Introduction

Since the first observations on increased monovalent ion exchange in vascular smooth muscle rings (VSMR) [25] and red blood cells [51] from spontaneously hypertensive rats, several research teams have reported that, at least partially, these abnormalities are caused by enhanced electroneutral Na+,K+,2Cl−cotransport (NKCC) (for review see [41, 47]). Two isoforms of this carrier have been cloned from vertebrate cDNA libraries. A ubiquitous NKCC1 isoform has been detected in all types of cells studied so far, whereas expression of the renal-specific NKCC2 isoform is limited to the apical membrane of epithelial cells from the thick ascending limb and macula densa [37, 53].

The hypothesis on NKCC1 involvement in the pathogenesis of hypertension is based on three major observations. First, in erythrocytes from F2 hybrids of spontaneously hypertensive and normotensive rats, NKCC activity correlated positively with blood pressure [10, 28]. Second, NKCC1−/− knockout mice exhibited decreased blood pressure as well as attenuated vascular tone and constriction of VSMR treated with the α-adrenoceptor agonist phenylephrine (PE) [16, 35]. Third, NKCC inhibition with bumetanide reduced contractions of rat VSMR and the guinea pig ureter triggered by K+o-induced depolarization, PE and electrical stimulation [4, 29]. Brown et al. were the first to suggest that NKCC contributes to the maintenance of vascular tone via adjustment of the transmembrane Cl− gradient [11]. Indeed, bumetanide decreased [Cl−]i, hyperpolarized vascular smooth muscle cells (VSMC) [15] and abolished differences in these parameters between normotensive and DOCA-salt-hypertensive rats [11].

In experiments on bone marrow transplantation from progenitors to X-ray-irradiated F1 hybrids of spontaneously hypertensive and normotensive rats, Bianchi et al. showed that enhanced NKCC detected in this experimental model of primary hypertension is genetically determined rather than a consequence of the hypertensive milieu [10]. On the other hand, other researchers detected augmented NKCC in VSMR from rodents with secondary hypertension [15, 24] and enhanced content of endogenous NKCC inhibitor in the plasma of salt-sensitive rats [5], suggesting that the carrier is subjected to cell-type-specific regulation by diverse stimuli. Indeed, no systematic involvement of Ca2+, cAMP and cGMP has been detected in studies of NKCC in human and rat erythrocytes [17], whereas in VSMR and cultured VSMC, this carrier was activated by Ca2+i-raising vasoconstrictors, including angiotensin II, and was inhibited by cAMP- and cGMP-mediated vasodilators [3, 4, 42, 49, 55, 57]. Opposing regulation by vasodilators was documented in a study of Na+-independent K+,Cl− cotransport (KCC) [2], i.e. another member of the Cl−-coupled cotransporter superfamily contributing to outwardly directed Cl− transport [1].

Similarly to “ying-yang” regulation by vasodilators, NKCC and KCC are also affected reciprocally by cell volume modulation. Indeed, shrinkage-induced activation of inwardly directed NKCC, Na+/H+ exchange, and Na+-coupled transport of organic osmolytes contributes to regulatory volume increase (RVI), whereas swelling-induced and outwardly directed KCC, K+ and anion channels provide regulatory volume decrease (RVD) [20, 31, 36]. It is important to underline that cell shrinkage activates NKCC1 in all types of nucleated cells studied so far [39]. Keeping this in mind, we employed hyperosmotic and isosmotic shrinkage to further explore the role of NKCC in VSMC contraction. The results of our study were reported in part at the 13th European Meeting on Hypertension [7].

Materials and methods

Preparation of VSMR

Endothelium-denuded VSMR were obtained from the thoracic aorta of 11- to 13-week-old Wistar rats euthanized under deep intraperitoneal anesthesia with sodium pentobarbital (Nembutal, 70 mg/kg) in accordance with institutional animal care guidelines. The isolated aorta was placed in physiologically balanced salt solution (PSS). Connective tissue and fat were taken out with scissors, whereas the endothelium was removed by careful rotation of a wooden manipulator inside the VSMR lumen just before the experiments. The 2- to 3-mm VSMR were either used immediately or stored at 4°C for up to 24 h. In preliminary experiments, we documented that 24 h storage did not affect VSMR contractile responses.

Isometric VSMR contraction

VSMR were mounted in 1-ml baths with stainless steel hooks inserted into the vascular ring orifice. One hook was fastened to a mechanical force transducer MX2B (Tomsk, Russia) with silk thread; the other served as an anchor. The tissues were bathed in PSS, buffered with TRIS at pH 7.4 (37°C) and bubbled with 95%O2/5% CO2 at a volumetric speed of ~1 ml/min. To control the contractile response, the VSMR were equilibrated for 1 h at tension of 0.5–1 g and exposed to K+o-induced depolarization caused by isosmotic substitution of 30 mM NaCl with KCl. The VSMR were then exposed to modified PSS containing either altered ions concentration or sucrose in the presence or absence of the test compounds. Isometric changes in VSMR tension were recorded with an XY recorder (Carl Zeiss, Jena, Germany).

