Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells

Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells. Am aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for speciﬁc cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb (cid:2) (a K (cid:2) congener) inﬂux was determined as Cl (cid:3) -dependent Rb (cid:2) inﬂux at different external Rb (cid:2) and Cl (cid:3) ion concentrations, [Rb (cid:2) ] o and [Cl (cid:3) ] o . Expressions of the cytoskeletal proteins (cid:4) -actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( V m ), and apparent Cl (cid:3) and Rb (cid:2) afﬁnities ( K m ), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. V m values of both Rb (cid:2) - and Cl (cid:3) -dependent inﬂuxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefﬁcients for Rb (cid:2) and Cl (cid:3) correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, K m values for [Cl (cid:3) ] o were strikingly bimodal with 60–80 mM in early, ~20–30 mM in medium, and 60 mM in late passage cells. In contrast, K m values for [Rb (cid:2) ] o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.


INTRODUCTION
Cardiovascular disease (CVD), including atherosclerosis, results in significant morbidity and mortality worldwide (55), and in the United States atherosclerosis accounts for more than 400,000 deaths annually (60,68). CVD involves buildup of plaques in blood vessel walls restricting blood flow. Normally, blood vessel walls display ordered concentric layers of endo-thelial cells (intima), smooth muscle cells (media), and fibroblasts (adventitia) (46,66). Proliferation of vascular smooth muscle cells (VSMCs) and migration from the media to the intima augment enlargement and restructuring of the atherosclerotic lesion in the blood vessel wall (69,85). Narrowing of blood vessels caused by atherosclerosis is a chronic inflammatory response with recruitment of macrophages, lymphocytes, and platelets from circulating blood (67). All these factors restrict blood flow and significantly increase the likelihood of a heart attack or stroke (57,68).
Phenotypically, VSMCs are heterogeneous as characterized by their shape, size, motility, and proliferative capacity (64). During growth of the atherosclerotic lesion, VSMCs transition from a contractile or differentiated physiological to a synthetic or dedifferentiated pathological phenotype (15,62,71,79). The term "dedifferentiation" in VSMCs does not imply reversal to an early multipotent stage, but rather a transition from contractile to the highly mitotic synthetic phenotype (15,34).
Therefore, in the present longitudinal study, the hypothesis was explored whether there exists a correlation between cell passage (P) number and different functional parameters of Cl Ϫ -dependent K ϩ transport or KCC, such as maximum velocity, V m , relative affinity, [K m ], for external K ϩ concentration ([K ϩ ] o ; using Rb ϩ as K ϩ congener) and external Cl Ϫ ion concentration ([Cl] o ), and ligand transport cooperativity in different synthetic VSMC phenotypes. Results revealed similar V m values for the two ions, i.e., stoichiometry of unity, as well as Hill cooperativity coefficients of Ͼunity, both increasing with cell passage, and a striking longitudinal difference between the apparent external affinities for the transported ions. Putative implications for structure/function changes are offered for the KCC signalosome, a term used here for the first time for a purported KCC multifunctional protein complex consisting of any combination of its 1, 3, and 4 isoforms present in VSMCs and their docking regulatory protein phosphokinases and phosphatases and other cofactors.
Balanced salt solutions for Rb ϩ transport studies. All stock salt solutions were prepared in deionized water. Balanced salt solution (BSS-NaCl) contained 20 mM HEPES-Tris buffer (pH 7.4 at 37°C) consisting of (in mM) 130 NaCl, 5 KCl, 2 CaCl 2, 1 MgCl2, 10 glucose. The preincubation solution contained 0.1% BSA in BSS, and the flux solution replaced 5 mM K ϩ with 10 mM Rb ϩ . The Cl Ϫ -free media contained sulfamate (Sf Ϫ ) in K ϩ , Rb ϩ , and Na ϩ salts, and gluconate in Mg 2ϩ and Ca 2ϩ salts. A wash buffer to terminate Rb ϩ uptake contained 10 mM MOPS-Tris and 108 mM MgCl2, pH 7.4 (300 mosmol/kgH2O). Stock solutions of ouabain (400 mM), bumetanide (400 M), and N-ethylmaleimide (NEM; 100 mM) were prepared in DMSO as a solvent so that the final DMSO concentration added to the cultures was Ͻ0.25%. The osmolalities (mosmol/kgH 2O) of the salt solutions were measured with an Advanced Micro-Osmometer (Norwood, MA).
