INTRODUCTION

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LOWERED OXYGEN TENSION (hypoxia) has generally been found to rapidly decrease contractile force of vascular smooth muscle, a reaction likely to be important for the local regulation of blood flow in response to tissue metabolic demands (see Ref. 30 for a review). Several processes involved in excitation and contraction may be affected by hypoxia, e.g., Ca²⁺ homeostasis, ion channel properties, and the cross-bridge interaction. However, the long-term effects of lowered PO₂ on contractility and metabolic patterns are less well known, despite the clinical importance of vascular alterations associated with tissue hypoxia and ischemia. In particular, chronic hypoxia has been implicated in the development of atherosclerosis as a result of intimal thickening or occlusion of vascular supply to the arterial wall (3, 20).

Experimentally induced chronic hypoxia in vivo has been shown to attenuate vasoreactivity to various contractile agents (1, 5, 12), to decrease production of D-myo-inositol 1,4,5-trisphosphate (32), to decrease Ca²⁺ sensitivity of arterial myofilaments (35), and to decrease collagen synthesis (10). In cultured arterial tissue, _1B-adrenoreceptor mRNA was increased in response to long-term hypoxia (8). Cells exposed to prolonged hypoxia adapt by inhibition of ATP-consuming processes, e.g., protein synthesis and Na⁺/K⁺ pumping (11, 25). However, limited information is available regarding metabolically dependent processes that may be inhibited by chronic hypoxia in intact smooth muscle.

To investigate chronic effects of hypoxia on vascular smooth muscle, we utilized a tissue culture model previously established to preserve phenotypic differentiation and contractility of vascular preparations over several days (16). Oxygen consumption (JO₂) and lactate production (J_lac) were determined under basal and stimulated conditions in cultured as well as fresh preparations, allowing determination of basal metabolic rates and energetic tension cost. Responses to hypoxia and metabolic inhibitors, as well as the occurrence and frequency of intracellular Ca²⁺ waves, were studied to investigate the functional consequences of metabolic adaptation to culture under normoxia or hypoxia.

	MATERIALS AND METHODS

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Organ culture. Female Sprague-Dawley rats weighing ~200 g were euthanized by cervical dislocation. The experimental procedures were approved by the Animal Ethics Committee of Lund University. A 5-cm segment of the tail artery, beginning ~1 cm distal to the radix, was dissected free under sterile conditions and transferred to a petri dish containing culture medium composed of Dulbecco's modified Eagle's medium and Ham's F-12 (1:1; Biochrom; Berlin, Germany) with the addition of antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin). The vessel segment had a diameter of ~0.5 mm along its entire length and was cut into 0.5-mm-thick rings under a dissection microscope. The rings were transferred to culture dishes containing medium as described above, with supplements as indicated for the respective experiments. The dishes were placed in a water-jacketed cell incubator at 37°C for 4 days. Rings cultured under hypoxic conditions were incubated under 95% N₂-5% CO₂. The PO₂ in the medium after 4 days was 14 mmHg, as determined with an oxygen electrode. To estimate the PO₂ in the tissue, we first calculated the drop in PO₂ from the liquid surface to the arterial preparation by solving the diffusion equation for the flux of oxygen into the tissue, giving a PO₂ at the tissue surface of ~7 mmHg. The PO₂ distribution across the arterial wall was then calculated as previously described (33) for an inside radius of 150 µm and an outer radius of 250 µm. Assuming JO₂ in the tissue was 6 × 10⁵ ml O₂ ml¹ · s¹ and Krogh's constant of 3.17 × 10¹⁰ ml O₂ cm¹ · mmHg¹ · s¹, the PO₂ in the middle of the arterial wall was found to be ~3 mmHg.