Cultured VSMC

The precise measurement of cell volume and inward ion fluxes in VSMR are complicated by a relatively large extracellular space, the presence of fibroblasts, and VSMC heterogeneity [18]. On the other hand, cultured VSMC rapidly downregulate the expression of several specific genes that define their contractile phenotype in vivo. Keeping this in mind, we used a low-density seeding strategy for VSMC derived from the aortae of Wistar-Kyoto (WKY) rats to select a cell line possessing the highest expression of smooth muscle-specific α-actin, SM22 protein and myosin light chain kinase. This cell line, WKY-7, also possessed the highest sensitivity to angiotensin II and endothelin-1, measured by mitogen-activated protein kinase ERK1/2 phosphorylation [14]. In the present study, WKY-7 cells were grown for 48–72 h in DMEM supplemented with fetal and newborn calf serum (10% of each), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). To establish quiescence, VSMC were incubated before experiments during 48 h in the presence of 0.2% calf serum.

NKCC activity

NKCC activity was measured as a bumetanide-sensitive component of the 86Rb influx rate. WKY-7 cells seeded in 24-well plates were washed twice with 2-ml aliquots of PSS. The medium was then aspirated, and 0.25 ml of PSS with 1 μCi/ml 86Rb and 1 mM ouabain with or without bumetanide was added. After 5-min incubation at 37°C, isotope uptake was terminated by the addition of 2 ml of ice-cold medium W containing 100 mM MgCl2 and 10 mM HEPES-TRIS buffer (pH 7.4). Radioactivity of the incubation medium and cell lysate was measured with a liquid scintillation analyzer, and the rate of 86Rb influx (V, nmol per mg of protein per 5 min) was calculated as V=A/am, where A was the radioactivity of the samples (cpm), a was the specific radioactivity of K+ (86Rb) in the medium (cpm/nmol), and m was protein content measured with the modified Lowry method. For more details see [46].

Intracellular water volume in WKY-7 cells seeded in 12-well plates was measured as [14C]-urea available space according to a previously described protocol [46] and calculated as V=Ac/Amm, where Ac was the radioactivity of the cells after 30 min incubation with 2 μCi/ml [14C]-urea (dpm), Am was the radioactivity of the incubation medium (dmp/μl), and m was protein content in the cell lysate (mg).

Intracellular Cl− content in WKY-7 cells was measured as the steady-state distribution of 36Cl as described previously in detail [44]. To calculate [Cl−]i, the volume of intracellular water was quantified in parallel experiments with the protocol presented above. To measure Cl−i content in VSMR, the rings were subjected to 5 h preincubation in PSS containing 2 μCi/ml 36Cl; in some experiments, bumetanide and sucrose were added in the last 30 min of incubation. The VSMR were then rinsed in 3×50-ml aliquots of ice-cold PSS and solubilized in scintillation cocktail containing Triton X-100:toluene 1:2 (v/v), 4 g/l 2,5-diphenyl-1,3,4-oxadiazole (PPO) and 0.1 g/l 1,4-bis[5-phenyl-2-oxazolyl]-benzene; 2–2′-p-phenylene-bis[5-phenyloxazole] (POPOP). Intracellular Cl− content (nmol/mg) was calculated as [Cl−]i=A/am, where A was the radioactivity of the samples (cpm), a was the specific radioactivity of Cl− in the medium (cpm/nmol), and m was the VSMR wet weight (mg) measured before incubation.

Voltage-gated L-type Ca2+ channel activity was estimated as verapamil- or nifedipine-sensitive component of the 45Ca influx rate. WKY-7 cells seeded in 24-well plates were preincubated for 30 min with PSS. Then, this medium was aspirated, and 0.25 ml of PSS containing 0.1 mM CaCl2 with or without nifedipine and verapamil was added. Isotope uptake was initiated by the addition of 0.25 ml of the same PSS with 3 μCi/ml 45Ca, and terminated in 5 min as indicated above. To induce cell depolarization, KCl in isotope-containing medium was increased up to 60 mM by equimolar substitution of NaCl. For more details see [45].

Solutions and chemicals

PSS contained 120.4 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 5.5 mM glucose, and 15 mM TRIS. Ca2+-free PSS contained 0.5 mM EGTA and 3.6 mM MgCl2. To avoid the impact of Cl−/HCO3 exchange on intracellular Cl− handling, bicarbonate-free PSS was employed in all experiments, and pH was adjusted to 7.4 by the addition of 0.1 N HCl. PSS osmolality was increased by the addition of 50–300 mM sucrose as a cell-impermeable osmolyte. In Cl−-free PSS, KCl, NaCl and MgCl2 were substituted with Na-gluconate, K-gluconate and MgSO4, respectively, whereas CaCl2 was omitted. Previously, it was shown that the absence of Ca2+o does not affect volume-dependent regulation of ouabain-resistant 86Rb fluxes in VSMC [43]. Chemicals were obtained from Sigma (St. Louis, Mo., USA) with the exception of EGTA, POPOP, PPO (Serva, Heidelberg, Germany), verapamil (Orion, Helsinki, Finland) and cell culture medium (Gibco BRL, Gaithersburg, Mo., USA). Radiochemicals were from New England Nuclear (Boston, Mass.) and Amersham (Mississauga, Ont.). Stock solutions of bumetanide were prepared in DMSO, whereas nifedipine was dissolved in 70% ethanol. Neither DMSO nor ethanol at a final concentration of 0.1% (v/v), respectively, affected the parameters measured.