Measurement of Rb ϩ transport in cultured cells. The flux protocol followed previous publications (6,90). VSMCs cultured to 90% confluence in 12-well plates were washed three times with BSS and then equilibrated for 10 min in preincubation media. Rb ϩ uptake required replacing preincubation solution with flux solution for a specific time at 37°C. Unless otherwise indicated, ouabain and bumetanide were added to both the preincubation and flux solutions to block the Na ϩ /K ϩ pump (NKP) and Na ϩ -K ϩ -2Cl Ϫ cotransporter (NKCC), respectively. NEM was added to stimulate KCC activity (7,47,52). Unidirectional Rb ϩ transport was terminated by washing the cells five times with an ice-cold isotonic MgCl 2/MOPS-Tris wash buffer. Intracellular Rb ϩ content was determined by extracting Rb ϩ ions with 5% PCA and 4 mM CsCl at 4°C for 15 min, and then measured by flame emission spectrophotometry in a PerkinElmer 5000 atomic absorption spectrophotometer as described elsewhere (7,90). Total protein content was determined by solubilizing the residual cellular matter with 1 N NaOH and using the BCA protein assay as previously described (25). Rb ϩ influx is expressed in nanomoles per milligram of total protein per unit time.
Western blot analysis. The protocol for Western blot analysis was adapted from former publications (3,50,89). The cytosolic proteins desmin, vimentin, and ␣-actin were resolved in 8.5% SDS-PAGE before transfer to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 10% nonfat dry milk or 5% BSA in Tris·HCl-buffered saline tween (TBS-T) for 1 h at room temperature (RT) and then exposed overnight at 4°C to the following primary antibodies: ms anti-␣-actin (1:1,000), rb anti-vimentin (1:1,000), ms anti-desmin (1:1,000), and ms anti-␤-actin (1:1,000) as an internal positive control. The PVDF membranes were then washed extensively in TBS-T and incubated for 2 h with the appropriate HRP-conjugated secondary antibodies against ms or rb IgG. Membranes were washed three times in TBS-T before signal detection with SuperSignal West Pico PLUS Chemiluminescence substrate (Thermo Scientific) in a Fuji LAS300 CCD camera under high-sensitivity/resolution settings. Densitometry of the protein signals was analyzed by computer software attached to a Kodak camera and/or J software.
Immunofluorescence of cytoskeletal markers. The immunofluorescence procedure was described elsewhere (3,50). VSMCs were seeded in eight-well chamber slides (Laboratory-Tech; NUNC) at a density of 8 ϫ 10 4 cells/well and grown until 25% confluence. Cells were washed with ice-cold 1ϫ PBS, fixed with 0.5 ml/well of 4% paraformaldehydeϩsaponin for 30 min at 4°C, then incubated in 0.5 ml/well of 3% normal goat serum (NGS) for 1 h at 4°C to block nonspecific immune staining. Cells were incubated with ms primary monoclonal anti-␣-actin (1:250) IgG antibodies in 3% NGS overnight at 4°C. Cells were then washed twice with ice-cold PBS then incubated with a sheep anti-ms IgG-FITC (1:1,000) in NGS for 2 h RT followed by final washes in PBS and deionized water. After growth chamber removal, a coverslip was mounted on the slide with Vectashield containing the nuclear stain 4=,6-diamidino-2-phenylindole (DAPI). Slides were imaged with an inverted Nikon E400 epifluorescence microscope using ϫ100 (oil) objectives, images were collected with a Cannon Rebel Model T1i camera system, the background was adjusted and then superimposed using GIMP software.
Statistical analysis. GraphPad Prism 5 software (La Jolla, CA) was utilized to determine statistical significance for the increase or decrease in protein expression level (expressing means Ϯ SD). For transport assays, bar or line graphs were created using Origin 7.0 (Origin Laboratories, Northampton, MA) and STATISTIX 7 software (Analytical Software, Tallahassee, FL), and data are reported as means Ϯ SD or SE for N ϭ number of independent experiments and n ϭ number of individual determinations. Statistical analysis was done by one-way ANOVA followed by a post hoc Tukey test and unpaired Student's t-test, with significance considered at P Ͻ 0.05.

