INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE PRODUCTION of osmotically active components within a cell, as well as the entry and exit of ions, must be tightly compensated if a cell is to maintain size and shape. One component of this homeostatic process is Na⁺-K⁺-2Cl cotransporter (NKCC) activity (7). This family of electrically neutral, bidirectional cotransporters is thought to provide facile movement of osmotic equivalents and is probably best known for its function in the kidney and as a target for diuretic compounds.

One member of the NKCC family [NKCC1, also known as bumetanide-sensitive cotransporter 2 (BSC2)] is thought to be ubiquitously expressed (2), giving strength to the hypothesis that this type of transport is important for cellular volume regulation (6, 7, 27). In intact skeletal muscle, however, there is some question about whether there is indeed functional expression of this cotransporter. Studies of muscle cells in culture indicate that NKCC is present (1, 13, 14, 20); however, culturing has also been shown to induce the expression of NKCC activity (23). Several recent publications demonstrate bumetanide-sensitive, ouabain-insensitive rubidium, thallium, and cesium uptake in rat soleus muscle, but a series of novel experiments suggest that ⁴²K uptake was not bumetanide sensitive (3, 4). Furthermore, bumetanide-sensitive ²²Na uptake in the rat soleus muscle also does not appear to be sensitive to extracellular potassium concentration, as would be expected if an NKCC were present (4). However, firm molecular evidence has yet to be presented to support the presence or absence of a member of the NKCC family in skeletal muscle.

In this study, we present molecular and functional evidence that NKCCs are expressed and active in rat slow-twitch skeletal muscle. We show that PCR amplification and cloning of a region of an mRNA expressed in soleus muscle is at least 90% homologous with the known rat and mouse NKCC1 mRNA (2); nuclease protection assays further demonstrate the presence of the mRNA in the soleus muscle. Furthermore, we show that the soleus expresses protein that is recognized by antibodies to NKCC proteins, and this protein is primarily located in the muscle fibers. Finally, we demonstrate that the soleus muscle exhibits cotransporter activity using radiotracer influx assays.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal care. Both male and female Sprague-Dawley and Wistar rats (200-250 g) were used for all experiments; no sex-related or strain-related differences in the data were observed. Animals were housed in light- and temperature-controlled quarters where they received food and water ad libitum. Animals were randomly assigned to experimental groups, and all animals were handled identically. Animals were anesthetized for tissue removal with pentobarbital sodium (40 mg/kg ip). All procedures were approved by the Animal Care and Use Committee of the University of Tennessee, Memphis.