Statistics

The data, presented as mean±SE, were analyzed by Student’s t-test or the t-test for dependent samples, as appropriate. Significance was defined as P<0.05.

Results

Cell volume modulation in hypo- and isosmotic media

Ten minutes incubation of WKY-7 cells was sufficient to establish the steady-state distribution of [14C]-urea between the extra- and intracellular compartment (Fig. 1A, curve 1). In the presence of 150 mM sucrose, VSMC volume was decreased by ~20% in the first 5 min, and did not change significantly during the next 40 min of incubation (Fig. 1a, curve 2). Figure 1b shows the dependence of cell volume on sucrose added to isosmotic PSS at concentrations from 50 to 150 mM.

Fig. 1
figure 1

Cell volume modulation in hyperosmotically (a, b) and isosmotically (c) shrunken vascular smooth muscle rings (VSMC). a Kinetics of cell volume modulation in hyperosmotic medium. WKY-7 cells were incubated for time intervals indicated on the x-axis in 0.5 ml physiologically balanced salt solution (PSS) containing 2 μCi/ml [14C]-urea. At the time point indicated by the arrow, 0.5 ml of PSS (1) or PSS containing 300 mM sucrose (2) was added. In both cases, the media contained 2 μCi/ml [14C]-urea. b Dependence of cell volume on medium osmolality. WKY-7 cells were incubated for 30 min in PSS containing 2 μCi/ml [14C]-urea and sucrose at concentrations indicated on the x-axis. c Kinetics of cell volume modulation under isosmotic shrinkage. The cells were incubated for 45 min in hyposmotic PSS containing 40 mM NaCl and 2 μCi/ml [14C]-urea. At the time point indicated by the arrow, this medium was aspirated, and isosmotic PSS (120.4 mM NaCl) with 2 μCi/ml [14C]-urea was added. Mean±SE from experiments performed in triplicate are shown

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In additional experiments, we triggered the loss of intracellular osmolytes by preincubation of WKY-7 cells in hyposmotic medium, and then transferred them to isosmotic PSS. This approach is commonly taken to verify the role of isosmotic shrinkage in the regulation of cellular functions [40]. In contrast to hyperosmotic shrinkage, transfer from hypo- to isosmotic medium transiently decreased WKY-7 cell volume that was normalized in 25 min (Fig. 1c). This observation is consistent with previous studies revealed RVI in isosmotically-shrunken VSMC only [46, 48].

NKCC activity

In the presence of the Na+,K+-ATPase inhibitor ouabain, the addition of 10 μM bumetanide decreased the K+ (86Rb) uptake rate by threefold. Further elevation of this compound up to 100 μM did not significantly affect the rate of K+ influx (data not shown) that is consistent with ID50 values of ~1 μM reported in studies on the effect of bumetanide on NKCC1 activity in other cell types [21, 22, 53].

The elevation of medium osmolality by the addition of up to 150 mM of sucrose led to ca. threefold activation of NKCC, measured as a component of ouabain-resistant 86Rb influx inhibited by 10 μM bumetanide (Fig. 2a, curve 1). Suppression of NKCC detected at a sucrose concentration of 300 mM is probably caused by feedback inhibition of this carrier with Cl−i [37, 53]. We also noted that elevation of medium osmolality decreased the (ouabain+bumetanide)-resistant K+ influx rate from ~7 to 2 nmol mg protein−1 5 min−1 (Fig. 2a, curve 2). This effect was completely abolished in Cl−-free medium (Fig. 2a, curve 3). These results suggest that suppression of bumetanide-resistant K+ influx in hyperosmotic medium is caused by the inhibition of KCC reciprocally regulated by cell volume and detected in our previous studies [6, 43].