RESULTS
Growth rate of contractile and synthetic VSMCs as a function of passage number. Cultured VSMCs were chosen as a model system to uncover further characteristics of their phenotypic differences that may be a useful contribution to our understanding of the progression of CVD (70). Several earlier reports correlated VSMCs' passage number with their respective phenotypes. At P0 -4, VSMCs are defined as predominantly contractile, at P5 as intermediate, and at P6 and later as synthetic (12,43,54). An important factor in characterizing VSMC properties is the time required for their proliferation in culture. When propagated at a constant seeding density, an inverse correlation between time to reach confluence and VSMC P number was observed. In accordance, Fig. 1 classifies P0 -4 VSMCs as contractile (C) when reaching confluence after 10 -14 days, P5-19 as early (E) synthetic between 4 and 7 days, P20 -69 as medium (M), and P70 -90 as late (L) synthetic phenotype cells consistently in 3 and 2 days, respectively. The statistics for the inset in Fig. 1 and accompanying legend define the contractile, early, medium, and late synthetic phenotypes.
K ϩ influx pathways in VSMCs. Rubidium ( 85 Rb ϩ ) has been widely used as a potassium (K ϩ ) congener in ion flux studies of rat VSMCs (6,90), rat C6 glioma cells (31), and sheep red blood cells (4,21,52). It is well known that VSMC have a sizable "leak flux" through ion channels (18) that, in our past work and in contrast to low ion channel activity in unaltered red blood cells (49), always interfered with facile measurements of Rb ϩ through KCC, a variable fraction of the leak flux. Furthermore, the relative ouabain-insensitive ␣1 Na-K-ATPase isoform dominates in rat VSMCs (11). Thus, to rule out these issues potentially affecting a more detailed kinetic study of KCC activity in VSMCs, the concentrations of ouabain and bumetanide to block Rb ϩ influx through NKP and NKCC, respectively, were assessed. Figure 3A displays that 2 mM ouabain achieved maximal inhibition of the OS-sensitive (OS) NKP flux, which, at zero ouabain, was~50% of the entire Rb ϩ influx, the remaining 40 -50% constituting the ouabain-insensitive (OI) Rb ϩ influx through NKCC, and the leak component containing KCC. An estimate of the IC 50 yielded~0.5 mM ouabain, suggesting the presence of predominantly ␣1 catalytic subunits. Figure 3B shows that, with 2 mM ouabain blocking NKP, 1 M bumetanide asymptotically abolished 70% of the OI flux remaining (Fig. 3A), consistent with a high affinity (~10 Ϫ7 M) of NKCC1 for this drug (40,41). The remainder of the uninhibited Rb ϩ influx constituted influx components through both ion channels and KCC. Thus the optimal ouabain and bumetanide concentrations used here were 2 mM and 2 M, respectively.
A further necessity to determine Rb ϩ uptake through KCC was to measure the former in Cl Ϫ -and Sf Ϫ -based media and calculate the difference (i.e., the Cl Ϫ -dependent Rb ϩ uptake). Figure 4A shows the Rb ϩ influx, measured at initial velocity over 30 min, in Cl Ϫ (black column) and Sf Ϫ (grey column). The calculated Cl Ϫ -dependent, KCC-mediated influx, 1 nmol/mg ϫ min (white column), was~70% of the total Rb ϩ influx in Cl Ϫ in the presence of 2 mM ouabain and 2 M bumetanide, which agrees with the data in Fig. 3B. A final test, evidencing Cl Ϫ -dependent Rb ϩ influx via KCC, was to determine its volume and NEM activation (4,6,27). As shown in Fig. 4B, KCC activity under hypotonic stimulus more than doubled (with respect to isotonic control, P Ͻ 0.01), and 0.05 mM NEM activated KCC by threefold (P Ͻ 0.001). The latter mode of activation, initially discovered by us in erythrocytes, has since become a diagnostic hallmark for the presence of KCC activity, ascribed earlier to thiol group modification (47,52).
