The anorectic cobalt protoporphyrin (CoPP) is known to elicit short-term hypophagia and long-term weight loss through unknown mechanisms in the brains of experimental animals. The goal of this work was to determine 1) if the prolonged duration of action of CoPP is related to its prolonged retention within the brain; and 2) with the use of immunohistochemical detection of Fos, the product of the early-immediate gene c-fos, which cells are activated after exposure to CoPP. These studies were carried out in male rats after intracerebroventricular administration of CoPP, 0.4 μmol/kg body wt, given under light halothane anesthesia. Residence of CoPP in the brain was determined by residual counts in dissected brains of 57CoPP-injected rats. Fos immunoreactivity was mapped in coronal sections of rat brains 4–6 h after injection with CoPP. The results showed that 57CoPP was retained in the hypothalamus preferentially compared with the cortex of the brain and could be detected in the hypothalamus for in excess of 5 wk. Fos activation was increased by CoPP, detected predominantly in neuronal rather than glial cells, and was markedly more robust in the hypothalamus than in other brain areas. Thus CoPP remains in the hypothalamus for prolonged periods and activates Fos expression in the hypothalamus.
- synthetic metalloporphyrin
- early-immediate gene
- cerebroventricular system
- central nervous system
iron (Fe2+) protoporphyrin IX (heme), highly conserved throughout evolution, is a prosthetic group essential to the functioning of a wide range of enzymes and metabolic processes. Substitution of its central iron atom with cobalt yields the synthetic metalloporphyrin cobalt (Co3+) protoporphyrin IX (CoPP). Unexpectedly, it was found that subcutaneous administration of CoPP elicits a dose-dependent transient decrease in food intake and a prolonged loss of body weight in rats, mice, chickens, and dogs (5–8). Similar effects observed after intracerebroventricular administration of CoPP to rats required only ∼1% of the subcutaneous dosage. In contrast, intracerebroventricular administration of equivalent doses of cobalt chloride, protoporphyrin or iron-, nickel-, cadmium-, manganese-, magnesium-, zinc- or tin-protoporphyrins were without effect on appetite or body weight (5). A remarkable aspect of these unpredicted biological effects of CoPP is the unprecedented magnitude and duration of action; 300 days after a single intracerebroventricular dose of 0.4 μmol/kg body wt, treated rats weighed 200 g less than saline-treated control rats (450 vs. 650 g) (5). It is known that CoPP, as measured by atomic absorption spectroscopy of its central cobalt atom, can be found in the brain and the hypothalamus (although not the pituitary) 2 days after intracerebroventricular injection (5), but it is not known how long CoPP remains within the central nervous system (CNS).
Cobalt protoporphyrin is known to be a potent inducer of heme oxygenase (HO) activity both in the CNS and in many peripheral tissues (3). HO is the rate-limiting enzyme involved in the catabolism of heme to biliverdin, iron, and carbon monoxide (18). These reaction products have recently been implicated in various biological functions but do not appear to be involved in eliciting the effects of CoPP on weight. This is evidenced by experiments in which the elevations in HO activity in brain and hypothalamus caused by intracerebroventricular administration of CoPP were normalized by coadministration of tin protoporphyrin (a competitive inhibitor of HO activity) without in any way altering the observed decrements in food intake or weight (7). Currently, the mechanism of action of CoPP in eliciting weight changes remains unknown.
Despite this, it is clear that the CNS is the organ in which CoPP acts to decrease food intake and regulate body weight (among other evidence, the dosage that is effective by the intracerebroventricular route is without effect when administered by the subcutaneous route). Moreover, using intrahypothalamic injection of even smaller doses of CoPP than used with the intracerebroventricular route, weight loss and/or decreased food intake were observed after injection into the paraventricular, dorsomedial, and ventromedial nuclei of the hypothalamus; injection into the thalamus or the lateral hypothalamic area was without effect (9). However, which cell types actually respond to CoPP remains unknown.