Fig. 2a, b
figure 2

Volume-dependent modulation of Na+,K+,Cl− cotransport (NKCC) in VSMC. a Dependence of NKCC activity (1) and the bumetanide-resistant component of 86Rb influx (2 and 3) on medium osmolality. Curves 1, 2: cells were incubated for 5 min in PSS containing 1 mM ouabain, 1 μCi/ml 86Rb±10 μM bumetanide. Curve 3: cells were incubated for 5 min in Cl−-free PSS containing 1 mM ouabain, 1 μCi/ml 86Rb and 10 μM bumetanide. The concentration of sucrose added to control (1 and 2) or Cl−-free (3) PSS is indicated on the x-axis. b Kinetics of NKCC modulation in hyperosmotically (2) and isosmotically (3) shrunken cells. Cells were incubated for 45 min in isosmotic (1, 2) or hyposmotic (40 mM NaCl) (3) PSS. These media were then aspirated, and 0.25 ml of isosmotic PSS (1 and 3) or PSS containing 150 mM sucrose (2) was added. At the indicated time points, isotope uptake was initiated by the addition of 0.25-ml aliquots of the same media containing 1 mM ouabain, 1 μCi/ml 86Rb±10 μM bumetanide and terminated in the next 5 min. Mean±SE from experiments performed in quadruplicate are shown

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The increment of NKCC detected after 5 min of 150 mM sucrose addition was preserved with up to 30 min of incubation in hyperosmotic medium (Fig. 2b, curve 2). In contrast, isosmotic shrinkage led to transient activation of this carrier that was completely abolished in 30 min of cell transfer from hypo- to isosmotic medium (Fig. 2b, curve 3).

Contractions of hyperosmotically and isosmotically shrunken VSMR: role of NKCC

Elevation of sucrose concentration from 50 to 200 mM led to dose-dependent VSMR contractions (Fig. 3, curve 2). The contractile response triggered by 150 mM sucrose (curve 3) remained stable for at least three subsequent additions made with 30-min washout intervals, and its amplitude with 20 min of sucrose addition was 51.8±9.0% (n=78) of the maximal contraction detected in the presence of 36 mM KCl (curve 1).

Fig. 3
figure 3

Isometric contractile force recording from vascular smooth muscle rings (VSMR) under hyperosmotic shrinkage. VSMR were subjected to K+o-induced depolarization (curve 1), subsequent elevation of sucrose concentration up to 300 mM (curve 2), and by the addition of 150 mM sucrose in the absence (curve 3) and presence (curve 4) of bumetanide. Bumetanide was added at a concentration of 10 μM 5 min before sucrose

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Figure 4 shows representative tracing of the contractile response of depolarized and PE-treated VSMR in control and hyperosmotic medium. In these experiments, we observed that contractions triggered by equimolar substitution of 30 mM NaCl with KCl and by 1 μM PE were suppressed after 20 min of sucrose addition by 89.6±8.8% and 82.8±10.8% (n=6, P<0.00001), compared to contractions, triggered by these stimuli in isosmotic PSS.

Fig. 4
figure 4

Effect of hyperosmotic shrinkage on VSMR contractions triggered by K+o-depolarization (a) and phenylephrine (PE) (b)

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To study the effect of isosmotic shrinkage on VSMR contraction, we applied a protocol developed with cultured cells (Fig. 1c). Application of the hyposmotic solution resulted in VSMR contractions that were completely abolished in 30–45 min (Fig. 5, curve 2). Subsequent transfer of VSMR to isosmotic medium led to transient contractions (curve 3) with a maximal amplitude of 21.6±8.7% (n=7) compared to K+o-induced contractions. Like hyperosmotic shrinkage, presumed isosmotic shrinkage of VSMR sharply attenuated contraction triggered by K+o depolarization and PE (Fig. 6).

Fig. 5
figure 5

Isometric contractile force recording from VSMR under isosmotic shrinkage. VSMR were subjected to K+o-induced depolarization (curve 1), reduced osmolality (PSS containing 40 mM NaCl, curve 2), and transfer from hypo- to isosmotic medium (PSS) in the absence (curve 3) or presence (curve 4) of 10 μM bumetanide . For more details see text

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Fig. 6
figure 6

Effect of isosmotic shrinkage on VSMR contractions triggered by K+o-depolarization (a) and PE (b). Broken lines show contractions in the absence of stimuli mentioned above

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Five minutes of pretreatment with 10 µM bumetanide led to 12% inhibition of sucrose-induced contractions (Fig. 3, curve 4). It should be underlined that in contrast to rapid inhibition of 86Rb influx detected with 10 μM bumetanide in WKY-7 cells (Fig. 2a), its action on sucrose-induced VSMR contractions increased with time (Fig. 7a) and reached 33% of inhibition after 30-min preincubation (n=4, P<0.005). In contrast to hyperosmotic shrinkage, both maximal amplitude and duration of contractions detected in isosmotically shrunken VSMR were potentiated rather than attenuated by bumetanide (Fig. 5, curve 4 versus 3, and Table 1).