KCC kinetic parameters in synthetic VSMCs. Previous studies from this laboratory have focused on characterizing the activity and expression of the different KCC isoforms as well as their regulation in contractile or early synthetic VSMCs (2-4, 6, 7, 22, 25, 89, 90). The focus of this study was to determine the relationship between KCC activity and VSMCs' phenotypic transition. Due to the rather extensive manipulations mandated by protocol, VSMCs frequently detached from their plates. Poly-D-lysine, commonly employed to improve cell attachment in culture, changes their ion transport properties and hence was not used to improve cell attachment.   Fig. 5, B-D, respectively. The Hill coefficient "N" was 1.9, i.e., different from unity (Fig. 5D). Figure 6A shows a Lorentzian adaptation of the saturating Rb ϩ influx through KCC, corrected for ion channel-mediated Rb ϩ influx, as a function of increasing [Cl Ϫ ] o in P39 cells (used above in the Rb ϩ -kinetic experiment), i.e., medium  4). B: maximal activation of KCC by hypotonicity and N-ethylmaleimide (NEM) in P3 VSMCs, respectively, normalized to baseline. Percent (%) Cl Ϫ -dependent Rb ϩ influx (KCC) in isotonic (control; 300 mosmol/kgH2O) and hypotonic (120 mosmol/kgH2O) media and after exposure to the thiol-modifying agent NEM (isotonic; 300 mosmol/kgH2O). NaCl was used to vary the osmolality. Ouabain and bumetanide were present in both preincubation and flux solutions to inhibit NKP and NKCC, respectively; see Fig. 3. Data represent average of two independent experiments, each done in triplicate (n ϭ 3), expressed as means Ϯ SE (n ϭ 6 individual determinations). **P Ͻ 0.05 and ***P Ͻ 0.01 vs. control group.  Fig. 6, B and C and Ͻ0.001 in Fig. 6D, respectively. The V m values for KCC were 2.0 and 1.8 nmol/(mg protein ϫ min) in Fig. 6, B and C and thus on the order of the V m values shown above for Rb ϩ . The apparent binding affinities for Cl Ϫ were calculated as 39, 29, and 21 mM for Fig. 6, B, C, and D, respectively. The Hill coefficient N was 1.6 and thus again different from unity, suggesting cooperativity of KCC-mediated transport. A summary plot of the KCC influx kinetic results obtained as a function of cell passage measured in eight separate experiments is shown in Fig. 7 Fig. 7A, the V m values for Cl Ϫ dependence of Rb ϩ influx via KCC increased by 3-fold over an interval of 80 passages as estimated by both LB and HW analysis, whereas the behavior of the V m values for Rb ϩ dependence of KCC at higher passages is uncertain due to lack of data. Concomitantly, the Hill coefficient N rose, at least for Cl Ϫ -dependent Rb ϩ influx, by twofold and seemed to increase moderately also for Rb ϩ dependence (Fig. 7B). The K m values for [Rb ϩ ] o (Fig. 7C) remained uninterestingly~17 mM. In sharp contrast, the K m values (Fig. 7D) for [Cl Ϫ ] o were much higher in the early passages, then fell to about one-fourth before rising again by threefold at P86 cells. One-way ANOVA followed by a post hoc Tuckey test on the three data points per passage revealed a statistical difference between the K m of P6 vs. P16 (**P Ͻ 0.01), P6 vs. P39 (*P Ͻ 0.05), of P86 vs. P16 [(*P Ͻ 0.05, at P16 vs. P86 (P Ͻ 0.05)], and no significance for P6 vs. P86. One-way ANOVA showed no significant difference between the four cell passages for [Rb ϩ ] o .

DISCUSSION
The novelty of the results of this study are prioritized as follows. 1) The apparent Cl Ϫ affinity of KCC was bimodal during VSMC phenotype progression with statistically highly significant differences between early, intermediate, and late synthetic phenotypes. 2) In sharp contrast, the apparent external K ϩ affinity as a function of cell passage was statistically not significantly different.
3) The calculated V m values for both Rb ϩ and Cl Ϫ dependence of Rb ϩ influx through KCC were stoichiometrically consistent with a 1:1 flux ratio. 4) Hill coefficients were greater than unity, suggesting an increase in cooperativity of ligand transport as a function of time, and they were accompanied by elevated KCC transport rates, V m , at higher cell passages. Such a precise longitudinal study has not been hitherto done and, therefore, is an important contribution to our understanding of animal VSMCs as models for CVD, requiring further analysis in terms of kinetic implications and perhaps structural or signaling events within the KCC signalosome.