This manuscript reports experiments undertaken to further examine the kinetics of CoPP localization within the brain using radiolabeled CoPP, a more sensitive and specific assay than atomic absorption spectroscopy, which also measures intrinsic cobalt such as that contained in vitamin B12. It also describes the results of studies examining the distribution of cell activation after intracerebroventricular administration of CoPP using immunohistochemical detection of Fos, the protein product of the c-fos gene. Fos combines with members of the Jun family of proteins to form heterodimeric activator protein 1 complexes that can bind to regulatory sequences in various genes and thereby activate or in some cases suppress transcription of the target gene(s) (11). Fos expression is widely used, especially in the CNS, to detect cells that are activated (i.e., react to a stimulus, in this case CoPP, by synthesizing components of the transcriptional regulating systems) in response to a given stimulus (2, 17). For example, many of the peptides, hormones, and transmitters that modulate the complex behavior of food ingestion elicit different Fos activation patterns in different areas in the brain.
MATERIALS AND METHODS
Cobaltic protoporphyrin IX and protoporphyrin IX were purchased from Porphyrin Products (Logan, UT). 57Cobalt was purchased from New England Nuclear (Boston, Massachusetts). All other chemicals were of the highest reagent grade commercially available.
Synthesis of 57cobalt protoporphyrin.
57Cobalt protoporphyrin (57CoPP) was synthesized nonenzymatically from the disodium salt of protoporphyrin IX, dissolved in pyridine, and 57cobaltous chloride (carrier free, 99%) in glacial acetic acid at 45°C, under a nitrogen atmosphere for 6 h. The reaction mixture was transferred to a separatory funnel, and after addition of an equal volume of ethyl acetate, was washed three times with water, three times with 6 N HCl, and twice more with water. The remaining organic phase was aliquoted and dried down under a stream of nitrogen. A small aliquot of 57CoPP was dissolved in 0.2 N NaOH, and purity was assessed by thin-layer chromatography on silica gel plates using authentic CoPP as standard. The mobile phase was 2,6-lutidine-water (5:3; vol:vol), presaturated for 30 min at room temperature with ammonia vapor. Free protoporphyrin was not detected (as indicated by the absence of fluorescence under ultraviolet light). Autoradiography revealed a single band of 57CoPP with the same retardation factor as standard, which was visualized under ambient light. The specific activity of synthesized 57CoPP was calculated from the concentration of 57CoPP measured by the pyridine-hemochromagen assay (16) and the radioactive decay as assessed by counting in a Packard Gamma Spectrometer in Aquasol. For the pyridine-hemochromagen assay, a small aliquot of 57CoPP was dissolved in 0.05 N KOH-MeOH (1:1; vol:vol), mixed with pyridine-1 N NaOH-water (2:1:2; vol:vol:vol) and divided into two cuvettes. One cuvette was oxidized with a few grains of Na2S2O4 and the other reduced with a drop of 3 mM K3Fe(CN)6. Difference spectra were recorded on a scanning spectrophotometer and the concentration of 57CoPP calculated as the difference in absorption recorded at wavelengths of 557 and 541 nm (ΔE557/541)÷20.7. Specific activity was 1.6 × 106 dpm/nmol, and yield was 11.3%.
Animal handling and treatment.
All animal handling was approved by and done in accordance with the guidelines and requirements of the University of Vermont Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats (mycoplasma free; 200 g) were purchased from Charles River (St. Constant, Quebec, Canada) and housed in an air-conditioned (23 ± 1°C) room with lights on for 12 h daily (starting at 6:00 AM) in the University of Vermont Animal Facility. Rats were housed singly in regular cages with free access to Purina Rat Chow (RMH 3000) and water for at least 5 days before surgery. Stereotactic surgery was used to implant chronic indwelling stainless steel cannulae into the third ventricle under fentanyl/droperidol anesthesia. Coordinates for cannula placement were obtained from the Konig and Klippel stereotactic surgery atlas (13); with the nose bar 2.4 mm below the intra-aural line and the vertical micrometer gantry tilted 9° toward the animal, the coordinates were 4.9 mm dorsal to the intra-aural line, 5 mm anterior to the intra-aural line, and 1.1 mm lateral to the midline (7). A minimum of 5 days later, animals were injected intracerebroventricularly with either 57CoPP or CoPP, which was dissolved in 0.2 N NaOH and the pH adjusted to 7.4–7.8. Control animals received equal volumes of vehicle. Volumes of intracerebroventricular injections never exceeded 10 μl per animal and were infused over a period of 5–10 s. Measures of body weight were made daily with a digital balance with a 6-s integration period to minimize the effects of animal movement.
Radiolabeled CoPP experiments.