RT-PCR. Total RNA was isolated from whole tissue by the guanidinium thiocyanate-cesium chloride method (17). Briefly, tissue was flash-frozen in liquid nitrogen and then homogenized in guanidinium thiocyanate buffer containing 4 M guanidinium isothiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M mercaptoethanol, and 0.5% Sarkosyl. The homogenate was centrifuged at room temperature for 10 min at 5,000 g. The supernatant was layered on a cushion of 5.7 M CsCl and 0.01 M EDTA (pH 7.5) and centrifuged overnight at 100,000 g at 20°C. The pellet was dissolved in Tris-EDTA [10 mM Tris · HCl (pH 7.6), 1 mM EDTA]. Total RNA was precipitated by adding 3 M sodium acetate (pH 5.2) and 20°C ethanol and collected by centrifugation. The resulting pellet was dissolved in sterile water. Total RNA (0.75 µg) was then reverse transcribed with 1 µg random hexamers and 800 U Superscript II RT (Life Technologies, Bethesda, MD) for 1 h at 42°C. This mixture was amplified by means of primers with the sequences: 3'-GCATCCCGAACAACACACACACGAAC and 5'-CACCCACACCAACACCTCTAC. These sequences correspond to regions in the 5' end of the highly conserved transmembrane-spanning domain of the NKCC and were chosen with the OSP program of Hillier and Green (9) and the mouse basolateral NKCC sequence (Genbank accession MMU13174). The PCR reaction mixture contained the following: 35.5 µl water, 5 µl reaction buffer (10×), 2 µl MgCl₂ (50 mM), 1 µl 10 mM dNTPs, 2 µl cDNA, 2 µl primers (0.25 µg/µl), and 2.5 U of Taq DNA polymerase. The cycling conditions were 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min 35 times followed by a final extension step of 10 min at 72°C. This mixture was digested with Rsa I at 37°C for 1 h and separated on a 2% agarose gel. When bovine adrenal RNA RT-PCR product is digested with Rsa I, the largest fragment produced is 588 bp. When rat RNA RT-PCR product is subjected to the same treatment, the largest fragment is 850 bp. This allowed for quantitation of the levels of RNA, as follows (Fig. 1). A specific amount of total bovine adrenal RNA was added to a specific amount of rat total RNA sample. This reaction mixture was reverse transcribed, amplified, and digested as described above. After digestion, the products could be separated by gel electrophoresis and quantified by means of video densitometry with the bovine adrenal product serving as an internal standard for the rat product. The data are expressed as ratio of rat RNA/bovine adrenal RNA.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   A: quantitative RT-PCR was performed by spiking each rat RNA sample with fixed amount of bovine adrenal RNA. Samples were reverse transcribed with random hexamers, and cDNA was amplified with primers that span conserved region of mRNA. Product from both species are same size (1,038 bp), but because of a slight difference in sequence, rat sequence lacks an Rsa I restriction site present in bovine sequence. Digestion with Rsa I (Rsa I digest) yields different size fragments from each, as shown in B. m gastroc, Medial gastrocnemius; adr, adrenal; glomer, glomerulosa; kid, kidney.

The PCR fragments were cloned and sequenced to verify the Rsa I restriction sites and the homology with known NKCC sequences. We obtained a sequence between the two primers for the rat soleus muscle (Genbank accession AF086758) that was 93% and 90% homologous with the same region in reported clones from rat parotid gland and mouse inner medullary collecting duct (Genbank accessions AF051561 and U13174, respectively).

RNase protection assay. An RNase protection assay was used to identify the presence of cotransporter-like mRNA. The PCR fragment generated above was cloned into the pGEM-T vector (Promega, Madison, WI) from which a radiolabeled RNA complementary to the mRNA was generated; RNA corresponding to the mRNA was generated by transcription in the reverse direction as a control. Digestion of the clone with Xba I allowed generation of a transcript of ~625 bases, of which a sequence of ~575 bases was complementary to the mRNA. The RNA probe was incubated with RNA isolated from the muscle, and a protected fragment was generated with the RPAII kit from Ambion (Austin, TX); -actin mRNA probe was included in the reaction as a control. The noncomplementary RNA probe was completely digested. The radiolabeled protected fragment was separated from degradation products on 7 M urea 7% polyacrylamide gels. The gels were dried and exposed to X-ray film with an intensifying screen at 80°C.