In healthy vessels, VSMCs undergo a change from the contractile to the synthetic phenotype, a process disrupted in the formation of atherosclerotic plaques. The equivalent transition of primary cultured VSMCs and the inverse relationship between time to confluence and passage number observed in this study corroborate previous reports (15). Western blot analysis (Fig. 2, A-C) of early, middle, and late synthetic VSMC lysates, demonstrating a significant loss of the contractile proteins ␣-actin, vimentin, and desmin, is consistent with previous studies showing similar changes in protein expression as VSMCs transition from contractile to synthetic phenotypes (29,79,88). Furthermore, immunocytochemistry (Fig. 2, D and E) revealed early synthetic VSMCs with abundant ␣-actin protein in a well-organized network, whereas late synthetic VSMCs displayed a severely disorganized and punctate expression of ␣-actin, suggesting problems with vesicular trafficking and an inability to move to their correspondent loci in the network. Another interpretation is that the cells of late passages are more secretory as their vesicles appear as punctuate and disorganized structures. Indeed, further magnification (not shown) supports this argument. Whether they are more secreting, or the punctuate expression signifies severe disorganization, is hard to discern because an image of the "normal" secretory VSMCs, which exist in equilibrium with the contractile ones in a healthy vessel, was not available. In the latter, the interchange depends on whether the vessel must contract/ relax or repair or grow more vessels. Taken together, our operational classification of primary cultured VSMCs into early, medium, and late synthetic phenotypes based on cell passage number is therefore quite appropriate and provides a useful framework to assess the role of KCC in VSMC phenotypic transition.
Attempting to kinetically characterize KCC of VSMCs during their phenotypic expression changes as a function of passage, nonlinear regression analyses (such as a Lorentzian function) were used to describe the raw experimental data (Figs. 5A and 6A), and three linear transformations (Figs. 5,  B-D and 6, B-D), each with their own limitations, to obtain estimates of the standard parameters such as V m , K m , and cooperativity, Hill N. In the LB approach of Figs. 5B and 6B, Based on the three independent kinetic approaches, it was inviting to perform statistical analyses on these values as shown in the longitudinal summary in Fig. 7. In Fig. 7A, the V m values increased, especially doubled in the case of KCC Rb ϩ   (9,39). Extrapolating from the low affinity for [Cl Ϫ ] o to a similar internally kinetic behavior of KCC may point to modulation of phosphorylation through WNK-controlled SPAK at the transporter's N-or C-terminal serine/threonine residues, a hypothesis that can be tested.
That the much harder to measure affinities for Cl Ϫ , but not for Rb ϩ , so strikingly changed, reminds one of the landmark review on "Chloride in Smooth Muscle" by Chipperfield and Harper; their classic paper pointed to the overriding presence of Cl Ϫ channels causing difficulties of measuring Cl Ϫ -dependent cation pathways (e.g., NKCC and KCC) in smooth muscle cells (18). However, as Figs. 4 -6 reveal, as well as the statistical behavior of K m values for [Cl Ϫ ] o in Fig. 7, special precautions were taken to define KCC in VSMCs. 1) Exposure to hyposmotic solution and to NEM caused up to threefold stimulation of KCC, both hallmark criteria for the presence of this transporter (7,13,31,48,52), causing its upregulation due to dephosphorylation of key threonine and serine residues within its C-terminal domain (5) by PP1/2 phosphatases (28,42,80). We have recently shown how the phosphorylation state of KCC3, for example, confers a potent molecular switch of transporter activity, cell volume, and K ϩ content (5). Indeed, the KCCs have been proposed by us in VSMCs (3) and found by others in different cell types, and especially in cancer and cell aging (16,17), playing important roles in cell proliferation, migration and malignancy. 2) Significantly higher ouabain concentrations (2 mM) were necessary to inhibit NKP activity in rat VSMCs compared with previous reports using different human cell types [e.g., human lens epithelial and embryonic kidney (36,51)]. This means rat VSMCs primarily express the ␣1 isoform. In human cells, three main ␣-subunits (␣1Ն␣3Ͼ␣2) of NKP have similar high ouabain affinities (19) as opposed to rodents, where the ␣2 isoform has a significantly higher ouabain affinity than ␣1 (44,61). We further determined that NKCC activity was fully inhibited with 1 M bumetanide, consistent with its high affinity for this cotransporter. Altogether, the ouabain-sensitive portion of the total Rb ϩ influx (NKP) was~50%, and NKCC constituted~40% of the remaining ouabain-resistant Rb ϩ influx, leaving 10% for the basal leak that includes ion channel and KCC activity.