In experiments utilizing 57CoPP, animals were injected intracerebroventricularly with identical dosages of 57CoPP and killed in groups of five at weekly intervals, for five successive weeks. Hypothalamic blocks were dissected from the brain as described (10), and both hypothalami and residual brain tissue were weighed and sonicated in water. After counting in a gamma spectrometer, counts were corrected for tissue weights to yield picograms of 57CoPP per gram of tissue (wet weight) and expressed as means ± SE.
In experiments involving immunohistochemistry, animals were injected intracerebroventricularly with CoPP, 0.4 μmol/kg body wt, or with vehicle. Four hours after injection, animals were anesthetized with halothane and perfused through the left ventricle of the heart, initially with oxygenated Krebs buffer and then with 4% paraformaldehyde in 0.1 M NaCl, pH 7.4, and PBS (∼350 ml). Brains were extracted from the skull, and hypothalamic blocks were dissected (by coronal cuts anterior and posterior to hypothalamic landmarks) and postfixed in 4% paraformaldehyde solution at 4°C overnight. The following day, the blocks were transferred to 30% sucrose and incubated overnight at 4°C. After freezing in Optimal Cutting Temperature Compound (Sakura, Japan), blocks were sectioned coronally at 40 μm on a cryostat, and sections were individually floated in multiwell dishes filled with PBS. Sections were treated for 30 min with 0.4% Triton detergent in potassium PBS to aid in antibody penetration. Primary antibodies were added; the plates were shaken gently for 1 h at room temperature and then incubated at 4°C for 72 h. Sections were then brought to room temperature while shaking, and the primary antibody solution was removed by washing three times with PBS buffer. After removal of the last wash, the secondary antibody solution was added and the plates were incubated for 2 h at room temperature in the dark. Removal of the second antibody was achieved by washing three times with PBS buffer before mounting on subbed slides. They were allowed to dry, rewetted with PBS buffer, and coverslipped with Citifluor (Ted Pella, Reading, CA).
Fos studies were performed either using peroxidase staining or indirect fluorescence visualization. The former utilized pan-Fos antisera (1:10,000; Genosys Biotechnologies, The Woodlands, TX), avidin-biotin horseradish peroxidase complex using Vectastain ABC Kits (Vector Laboratories, Burlingame, CA), and diaminobenzidine. Tissues were examined using an Olympus Photomicroscope and Fos staining either photographed with Ectochrome Elite II film or counted by region based on the rat brain atlas (15). The fluorescence method utilized rabbit c-Fos-AB5 (1:2,000; Oncogene Research Products, Cambridge, MA) with goat anti-rabbit Cy3 (1:500; Jackson Immuno Research Laboratories, West Grove, PA) as the secondary antibody. Double-labeling experiments to identify cell types utilized anti-neuronal nuclei (Neu-N) antibody (1:500; Chemicon International; Temecula, CA) and anti-glial fibrillary acidic protein (GFAP) antibody (1:500; NeoMarkers, Freemont, CA). Sections were examined using a fluorescence microscope, and digital images were captured with MagnaFire 2.1 Software and imported into Paint Shop or Photoshop for printing. Tissue incubated in the absence of primary or secondary antisera showed no reaction products.
Assessment of positively stained Fos-immunoreactive nuclei.
Fos-positive cells were quantified using bright-field microscopy from coronal sections (1:3 series) in which Fos was visualized with diaminobenzidine. Brain sections were compared with those in the Paxinos and Watson atlas (15) to determine distribution of different hypothalamic nuclei. After counting, Fos was then mapped on representative sections from the atlas. Staining observed in experimental tissue was compared with that observed from experiment-matched negative controls. Nuclei exhibiting immunoreactivity that was greater than the background level observed in experiment-matched negative controls were considered positively stained. Counted positively stained nuclei were not further divided into categories of different staining intensities.