Fig. 7
figure 7

Kinetics of inhibition of sucrose- (a), K+o- (b) and PE- (c) induced contractions by bumetanide. VSMR were preincubated with 10 (1), 50 (2) or 100 μM (3) bumetanide, and then contractions were triggered by the addition of 150 mM sucrose, 1 μM PE or equimolar substitution of NaCl with 30 mM KCl. The amplitude of contractions in the absence of bumetanide was taken as 100%. Mean±SE from 4–7 experiments are shown

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Table 1 Effect of bumetanide on vascular smooth muscle ring (VSMR) contraction triggered by isosmotic shrinkage. Isosmotic shrinkage was triggered as indicated in the legend to Fig. 5. Bumetanide was added in the last 5 min of incubation in hyposmotic medium. The maximal amplitude of contractions triggered by K+o-induced depolarization was taken as 100%. Mean±SE obtained in n experiments is shown
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Keeping in mind the distinct effect of bumetanide in hyperosmotically and isosmotically shrunken VSMR, we examined its action on contractions triggered by 1 μM PE and by depolarization in modest- (36 mM) and high- (66 mM) K+ medium. Similarly to hyperosmotic shrinkage, contractions triggered by [K+]o elevation up to 36 mM were slightly inhibited by 5 min incubation with 10 μM bumetanide, reaching 35±12% and 53±10% of inhibition after 30 min preincubation with 10 or 100 μM bumetanide, respectively (Fig. 7b). A more striking action of bumetanide was detected in the study of PE-induced contractions. In this case, 30 min preincubation with 10 or 100 μM bumetanide suppressed contractions by 42% (n=4, P<0.02) and 89% (n=9, P>0.0000001) (Fig. 7c). In contrast to PE (Fig. 8a) and modest elevation of [K+]o (Fig. 8B), contractions in high-K+ medium were completely insensitive to bumetanide (Fig. 8c), and after 30 min preincubation with this compound at 100 μM concentration the maximal amplitude was 96±6% of control values (n=6).

Fig. 8
figure 8

Effect of bumetanide on contractile force generation by VSMR induced by PE (a) or by depolarization in moderate- (b) or high-K+ (c) medium

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Role of intracellular Cl−

Bumetanide did not affect cell volume in hyperosmotically shrunken VSMC, but suppressed RVI detected in VSMC subjected to isosmotic shrinkage (Table 2). Comparison of the sucrose effect on cell volume and Cl−i content shows that [Cl−]i elevation by ~40% was mainly caused by attenuation of cell volume rather than by NKCC activation. Indeed, NKCC inhibition with bumetanide decreased the sucrose-induced increment of [Cl−]i by only10–15%. After 15 min of isosmotic shrinkage, [Cl−]i was increased in the absence and presence of bumetanide by 75% and 30%, respectively, which was also consistent with the magnitude of volume modulation observed in control and bumetanide-treated, isosmotically shrunken VSMC (Table 2).

Table 2 Effect of hyper- and isosmotic shrinkage on intracellular water content and Cl−i concentration in WKY-7 cells. To establish the steady-state distribution of isotopes, cells were preincubated with [14C]-urea and 36Cl for 2 h. Hyperosmotic shrinkage was then induced by the addition of 150 mM sucrose, and isosmotic shrinkage was triggered as indicated in the legend to Fig. 1. Bumetanide was added 10 min before cell shrinkage. Mean±SE obtained in experiments performed in quadruplicate is shown
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Long-term maintenance in culture affects diverse cellular functions, including the activity of ion transporters [9, 52]. Thus, in VSMC subjected to more than ten passages and in the absence of any stimuli, such as cell shrinkage, NKCC operates as a (Na+,Cl−)-dependent K+/K+ exchanger [44]. These data are consistent with the modest effect of bumetanide on [Cl−]i in cultured WKY-7 cell line (Table 2) and contrast with the 30–40% decrease of [Cl−]i in freshly isolated rat femoral arteries [12]. Keeping this in mind, we studied the effect of bumetanide on [Cl−]i in control and shrunken aortic rings. Table 3 shows that 30 min preincubation with 100 μM bumetanide decreased Cl−i content by ~40%, and completely abolished the rise of this parameter caused by 20 min hyperosmotic shrinkage in the presence of 150 mM sucrose.

Table 3 Effect of bumetanide and sucrose on intracellular Cl− content in VSMR. Bumetanide was applied 5 or 30 min before sucrose addition. Mean±SE obtained in experiments performed in quadruplicate is shown. For more details of methodology see Materials and methods
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Role of L-type Ca2+ channels and extracellular Ca2+

It is generally accepted that electrical excitation leads to VSMC contraction caused by Ca2+ influx via voltage-gated long-lasting (L-type) Ca2+ channels (for references see [30]). K+o-induced depolarization of WKY-7 cells caused two- to threefold elevation of the 45Ca influx rate. Neither 1 μM nifedipine nor 30 μM verapamil, potent inhibitors of L-type Ca2+ channels, significantly affected baseline Ca2+ uptake, but completely abolished the depolarization-induced increment of the Ca2+ influx with a ID50 of ~0.01 and 1 μM, respectively (data not shown). These values are consistent with data obtained for L-type Ca2+ channels by the patch-clamp technique [34]. Neither hyperosmotic shrinkage nor hyposmotic swelling affected nifedipine-resistant Ca2+ uptake in control and depolarized VSMC (Fig. 9). Hyposmotic swelling sharply increased nifedipine-sensitive Ca2+ influx under baseline conditions, whereas hyperosmotic shrinkage inhibited by ~40% nifedipine-sensitive Ca2+ influx triggered by K+o-induced depolarization (Fig. 9).

