae possess and others lack this channel in close proximity.
In the study on Cav-enriched membranes from trachea (12), we found that calreticulin and calsequestrin were associated with the membranes and were immunoprecipitated with Cav-1. Although these proteins, especially calreticulin, are low affinity, high capacity Ca2+-binding sites in the SR interior of smooth muscle (16, 24, 27, 29, 32, 44, 48), there is evidence, at least for calreticulin, that it occurs in low concentrations in plasma and can bind to cell surfaces (45, 51). It is therefore a candidate for the extracellular binding site we require to explain our findings (33). It is also possible that one or both of these Ca2+-binding proteins exist bound to Cav, where they provide a basis to lower the free [Ca2+] in the region between the peripheral SR and caveolae. They would also provide a reservoir of Ca2+ to sustain SR Ca2+ uptake and recycling between SR and the caveolar extracellular space.
It is also possible, of course, that the presence of calreticulin and calsequestrin with Cav in immunoprecipitation experiments in trachea is the result of loss from the SR interior during cell disruption and membrane isolation, followed by binding to a consensus sequence of Cav-1 (12). Furthermore, it is possible that their apparent partial colocalization with Cav-1 or nNOS in this study is a result of lack of resolution in our microscopy methods. Such a possibility is supported by the fact that the SR Ca2+ pump also appears to be in part colocalized with nNOS, suggesting that the peripheral SR is so close to the caveolae as not to be resolved from them. However, our findings suggest explanations for phenomena observed in smooth muscle and raise important questions that open new possibilities for further study.
Finally, the ability of an antibody to Cav-1 to partially inhibit the outward currents when pipettes were filled with Ca2+ buffered at 200 nM requires comment. We cannot exclude that this is an artifact, but this is less likely because SNP restored outward currents. It is also possible that the Cav antibodies occupied the Cav molecules partially occluding sites at which nNOS binds. This could release nNOS into the cytosol making it unavailable in the region near caveolae where [Ca2+] may be higher and where NOS activation occurs. Provision of NO from an external donor would reverse any decrease in outward currents, as observed. Further studies on the interaction of Cav antibodies and nNOS activity are needed. The failure of calmodulin to affect outward currents significantly may be the result of our use of an inadequate concentration. There was a tendency for a reduction in outward currents both at 50 and 200 nM [Ca2+], and this might have been the result of a reduction in free [Ca2+] near nNOS in caveolae as a result of calmodulin binding it. Calmodulin has multiple interaction sites and additional data about its effects on outward current under patch-clamp conditions are needed.
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ACKNOWLEDGEMENTS |
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This study was supported by the Medical Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. E. Daniel, Dept. of Pharmacology, Univ. of Alberta, 9-70 Medical Sciences Bldg., Edmonton, AB T6G 2H7, Canada (E-mail: edaniel{at}ualberta.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 April 2001; accepted in final form 14 June 2001.
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