The studies involving 57CoPP in brain and hypothalamus were analyzed using ANOVA. Studies of Fos immunoreactivity in vehicle- and CoPP-treated rats were analyzed based on a repeated-measures ANOVA, with group (experimental vs. control) as the between-subjects factor and nucleus as the within-subjects factor. Two separate analyses were performed, one including the hypothalamic nuclei and one including only the nonhypothalamic nuclei. Hypothalamic nuclei consisted of anterior hypothalamic, arcuate hypothalamic, dorsal hypothalamic, dorsomedial hypothalamic, lateral hypothalamic, posterior hypothalamic, paraventricular hypothalamic, and ventromedial hypothalamic, while intermediodorsal thalamic, lateral posterior thalamic, premammillary dorsal, premammilary ventral, precommissural, supramammillary, tuberomammillary, tuberal magnocellular, ventral lateral geniculate magnocellular, and cingulum comprised the nonhypothalamic nuclei. Results are expressed as least-square means and SEs. These are the results of the ANOVA and represent the means and SE for each variable when controlling for the other variables in the model.
Time course of 57CoPP distribution in the CNS.
Data from the experiment utilizing radiolabeled CoPP are presented in Fig. 1. 57CoPP concentrations, as evidenced by gamma emissions at different time points, declined over the 5 wk of the experiment. However, it is noticeable that picograms of 57CoPP per gram of whole brain declined almost completely to 5% of initial values by week 3, at which time picograms of 57CoPP per gram of hypothalamus were still robust at 14% of initial values. It is also clear that at the termination of the experiment at week 5, marked residual 57CoPP was detectable in the hypothalamus in contrast to whole brain, which was essentially at trace levels (13.1 pg of 57CoPP/g hypothalamus vs. 0.4 pg of 57CoPP /g of brain). Levels of 57CoPP were statistically greater in the hypothalamus than in the brain (P = 0.05 by ANOVA).
Distribution of Fos expression.
The distribution of Fos immunoreactivity in sections of brains of rats treated intracerebroventricularly with CoPP, 0.4 μmol/kg body wt, or vehicle is displayed in Fig. 2. Basal mean Fos counts in sections from vehicle-treated rats were low or absent in both hypothalamic and extrahypothalamic sites. In contrast, mean Fos counts in CoPP-treated rat brains were increased diffusely in both hypothalamic and extrahypothalamic sites, but the degree of increase was markedly more robust in hypothalamic sites. The results for the hypothalamic nuclei indicate that the immunofluourescence in the control group (least squares mean ± least squares SE; 0.75 ± 1.11) was significantly different (P = 0.0001) from that in the experimental group (7.54 ± 1.11). In addition, there were differences in immunofluorescence among the hypothalamic nuclei (P = 0.0044); however, the pattern of immunofluorescence was consistent between the two groups, as indicated by the nonsignificant interaction (P = 0.0522). The results based on the nonhypothalamic nuclei suggest no difference in immunofluorescence between groups (experimental 0.20 ± 0.58; control 1.85 ± 0.58; P = 0.0513). In addition, there was no difference between nuclei (P = 0.7168), nor was there an interaction between group and nuclei (P = 0.6280). Representative examples of Fos immunoreactivity in CoPP- and vehicle-treated brain sections are shown in Fig. 3. This example shows the absence of parenchymal staining in the ventromedial and paraventricular hypothalamic nuclei of vehicle-treated rats (Fig. 3A) compared with the clear Fos immunoreactivity in CoPP-treated brains (Fig. 3B) and at higher power (Fig. 3C). Fos-immunoreactive nuclei of parenchymal cells are readily seen at this higher power.
Cellular location of Fos expression.
Experiments to determine which cell types express Fos after treatment with CoPP were carried out using dual-antibody incubations with subsequent identification using either green or red fluorescence (as described in materials and methods). Initial time courses (data not shown) revealed that optimum results were achieved if brains were harvested 4 h after intracerebroventricular treatment with CoPP, 0.4 μmol/kg body wt. Figure 4 shows representative photomicrographs from such an experiment using immunofluorescence to localize Fos and Neu-N, a marker for neuronal cells. As can be seen in Fig. 4, there are cells that clearly express the red fluorescence of Fos (Fig. 4A) and the green fluorescence of Neu-N (Fig. 4B). When both fluorescent probes are excited together (Fig. 4C), significant numbers of cells are yellow, which indicates colocalization of both Fos and Neu-N in the same cell type (white arrows in Fig. 4C). Figure 5 shows a photomicrograph of a double-stained section of the arcuate nucleus of the hypothalamus showing clear Neu-N fluorescence (green), Fos fluorescence (red), and colocalization (yellow) after intracerebroventricular treatment with CoPP. Thus Fos activation is apparent in some but not all neuronal cells in this area. Similar experiments were conducted to localize Fos and glial fibrillary acidic protein (GFAP), a marker of CNS cells of the glial lineage. Representative examples are shown in Fig. 6. Parenchymal red-staining Fos is evident near the III ventricle (Fig. 6A) and abundant green staining GFAP lines the III ventricle (Fig. 6B), but it is clear from the composite picture (Fig. 6C) that there is a paucity of colocalization. This either lack of or extremely low level of colocalization of Fos and GFAP was found throughout the brain in both hypothalamic and extrahypothalamic sites.