How finally can one reconcile an apparent concomitant or simultaneous change of in V m , Hill N, and K m parameters for [Cl Ϫ ] o without a translational change in KCC1, 3, and 4 protein expression in VSMCs as a function of cell passage (data not shown)? Regardless, whether the KCC proteins were cytosolic or membrane bound, since their actual mass did not change, the obvious answer is that a greater level of KCC dephosphorylation at the now well-established N-and C-terminal serine and threonine moieties, as reflected in kinetic parameter change, commenced as a function of cell passage (5). It is notable that VSMCs lose their PKC (12,25,56), an enzyme that belongs in the lipid signaling orbit in the process of changing from contractile to synthetic phenotypes. An alternate hypothesis cannot be excluded. It has been recently shown that C-terminal domains of prokaryotic Archea CCC contain ␣/␤ folds, also found in stress proteins, which participate in solution dimer formation governing homodimer structure/function relationships of full-length CCC transporters (86). Perhaps these protein equilibria are affected by the different Eisenman anion series coordinates of the three KCC isoforms 1, 3, and 4 that are not only transcribed in rat VSMCs into mRNA (23)(24)(25) but are also translated as proteins (data not shown).
The changes in KCC kinetics summarized in Fig. 7 may be indeed related to various additional mechanisms. For instance, changes in KCC isoforms' expressional composition in the late and medium synthetic phenotypes, or mutually exclusive KCC gene-expression profiles, may occur in VSMCs, as proposed earlier for the chronic effect of PDGF in early synthetic VSMCs (90). This is an important area of research that is out of the scope of the present study and requires further investigation. In addition, changes and distribution of the cytoskeleton could modulate membrane proteins and alter ion transport activity (38). Because cytoskeletal proteins regulate intracellular signaling cascades, their distribution and location may be important for determining the VSMC phenotype. Thus any reorganization of the cytoskeleton or signaling molecules could affect VSMC phenotypic modulation (88). Moreover, several cell culture models suggest that the cytoskeleton machinery is linked to cell migration (14,38,81). Further evidence indicates ion channels and transporters play a significant role in cell migration (3,16,73,74). During the cell migratory process, there is polarized distribution of ion channels and transporters due to structural reorganization of the cytoskeleton, as we previously suggested for VSMCs (3). Based on these findings, it is plausible that, as VSMCs undergo phenotypic transformation, an interaction between KCC and the cytoskeleton could enhance membrane expression and increase its transport activity (3). As discussed above and in support of the role KCC might play in VSMC phenotypic transition, recent studies on C281 K-Cl COTRANSPORT IN VASCULAR SMOOTH MUSCLE CELLS the expression of KCC isoforms in embryogenesis and dedifferentiated cancerous cells have linked KCC to tumorigenesis, cell proliferation, and migration (16,(75)(76)(77)87). Similarly, VSMC synthetic phenotypic transition is also associated with cell proliferation and migration (71). Thus differential expression (22)(23)(24)(25)90) and activity of the KCC isoforms in VSMCs could be driving this phenotype transition. Alternatively, it is also possible that the transition to a medium and, even later, synthetic phenotype may affect KCC regulation.
Summary. Concomitant with the process of transition from contractile to synthetic VSMC phenotypes, as defined here based on the passage numbers, significant changes in kinetic parameters such as V m , K m , and Hill N for the cotransported Rb ϩ and Cl Ϫ ions were detected that may contribute to our understanding of the pathological progression during CVD. These findings point and add to the growing information base that KCC and its tissue-specific isoforms may exert their participation through abnormalities or changes in their kinetic parameters in the development of vascular lesions, leading to vascular occlusion causative for heart attacks and strokes.