Western blots. The muscle was homogenized in 9 vol ice-cold PBS (pH 7.4) containing 175 mM sucrose, 0.7 mM EDTA, 10 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml antipain, and 1 µg/ml pepstatin A. The homogenate was centrifuged at 1,900 g for 5 min at 4°C, and the supernatant was centrifuged again at 48,000 g for 30 min at 4°C. The pellet was rehomogenized in the homogenate buffer. The protein content was estimated by means of the bicinchoninic acid assay (Pierce, Rockford, IL). One hundred micrograms of protein was mixed with SDS denaturing buffer, warmed to 95°C for 5 min, and electrophoresed overnight on a 7.5% SDS-PAGE gel. To test for cotransporter glycosylation, some protein samples were also treated using an N-glycosidase F deglycosylation kit (Roche Molecular Biochemicals USA, Indianapolis, IN) before denaturation and electrophoresis. The gels were electroblotted with the semidry blotter from Buchler Instruments (Fairfield, NJ). The membrane was incubated at room temperature for 1 h in the Western blocking buffer [PBS (pH 7.4) and 0.5% (vol/vol) Tween 20 (PBS-T) supplemented with 5% (wt/vol) nonfat dry milk]. Immunologic reaction was performed at room temperature for 1 h in PBS-T containing 5% (wt/vol) nonfat dry milk and the specific antibody. Either the T4 antibody raised against the 310 C-term residues of human colonic NKCC1 (from the Developmental Studies Hybridoma Bank, University of Iowa) (16) or a polyclonal antibody raised against the 453 C-term residues of rat parotid NKCC1 (the kind gift of Drs. Turner and Moore-Hoon) (18) were used at 1:50 or 1:10,000 dilution, respectively. The nonglycosylated form of each of these NKCC1 proteins has a calculated molecular weight of 130 kDa. The membrane was subsequently washed with PBS-T buffer and incubated for 1 h at room temperature with a horseradish peroxidase conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody. After extensive washing with PBS-T buffer, the immunocomplexes on the membrane were visualized by chemiluminescence (Renaissance kit, DuPont NEN Research Products, Boston, MA).

Immunohistochemistry. The soleus muscle was fixed with 4% paraformaldehyde in PBS by allowing it to soak for 1 h at 4°C; it was transferred to a sucrose solution [30% sucrose/2 mM MgCl₂ in PBS (pH 7.4)] and soaked overnight at 4°C. The tissue was embedded in Tissue-Tek OCT Compound (Miles, Elkhart, IN), frozen at 80°C, and then sectioned into ~50 µm slices with a microtome cryostat. These slices were fixed to 0.5% gelatin-coated slides by incubating for 30 min in 4% paraformaldehyde in PBS. The slices were permeabilized by incubating for 2 min in 20°C acetone and blocked for 30 min in 5% goat serum in 0.2% BSA. After diluting the primary antibody in PBS (0.2% BSA), the slices were incubated for 1 h, followed by incubation for 30 min with fluorescently labeled secondary antibody in PBS (0.2% BSA). The sections were visualized with the BioRad MRC1024 Confocal System Lasersharp version 3.0.

⁸⁶Rb uptake. The soleus muscles were removed from anesthetized rats and immediately placed in ice-cold Krebs-Ringer solution [in mM: 120.2 NaCl, 25.1 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂, 10 D-glucose (pH 7.4)]. A stock solution of bumetanide was made by dissolving it in DMSO and then in an equal volume of Krebs-Ringer solution (pH 7.4) to a final concentration of 10³ M. The bumetanide Krebs-Ringers solution was made by further diluting the stock bumetanide to a concentration of 10⁵ M with a solution of Krebs-Ringer containing 1 mM ouabain. The muscles were placed in oxygenated treatment Krebs-Ringer or control (DMSO vehicle, 1 mM ouabain) Krebs-Ringer incubation medium for 15 min at room temperature. After preincubation the soleus was divided into four parts by longitudinally teasing apart bundles of fibers so as to preserve fiber integrity; this was demonstrable by their contractile response to electrical stimulation. The fiber bundles were placed in oxygenated Krebs-Ringer solution containing treatment or vehicle plus ouabain and 1 µCi/ml ⁸⁶Rb. Vehicle treatment served as the control. Muscles were incubated for 0, 5, 15, or 60 min at 30°C. After incubation, the muscles were blotted, weighed, and homogenized in 2 ml of 0.3 M trichloroacetic acid for scintillation counting. The relative uptake of ⁸⁶Rb by the muscles was calculated by dividing the counts per minute in the muscle by the counts per minute of the incubation media and dividing this value by the weight of the muscle. This allowed the relative uptake to be expressed as milliliters of incubation medium ⁸⁶Rb activity taken up per gram of muscle wet weight (3). ⁸⁶Rb activity was then calculated by multiplying the relative ⁸⁶Rb uptake by the potassium concentration of the buffer and expressed as micromoles per gram wet weight. The treatment effect was calculated by subtracting the treatment-ouabain-treated muscle from the vehicle-ouabain-treated muscle at each time point. In addition to using bumetanide as a treatment, we also used furosemide at concentrations of 10⁵ M and 10⁴ M. In some experiments treatment consisted of varying the ionic composition of the incubation medium. In those experiments specific components of Krebs-Ringer solution were replaced, and ⁸⁶Rb uptake was measured after 60 min of incubation. These experiments compared the altered Krebs-Ringer-ouabain medium with the standard Krebs-Ringer-ouabain medium. For sodium-free Krebs-Ringer medium, sodium chloride was replaced with choline chloride, and sodium bicarbonate was replaced with choline bicarbonate. For chloride-free Krebs-Ringer medium, sodium chloride was replaced with sodium methyl sulfate, and calcium chloride was replaced with calcium acetate. For low-sodium Krebs-Ringer medium, 95.2 mmol/l of sodium chloride was replaced with choline chloride. For low-chloride Krebs-Ringer medium, 95.2 mmol/l of sodium chloride was replaced with sodium methyl sulfate. For chloride restoration Krebs-Ringer medium, 55.2 mmol/l of sodium chloride was replaced with sodium methyl sulfate.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