In summary, we have shown that radiolabeled CoPP was retained preferentially in the hypothalamus compared with the cortex of the brain for in excess of 5 wk after a single trace intracerebroventricular injection of 57CoPP. This preferential location was reflected in the distribution of Fos immunoreactivity 4–6 h after intracerebroventricular treatment with CoPP, which was increased compared with saline-injected animals and markedly more so in the hypothalamus compared with other areas that abut the cerebroventricular system elsewhere in the brain. The Fos immunoreactivity colocalized to cells that stained positively for the neuronal cell marker Neu-N, but there was little to no colocalization to glial cells identified with the marker GFAP.
It is known from previous work utilizing atomic absorption spectrometry to measure the concentration of cobalt in the tissues that CoPP can be detected in both the hypothalamus and the whole brain of rats 48 h after intracerebroventricular injection of CoPP, 0.4 μmol/kg body (5). That this is reflective of the tissue concentrations of CoPP is predicated on 1) the theoretical reasoning that there is no known in vivo mammalian system capable of generating sufficient reducing equivalents to open the tetrapyrrole ring when it contains cobalt and 2) the experimental observation that in vitro incubation of CoPP in tissue culture or in cellular extracts supplemented with NADPH (such as would be permissive, for instance, to measure HO activity) failed to detect free cobalt or protoporphyrin breakdown products (data not shown). Using identical logic, experiments were performed to study the time course of CoPP residence in the CNS using 57CoPP. Sensitivity was thus improved because naturally occurring cobalt (as for example, in vitamin B12) could not contribute to the signal and the 57CoPP was of high specific activity. For these experiments, the amount of 57CoPP injected was approximately three orders of magnitude lower than that which would normally be required to elicit weight loss, and this trace amount, predictably, had no effect on the weight of injected rats. However, it should be acknowledged that use of this tracer dose of 57CoPP might theoretically generate different kinetics and retention patterns than those observed after administration of the higher doses of CoPP that elicit weight loss. The results indicate that 57CoPP was detectable in the hypothalamus for at least 5 wk after intracerebroventricular injection. There was also a clear preferential retention of 57CoPP in the hypothalamus; the concentration remaining in whole brain at 5 wk was almost negligible compared with that detected in the hypothalamus (Fig. 1). The maximum duration of 57CoPP residence in the hypothalamus is unknown, but in preliminary unpublished data from our laboratory, 57CoPP has been detected in hypothalami of rats 4 mo after intracerebroventricular injection.
Although it is known that CoPP acts in the CNS to elicit changes in appetite and weight (5, 8), is 100-fold more active given intracerebroventricularly than subcutaneously (7), is sevenfold more active when given intrahypothalamically into specific nuclei than when given intracerebroventricularly (9), and as demonstrated above is retained preferentially by the hypothalamus for remarkably prolonged periods of time, it is unknown with which cell types CoPP interacts. To investigate this, the often used approach of looking for cells that are activated in response to a given stimulus by the surrogate endpoint of immunohistochemical detection of Fos protein in response to increased expression of the c-fos gene was adopted. These results indicate that intracerebroventricular CoPP induced a general activation of Fos expression in multiple hypothalamic nuclei (Fig. 2). In half of the areas in which Fos was quantified, there was no expression in vehicle-treated rats, and in those areas in which there was expression following vehicle, levels range from 15 to 50% of the corresponding values from CoPP-treated rats. However, the magnitude of the increase in Fos expression was overall greater in the nine hypothalamic sites that were counted compared with the extrahypothalamic sites. It is also notable that Fos expression in both hypothalamic and extrahypothalamic sites was generally observed in tissues located close to some aspect of the complex ventricular system that runs throughout the brain. Moreover, this was the case for Fos expression in both vehicle-treated and CoPP-treated animals, although the degree of expression was markedly less after treatment with vehicle than treatment with CoPP. These findings are consistent with the fact that in these experiments, both vehicle and CoPP solutions were administered intracerebroventricularly, into the dorsal III ventricle. Thus instillation of the relatively large volume (10 μl) of vehicle over 5–10 s could have induced a nonspecific activation of Fos (2) in those tissues close to the ventricular system, whereas the presence of CoPP in the same sites could have led to a more robust and specific activation of Fos expression.