Fig. 9
figure 9

Effect of hyperosmotic shrinkage and hyposmotic swelling on baseline (a) and depolarization-induced (b) 45Ca uptake by WKY-7 cells. Cells were incubated for 5 min with 45Ca (2–3 μCi/ml) in control (~6 mM KCl, 120 mM NaCl) or high-K+ PSS (~43 mM KCl, 83 mM NaCl) containing 0.1 mM CaCl2 with or without 1 μM nifedipine. To induced shrinkage, 150 mM sucrose was added. To induce swelling, NaCl concentration was decreased by 60 mM. Mean±SE from three experiments performed in triplicate are shown

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Keeping in mind the K+o-dependent effect of bumetanide on VSMR contractions (Fig. 8), we studied 45Ca uptake at three different K+o concentrations. Thirty minutes of preincubation with 10 μM bumetanide did not alter 45Ca uptake at [K+]o=6 and 60 mM, whereas at modest depolarization ([K+]o=30 mM) the nifedipine-sensitive component of 45Ca uptake was decreased in bumetanide-treated VSMC by twofold (Table 4).

Table 4 Effect of bumetanide on 45Ca uptake by WKY-7 cells. Cells were preincubated for 30 min in 0.25 ml physiologically balanced salt solution (PSS) containing 0.2 mM CaCl2 with or without 20 μM bumetanide. Aliquots of Ca2+-free PSS containing 3–4 μCi/ml 45Ca, with or without 2 μM nifedipine and with NaCl and KCl at final concentrations indicated in the left column, were then added. Mean±SE from experiments performed in quadruplicate is shown
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Consistent with previous data [19], both verapamil and nifedipine almost completely inhibited depolarization-induced VSMR contractions and partially suppressed the contractions triggered by PE (Table 5). We also observed sharp inhibition by L-type channel blockers of VSMR contractions in hypotonic medium. In contrast, neither verapamil nor nifedipine affected hyperosmotically and isosmotically shrunken VSMR contractions (Table 5).

Table 5 Effect of L-type channel blockers on the amplitude of VSMR contraction. VSMR were treated with Ca2+ channel blockers 15 min before the application of contractile stimuli. The maximal amplitude of K+o-induced contractions was taken as 100%. ND These parameters were not determined. Mean±SE fromn experiments is shown
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To further examine the role of Ca2+, we subjected VSMR to 1 h preincubation in Ca2+-free solution containing the Ca2+ chelator EGTA. In contrast to almost complete inhibition of K+o- and PE-induced contractions by Ca2+ depletion documented in previous studies [19] and confirmed in our experiments, this procedure attenuated the amplitude of contractions of hyperosmotically and isosmotically shrunken VSMR by ~50 and 35%, respectively (Table 6).

Table 6 Effect of extracellular Ca2+ on the maximal amplitude of VSMR contractions. The maximal amplitude of K+o-induced contractions was taken as 100%. Mean±SE fromn experiments is shown
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To determine whether or not the lack of complete suppression of shrinkage-induced contractions in Ca2+-free medium is caused by increased sensitivity of the contractile machinery to Ca2+, we compared the effect of extracellular Ca2+ on depolarization-induced contractions of control and shrunken VSMR. The addition of 1 mM CaCl2 led to the development of full-scale contraction of VSMR subjected to depolarization in Ca2+-free medium (Fig. 10). In contrast, [Ca2+]o elevation up to 10 mM did not trigger contractions of hyperosmotically shrunken VSMR subjected to subsequent depolarization (Fig. 10). Negative results were also obtained in isosmotically shrunken VSMR (Fig. 11).

Fig. 10
figure 10

Effect of extracellular Ca2+ on depolarization-induced contractions of control and hyperosmotically shrunken VSMR

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Fig. 11
figure 11

Effect of extracellular Ca2+ on depolarization-induced contractions of control and isosmotically shrunken VSMR

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Discussion

The data presented here drew us to a number of conclusions: (1) both cell shrinkage and swelling lead to VSMR contractions; (2) contractions triggered by shrinkage are not mediated by activation of L-type Ca2+ channels and are at least partially Ca2+o-independent. In contrast, contractions of swollen VSMR are accompanied by elevated Ca2+ uptake and abolished by L-type Ca2+ channel inhibitors; (3) both hyperosmotic and isosmotic shrinkage inhibits contractions triggered by K+o-induced depolarization and PE; (4) NKCC inhibition with bumetanide decreases [Cl−]i and contractions triggered by modest depolarization as well as contractions triggered by PE and hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR; (5) suppression by bumetanide of contractions evoked by modest [K+]o elevation is caused by partial inactivation of L-type Ca2+ channels, probably because of Cl−i-mediated membrane hyperpolarization; and (6) in isosmotically shrunken cells, NKCC inhibition enhances contractions via RVI suppression. Data supporting these conclusions are considered below.