RT-PCR of RNA isolated from the rat soleus muscle and rat kidney yields a fragment 1,038 bp in length, corresponding to a portion of the conserved transmembrane domain of known NKCCs (Fig. 1). This is expected, because the sequence that was amplified is conserved among tissues of diverse species, from shark rectal gland to bovine adrenal gland to mouse inner medullary collecting duct (21). When the PCR products were digested with Rsa I, quantitation of the product showed that the rat kidney expressed less than half as much mRNA as the rat soleus muscle (Fig. 2). The PCR products were cloned and sequencing of the PCR product from the soleus muscle of the rat (submitted to Genbank as accession AF086758) shows a 90-93% homology to the 5' end sequence of the transmembrane spanning domain of NKCC1 mRNAs expressed in rat parotid gland and mouse inner medullary collecting duct (2, 18). An RNase protection assay was also performed to verify the mRNA presence in the soleus muscle. By means of an RNA transcript made from the PCR product clone, an expected protected fragment of ~575 bp in length was observed (Fig. 3). Although these data are all indicative of NKCC expression in muscle, we were concerned that this amplification was the result of contamination from either the vasculature of the tissue or other nonmuscle cells.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   A: with bovine adrenal RNA as internal control, RT-PCR reaction efficiencies could be controlled. Rsa I digestion of PCR products yields fragments of different sizes. Ratio of these fragments allowed comparison among samples and gels. B: with relative expression value of 1.0 for cotransporter expression in rat adrenal gland, relative expression of cotransporter in other tissues was compared. Values are means ± SE; n = 5 samples for each determination. BAG, bovine adrenal glomerulosa.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   RNase protection assay of rat muscle mRNA with an RNA probe generated from clone of PCR product demonstrates that expression of mRNA sequence in muscle is not a PCR artifact. -Actin mRNA probe was also included in protection assay as a control.