That CoPP was taken up by the tissues is likely because Fos activation was detected in cells situated quite far from the ventricular system (see Fig. 3). It is possible that such activation could have resulted from some action or signaling from a site remote from the cell but which was more proximate to the CoPP contained within the ventricular system. However, the observation that CoPP elicited hypophagia and weight loss after microinjection into the hypothalamus but not the thalamus (9) tends to mitigate against this possibility. Indeed, the data suggest that CoPP needs to reside within or at least in very close proximity to target cells to elicit both biological sequelae and Fos activation. It is also clear from the data presented in the photomicrographs from Figs. 4–6 that expression of Fos 4 h after intracerebroventricular treatment with CoPP is found in neurons (identified as positive for both Neu-N and Fos) but minimally in glial cells (identified by the near absence of dual staining of Fos and GFAP). These dual-labeled neuronal cells were observed not only in hypothalamic nuclei involved in regulating feeding behavior, such as the arcuate nucleus (see Fig. 5), but also in extrahypothalamic sites such as the paraventricular thalamic nucleus and some parts of the hippocampus. It is not known if this extrahypothalamic neuronal Fos activation is related to the biological sequelae of hypophagia and weight loss from CoPP. Alternatively, it could result from the presumably higher concentrations of CoPP that occur near to the site of metalloporphyrin instillation into the dorsal III ventricle.
The effects of CoPP on appetite are relatively transitory, but the decrease in body weight compared with control animals can persist for days to months after treatment with CoPP (5, 7). The results from experiments with 57CoPP described in this manuscript indicate that CoPP is retained in the CNS, preferentially in the hypothalamus, for at least 5 wk after a single intracerebroventricular dose. Although the mechanism of action of CoPP in eliciting these effects on weight and appetite remains unknown, it is tempting to speculate that the longevity of its effects is in some way predicated on the longevity of its retention in the hypothalamus. In preliminary observations from our laboratory, Fos immunoreactivity has been detected in both hypothalamic and extrahypothalamic sites not only 4 h but also 2 and 7 days after intracerebroventricular injection of CoPP. It could be argued that such prolonged expression of Fos after intracerebroventricular injection may have resulted from continued stimulation of Fos expression induced by the continued presence of CoPP within or near the cells. This would be unusual in that Fos immunoreactivity, the product of the immediate-early gene c-fos, is often expressed maximally at 2–6 h and usually decays within 24 h (2). However, this pattern is not inviolate and might be altered by the continued presence of a stimulus (in this case CoPP) that is preferentially retained and is not subject to metabolism or degradation. There is also precedent in the literature for prolongation of fos expression in the CNS for 4 (12), 6 (14), or even 15 days (20) after various stimuli.
Previous observations (4, 19) led us to posit that CoPP must work distal to the level of at least some of the well-studied orexigenic peptides like neuropeptide Y (NPY). Other investigators have also reported that after intracerebroventricular CoPP, the feeding responses to intracerebroventricular NPY, galanin, and norepinephrine were all reduced compared with vehicle-treated rats (1). We have preliminary unpublished data that show there are no compensatory increases in orexins A or B, or melanin concentrating hormone after treatment with CoPP. These observations lend further support to the argument that CoPP must exert its influence, at least in part, at some point distal to the receptor-peptide interaction. Future work on the CoPP-activated Fos-positive neurons in the hypothalamus and possibly in the extrahypothalamic sites should help to clarify the mechanism of action of this unique metalloporphyrin in eliciting hypophagia and weight loss. If so, it may also serve to shed light on some of the feeding circuitry that exists distal to the currently known multiple peptide-receptor interactions.
These studies were supported by National Institutes of Health Grant R01 DK-53479 to R. A. Galbraith.
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- Copyright © 2004 the American Physiological Society