Cell volume modulation is sufficient to evoke VSMR contractions

Several lines of evidence indicate that VSMR contractions detected in anisosmotic media are caused by cell volume modulation rather than by altered medium osmolality per se. First, the magnitude of VSMR contractions triggered by sucrose at concentrations from 50 to 150 mM (Fig. 3) was in proportion to cell volume reduction measured as the volume of [14C]-available space (Fig. 1b). Second, the volume of hyposmotically swollen VSMC was rapidly normalized by RVD occurring via activation of K+ and Cl− channels [6]. These data are consistent with our results showing slight cell volume elevation after 45 min incubation of VSMC in hyposmotic medium (3.03±0.16 versus 2.85±0.18 µl/mg protein in control; n=3) and transient VSMR contractions (Fig. 5, curve 2). Third, sustained versus transient contractions detected in sucrose-treated (Fig. 3) and isosmotically shrunken VSMR (Fig. 5) are consistent with the dynamics of cell volume modulation observed under these conditions (Fig. 1a, c). Fourth, the transient kinetics of volume decrease in isosmotically shrunken VSMC are caused by NKCC-mediated RVI [46]. Both cell volume decrease (Table 2) and force generation in isosmotically shrunken VSMR were prolonged under NKCC inhibition with bumetanide (Table 2 and Fig. 5).

Contractions of shrunken and swollen VSMR: distinct impact of Ca2+-mediated signaling

Despite the monotonous impact on force generation, the cellular mechanisms underlying the contractile responses of shrunken and swollen VSMR are essentially different. Indeed, similarly to K+o-induced depolarization, hyposmotic swelling of VSMC augmented 45Ca influx inhibited by nifedipine (Fig. 9). Moreover, both nifedipine and verapamil abolished contractions of swollen VSMR (Table 5). These results strongly suggest that swelling-induced VSMR contractions were mediated by L-type Ca2+ channel activation (Fig. 12). In contrast, contractions triggered by hyper- and isosmotic shrinkage were resistant to L-type Ca2+ channels blockers (Table 5). Contractions of shrunken VSMR were also preserved in the absence of Ca2+o and depletion of intracellular Ca2+ stores by incubation in Ca2+-free, EGTA-containing medium, i.e. conditions abolishing K+o- and PE-induced contractions (Table 6).

Fig. 12
figure 12

Cross-talk of cell volume, Na+,K+,Cl−-cotransport, intracellular Cl−i and Ca2+ in the regulation of vascular smooth muscle contraction. 1 NKCC; 2 L-type Ca2+ channels; 3 calcium release-activated Ca2+ channels; 4 Ca2+-activated anion channels; Em electrical membrane potential; MLCK myosin light chain kinase; RVI regulatory volume increase; bum, ver, nif bumetanide, verapamil and nifedipine, respectively. +, − activatory and inhibitory stimuli; ? unknown intermediates of signal transduction. For more details see text

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In additional experiments, we observed that hyperosmotic shrinkage sharply attenuates rather than enhances contractions triggered by K+o-depolarization and PE (Fig. 4). This finding is consistent with data obtained on VSMR shrunken in the presence of 100 mM urea [58]. Inhibition of K+o- and PE-induced contractions was also seen in isosmotically shrunken VSMR (Fig. 6). Importantly, 30 min preincubation in hyposmotic medium preceding isosmotic shrinkage increased the magnitude of K+o-induced contractions (148.2±28.7 versus 100% in control; n=9, P<0.05) and did not influence contractions triggered by PE (100±14.4%, n=6). Viewed collectively, these results demonstrate the distinct mechanism of contractions triggered by cell shrinkage and physiological stimuli.

Several attractive hypotheses have been generated to explain the Ca2+-independent mechanism of excitation-contraction coupling detected in shrunken VSMR. Thus, Kravtsov and co-workers reported that K+o-induced contractions in Mg2+-free medium are not mediated by Ca2+-influx and occur in the presence of EDTA [30]. It should be underlined, however, that in contrast to shrunken VSMR, K+o-induced contractions in Mg2+-free medium were suppressed by L-type channel blockers [30]. It was shown that Ca2+o-independent VSMC contractions triggered by activators of protein kinase C, tyrosine and Rho-associated kinases were mediated by calcium sensitization of the contractile machinery [8, 23, 38]. Both myosin light chain phosphorylation and Rho kinase-dependent myosin II translocation have been detected in hyperosmotically shrunken cells [26, 27, 50, 54, 56]. However, in contrast to K+o- and PE-induced contractions, contractions of Ca2+-depleted, shrunken VSMR were not evoked by the addition of Ca2+ (Figs. 10, 11). Keeping in mind that the total content of macromolecules in shrunken cells is increased and that modest elevation of this parameter affects diverse intracellular proteins, including cytoskeleton organization [13, 33], the role of macromolecular crowding in contractions of shrunken VSMR deserves further investigation.