To verify the cotransporter in the muscle, we employed immunochemical techniques. Western blots with antibodies specific for the cotransporter showed bands in the range of 97-220 kDa (Fig. 4). The horseradish peroxidase-conjugated secondary antibodies alone did not cross-react with the blots, nor did preimmune serum for the polyclonal antibody (also a gift from Drs. Turner and Moore-Hoon). Deglycosylation of the samples before electrophoresis did not change the Western blot banding pattern, as has been observed with chloride-secreting epithelium (16). Immunohistochemical analysis with the antibodies showed that it is found primarily associated with muscle fibers rather than the vasculature or other cells (Fig. 5). However, the vasculature did show some staining, as would be expected (7, 19, 23), but the majority of staining is located in the myofibers. As with the Western blots, neither the fluorescent secondary antibodies nor the preimmune serum exhibited reactivity.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Western blots of soleus and plantaris muscle protein indicate both T4 monoclonal antibody (Ab) and polyclonal Ab recognize proteins in size range appropriate for Na+-K+-2Cl cotransporter (NKCC). However, each Ab apparently recognizes different proteins. In particular, polyclonal antibody recognizes protein expressed in soleus muscle that is not abundant in plantaris muscle. Protein sample probed with each Ab was same in each blot, and each was run on same gel.

View larger version (190K): [in this window] [in a new window]   Fig. 5.   Confocal microscopy of soleus muscle with each of the antibodies (A and C: T4 monoclonal antibody; B and D: polyclonal antibody) indicates reactive protein is primarily associated with muscle fibers. Arrows identify staining of skeletal muscle fibers (A-D), lack of staining in vasculature in case of T4 antibody (A), and staining of cells within vasculature in case of polyclonal antibody (B). Calibration bars are 20 (A and B) and 5 µm (C and D).

⁸⁶Rb uptake data indicated that the cotransporter was functional. In the presence of bumetanide, ~15% of the ⁸⁶Rb uptake was bumetanide sensitive and ouabain insensitive. Approximately 50% of the ⁸⁶Rb uptake was caused by the Na⁺-K⁺-ATPase activity, as measured by inhibition with 1 mM ouabain (Fig. 6). To verify that the ⁸⁶Rb transport activity exhibited classical cotransporter characteristics, several other treatments were used: chloride-free medium, sodium-free medium, 25 mM sodium, 25 mM chloride, 65 mM chloride, 10⁵ M furosemide, and 10⁴ M furosemide. Bumetanide-sensitive, ouabain-insensitive ⁸⁶Rb uptake is inhibited in chloride-free medium and sodium-free medium with the characteristic difference in sensitivity to sodium and chloride concentrations (Fig. 7). A much lower sodium concentration restores cotransport activity than is required for chloride restoration of cotransport activity (24, 26). Thus at 25 mM sodium the ⁸⁶Rb uptake was fully restored, whereas at 25 mM Cl the ⁸⁶Rb uptake remained depressed. At 65 mM Cl the ⁸⁶Rb uptake was fully restored. When treated with furosemide there was no inhibition at the concentrations used, as would be expected (data not shown). Furosemide has been shown to be a less potent inhibitor than bumetanide at similar concentrations. Also it should be noted that if the inhibition is occurring through the same mechanism, then the degree to which the ⁸⁶Rb uptake is inhibited should be similar. Indeed it was; with bumetanide and sodium- and chloride free mediums, 25 mM chloride ⁸⁶Rb uptake was inhibited to the same degree.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Radiotracer uptake by soleus muscle is both ouabain and bumetanide sensitive. Muscle was preincubated with inhibitors (105 M bumetanide, 1 mM ouabain) 15 min before 86Rb uptake. * P < 0.05 for bumetanide + ouabain-sensitive uptake vs. ouabain-sensitive uptake; n = 5 samples per point.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Soleus muscle ouabain-insensitive 86Rb uptake exhibits differential sodium and chloride sensitivity. Transport requires very little sodium, whereas chloride must be present in much higher concentrations. 86Rb uptake was measured after 60 min of incubation. n = 6 samples per point.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The existence and expression of a functionally active NKCC in various intact tissues has been established, most prominently in secretory epithelia. Evidence for its expression in nonepithelial cells and more specifically muscle cells in culture also has been established (4, 7, 14, 19, 20, 23). However, it has been shown that culturing cells can induce the expression of some genes, one of which is the NKCC (23). Before the present study, the molecular evidence of a functional NKCC in intact skeletal muscle was scant. In fact, a series of novel ⁴²K-uptake experiments indicated that the rat soleus muscle lacked the functional cotransporter (3). However, our experiments provide molecular evidence of NKCC expression and activity.

Several isoforms and splice variants of the cotransporter exist (21). We chose to design primers to a highly conserved sequence across species of the broadly expressed NKCC1 isoform (28). We felt that these primers would have the greatest likelihood of identifying the expression, if any, of an NKCC family member. RT-PCR of RNA isolated from the rat soleus muscle produced a fragment of predicted length. Sequencing of the PCR product demonstrated a 90-93% homology over the entire length of the fragment with the region of rat and mouse basolateral NKCC1 mRNAs flanked by the primers (2, 18). This region includes the coding sequence for the second membrane spanning region that is thought to be involved in potassium and bumetanide binding (10). Restriction mapping of the RT-PCR products indicate amplification of a single sequence. We were thus confident that the RT-PCR was amplifying part of an mRNA similar to known NKCC1 (BSC2) mRNAs.

However, we were unsure as to the location of the expression. Expression could have been caused by some contaminating nonmuscle cells or expression by the vasculature of the muscle. By means of antibodies specific for NKCC proteins, immunoblots from whole muscle homogenates showed bands of the expected size. However, different specific antibodies yielded strikingly different results. The polyclonal antibody (18) recognized several proteins that ranged in size from 97 to 200 kDa, whereas the monoclonal antibody (T4,16) recognized two bands in the range of 180-200 kDa. Although both antibodies were raised to the C-terminus of NKCCs, the epitopes were from different species. So even one amino acid difference could alter the specificity of the antibody. A possibility is that the antibodies recognize different isoforms of the cotransporter, or they recognize different processed, unprocessed, active, or inactive forms of the cotransporter. In support of the possibility that the polyclonal antibody is recognizing different forms of the cotransporter is the appearance of the immunoreactivity in Western blots of the predominantly slow-twitch soleus muscle but very little in the predominantly fast-twitch plantaris muscle (Fig. 4), whereas the monoclonal T4 antibody recognizes cotransporter protein in both muscles. Also, cloning of the 5' end of a putative NKCC mRNA expressed in soleus muscle indicates that unique isoforms may be expressed (5). Whether the polyclonal antibody is recognizing a true isoform (genetic or alternatively spliced) or a posttranslational modification of the cotransporter remains to be determined, but both possibilities exist. For example, it is known that alternatively spliced isoforms of a secretory form of the cotransporter are expressed in different locations within the kidney, each with putative posttranslational modification sites (22).

The purpose of the immunohistochemical analysis was to identify whether a cotransporter protein was localized to muscle fibers or to other cells in the muscle bed. Immunohistochemistry with either the monoclonal or polyclonal antibody revealed that a cotransporter was associated with the skeletal muscle fibers; the polyclonal antibody recognized protein in the vascular endothelium as well. Although antibody binding was associated with the sarcolemmal margins of the fibers, significant binding also appeared to be located within the fibers. This observation raises the possibility of a potentially important mechanism for cotransporter activity regulation: rapid activation of cotransporter activity could be by translocation of the protein from intracellular pools to the sarcolemma, as has been offered as a possibility in other cell types (11, 12, 15). It is also possible that a cotransporter may have both sarcolemmal and intracellular membrane functions. Nonetheless, the data presented here indicate that the majority of the immunoreactive protein is associated with muscle fibers and not with other cell types.