Role of NKCC and intracellular Cl−

Unlike the dominant contribution of K+ permeability to resting membrane potential in skeletal and cardiac muscles, the PK/PCl ratio in VSMC varies from 0.8 to 0.5 [12]. These data led Brown et al. to suggest that NKCC contributes to vascular tone via the maintenance of [Cl− ]i [11]. Later on, it was shown that NKCC inhibition with bumetanide decreases [Cl−]i, and hyperpolarizes the sarcolemma in freshly isolated VSMR [15]. These data suggest that NKCC inhibition will decrease VSMC contractions triggered by modest depolarization, but will not affect contractions in sharply depolarized cells (Fig. 12). Indeed, 30 min pretreatment with bumetanide reduced the increment of nifedipine-sensitive Ca2+ uptake and VSMR contractions evoked by 30 mM KCl but did not affect these parameters at [K+]o=60 mM (Table 4, Fig. 8). It may be assumed that the lack of effect of bumetanide in high-K+ medium is caused by partial inactivation of NKCC due to reduction of [Na+]o from 120 to 66 mM. However, the high affinity of VSMC NKCC for Na+o, documented in our previous study (K0.5~25 mM) [44] did not support this assumption.

We observed that at 100 μM concentration bumetanide almost completely blocked the contractions triggered by PE (Fig. 8). Akar et al. reported that at 10 μM and 20 min preincubation, bumetanide inhibited VSMR contractions triggered by 1 μM PE by only 5–10% [4]. Several explanations for this controversy might be proposed. First, complete and rapid inhibition of NKCC, detected in cultured VSMC in the presence of 10 μM bumetanide (Fig. 2), does not mean that this concentration will lead to rapid hyperpolarization. Indeed, the effect of bumetanide on K+o-induced contractions was increased with concentration and time (Fig. 7b). We also detected that 30 min of incubation with 10 or 100 μM bumetanide decreased [Cl−]i by 18% and 38%, respectively (Table 3). Second, the above-mentioned discrepancy is caused by differences between rat strains. Indeed, the PE EC50 of 10−6.9 M, detected in VSMR from rats in our study (data not shown), was an order of magnitude higher than values reported by Akar et al. [4]. Third, at high concentrations, bumetanide affects VSMR contractions independently of NKCC inhibition. This assumption makes sense in analyzing the mechanisms of almost complete inhibition of PE-induced contractions after 30 min preincubation with 100 μM bumetanide (Fig. 8a). However, keeping in mind the lack of effect of bumetanide on contractions triggered by high [K+]o (Fig. 8c), upstream PE-specific intermediates of signaling, such as α-adrenoceptors, phospholipase C, calcium release-activated Ca2+ channels, rather than downstream elements of the contractile machinery, should be examined as potential targets for high-ceiling diuretics. Fourth, at high concentrations loop diuretics partially suppress KCC [32]. Recent data suggest that KCC activation contributes to vasodilatory action of nitric oxide derivatives [1]. The role of this carrier in modulation of VSMR contraction caused by 100 μM bumetanide should be examined further.

Rapid elevation of [Cl−]i in sucrose-treated VSMC cells is a direct consequence of cell shrinkage. In contrast, NKCC activation is a major determinant of [Cl−]i elevation in isosmotically shrunken cells subjected to preliminary Cl− depletion in hyposmotic medium. A key role of NKCC in [Cl−]i adjustment is consistent with a rapid decline of this parameter in isosmotically shrunken VSMC treated with bumetanide (Table 2). Thus, it can be suggested that shrinkage-induced contraction is caused by [Cl−]i elevation and membrane depolarization. It should be underlined, however, that the lack of involvement of L-type Ca2+ channels in contractions of isosmotically and hyperosmotically shrunken VSMR (Table 6) contradicts this assumption. Moreover, we observed that inhibition of NKCC providing inwardly directed Cl− transport potentiates rather than inhibits the contraction of isosmotically shrunken VSMR (Fig. 5). Prolongation of isosmotically shrunken VSMR contractions by bumetanide is probably caused by suppression of NKCC-mediated RVI (Figs. 1c, 2c, Table 2). Two hypotheses can be offered to explain the partial inhibition with bumetanide of nifedipine-insensitive contraction of hyperosmotically shrunken VSMR (Fig. 7A). First, hyperpolarization developing under NKCC inhibition suppressed the activity of inwardly directed Ca2+ transporters distinct from L-type channels. It should be mentioned, however, that contractions of shrunken VSMR were only partially suppressed in Ca2+-free medium (Table 6). Second, [Cl−]i modulation affects intracellular proteins involved in assembly of the contractile machinery independently of the regulation of membrane potential.

In conclusion, our results show that both swelling and shrinkage lead to VSMR contractions. Swelling-induced contractions are caused by activation of L-type Ca2+ channels. In contrast, contractions of shrunken VSMR are insensitive to the presence of L-type channel inhibitors and occur in the absence of Ca2+o. Augmented contractions detected in isosmotically shrunken VSMR in the presence of bumetanide are probably caused by suppression of NKCC-mediated RVI. The mechanism of bumetanide-sensitive, Ca2+-independent contractions in hyperosmotically shrunken VSMR deserves further investigations.