There are some uptake studies that indicate that a functional cotransporter is not present in slow-twitch skeletal muscle. Using ⁴²K as the tracer, Dørup and Clausen (3, 4) were unable to show significant bumetanide suppression of ⁴²K influx by the rat soleus muscle during 10 min of uptake. One of these studies also showed a chloride-sensitive, bumetanide-sensitive ²²Na uptake in the soleus muscle (4). The conclusion drawn was that a bumetanide-sensitive NKCC activity is not detectable in the soleus muscle. However, in agreement with these studies, we showed that ⁸⁶Rb uptake by the rat soleus muscle was decreased after incubation with bumetanide. Approximately 15% of the Rb uptake was bumetanide sensitive and ouabain insensitive, and ~50% was ouabain sensitive, thus 35% was a result of channels or uncharacterized mechanisms. A significant suppression of the uptake of ⁸⁶Rb was also seen when the muscle was treated with chloride-free medium and sodium-free medium. This ion dependence is characteristic of NKCC activity (24, 26). When the muscle was treated with both chloride-free medium and bumetanide or just chloride-free medium alone, there was no significant difference in the degree of inhibition of ⁸⁶Rb uptake. Therefore, it appears that the chloride dependence and inhibition by bumetanide of ⁸⁶Rb uptake work through the same mechanism. We have interpreted these results, combined with our molecular data, as evidence for a functioning cotransporter in rat soleus muscle fibers; we must be aware, though, that both muscle and nonmuscle cells may be contributing to the ⁸⁶Rb uptake. Why then do ⁴²K and ⁸⁶Rb show differences in their bumetanide-sensitive uptake, as reported by Dørup and Clausen (3, 4)? One simple possibility is the incubation time; we required 15 min of incubation before a bumetanide-sensitive uptake was detectable, whereas the cited studies used a 10-min incubation time. This alone would not explain the difference between ⁴²K and ⁸⁶Rb unless other factors contributed to measurement difference in uptake, i.e., increased uptake rate for Rb or greater measurement error for ⁴²K uptake. The former is not supported by their rate data (3) and the latter is unlikely given the careful controls with which their studies were undertaken. Another possibility raised in one of the studies (3) is that the NKCC system in muscle is slightly different from the classical cotransporters in that the cotransport of the larger potassium congeners, Rb, Cs, and Tl, can be inhibited, but potassium escapes inhibition. Indeed, the immunoreactivity data present here raise the possibility that the NKCC system in the soleus muscle may have added complexity.

We and others have used bumetanide as a pharmacological means to measure cotransporter activity against a background of other ion fluxes. In part, the imprecision and disparate results from the use of this tool have obscured the physiological question of the significance of the cotransporter activity in skeletal muscle. Although the cotransporter is bidirectional, the thermodynamically favorable direction in resting muscle is inward on the order of 4 to 6 kcal. Given this favorable transport direction, it could be argued that the mass of skeletal muscle would provide a huge sink for potassium, sodium, and chloride, and therefore the unidirectional flux would be physiologically dangerous. However, this flux is apparently balanced by leakage and active transport. For potassium, the large mass of skeletal muscle exhibiting inward passive cotransport combined with active transport of sodium and potassium may provide an important mechanism for potassium recovery and buffering. For example, working muscle loses a large amount of potassium into the general circulation (8, 25). The NKCC activity provides a possible means for recovering part of the lost potassium. Likewise, ingestion of food often produces a significant potassium load that, if placed in the general circulation, could cause a dangerous hyperkalemia. NKCC activity in a large muscle mass would contribute to the buffering of the ingested potassium. Thus in combination with the active transport of potassium, passive transport coupled to thermodynamically favorable gradients could provide an additional mechanism to sequester potassium within muscle cells.

In summary, although there has been some debate over the presence of a functional NKCC activity in slow-twitch skeletal muscle, the molecular and functional evidence presented here indicate that it is expressed. The mRNA of a conserved sequence of NKCCs that can be detected by RT-PCR and RNase protection assay, immunoblots, and immunohistochemistry all indicate that the muscle is expressing a cotransporter protein. Furthermore, the ⁸⁶Rb uptake data, with its characteristic sensitivity to bumetanide, sodium concentration, and chloride concentration, indicate the muscle has a functional NKCC activity.

Perspectives