Force registration. Arterial rings were relaxed for 20 min in a nominally Ca²⁺-free Krebs solution. The rings were then mounted on parallel stainless steel wires (diameter 0.25 mm), one of which was connected to a force transducer (model AE 801, SensoNor; Horten, Norway) and the other to an adjustable support. Preparations were immersed in a modified Krebs solution in exchangeable Plexiglas cups (0.4 ml) fitted into a thermostated metal block (37°C) under normoxic conditions. The solution had the following composition (in mM): 135.5 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 11.6 HEPES, and 11.5 glucose. The rings were allowed to equilibrate for 20 min, stretched to optimal length as described previously (16), and activated twice with high-K⁺ solution, prepared by isomolar exchange of NaCl for KCl. Concentration-response curves to norepinephrine (NE; 1 nM-30 µM) were obtained in a cumulative manner.

Measurements of metabolic rates. JO₂ was recorded using a system described by Lövgren and Hellstrand (17). Modified Krebs solution containing antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) was introduced into a glass chamber (0.25 ml vol) from a reservoir where it was equilibrated with air. The decrease in PO₂ with time was recorded using a polarographic O₂ electrode (Eschweiler; Kiel, Germany). Background JO₂ was recorded after each experiment. In cases where a background was detected, it was subtracted from the measured JO₂. All measurements of JO₂ were carried out under normoxic conditions.

Confocal microscopy. For detection of intracellular Ca²⁺ concentration ([Ca²⁺]_i) waves, rings were mounted inside-out on thin glass capillaries (diameter 250 µm) and incubated with the [Ca²⁺]_i indicator Fluo-4-acetoxymethyl ester (10 µM) and pluronic F-127 (0.05%, both from Molecular Probes) at room temperature for 80 min. Exciting light was at 488 nm and emitted light was detected at >505 nm using a laser scanning confocal microscope (model 510LSM, Zeiss; Jena, Germany). Preparations were washed in modified Krebs solution (22°C) for at least 15 min before NE (0.1 µM) was added. Images were obtained every 0.37 s for 30 s, and wave activity was assessed in all distinguishable cells with the use of Zeiss 510LSM software.

Protein synthesis. Protein synthesis in freshly dissected or cultured arterial rings was determined by measuring L-[4,5-³H]leucine (Amersham Pharmacia Biotech; Little Chalfont, UK) incorporation. Segments of rat tail artery were cultured as described above. After 4 days, the segments were transferred to normoxic medium and allowed to equilibrate for 1 h. The preparations were incubated with 1 µCi/ml of L-[4,5-³H]leucine for 1 h. The reaction was stopped by placement of the culture dishes on ice. The tissue was frozen in liquid nitrogen, transferred to test tubes containing 5 mM NaOH, and homogenized by sonication. An aliquot of the homogenate was precipitated with 5% trichloroacetic acid and centrifuged at 13,200 g for 2 min at 4°C. The pellet was washed once with trichloroacetic acid and dissolved in Soluene (Packard Instrument, UK). A liquid scintillation cocktail (Opti-Phase HiSafe2; Wallac Scintillation Products) was added and the radioactivity was measured using a scintillation counter (model LS6500, Beckman Coulter; Fullerton, CA). Total protein content was determined using a Bio-Rad protein assay.

Statistics. Summarized data are expressed as means ± SE. Student's t-test was used to evaluate statistical significance. For multiple comparisons, one-way ANOVA was used. P < 0.05 was considered statistically significant.

	RESULTS

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Effects of hypoxic culture on force development of arterial rings. Culture of tail arterial rings in serum-free medium does not affect maximal force in response to NE, as demonstrated earlier (15). Rings cultured under continuous hypoxia (PO₂ = 14 mmHg) showed unimpaired maximal force response in oxygenated medium compared with rings cultured under normoxia (Fig. 1A), although the EC₅₀ for NE was increased (179 ± 27 vs. 73 ± 15 nM, P < 0.05, n = 3). In contrast to NE-induced responses, force development in high-K⁺ solution is decreased after culture, irrespective of conditions used (Fig. 1B). Tension transients in response to caffeine (20 mM) in Ca²⁺-free solution were larger after hypoxic than after normoxic culture (Fig. 1C).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effects of culture under normoxic and hypoxic conditions on tension development of tail arterial segments. A: shows stimulation with increasing concentrations of norepinephrine (NE). B: tension responses to stimulation with high-K+ solution in Ca2+ (2.5 mM)-containing medium. C: tension transients in response to caffeine (20 mM) in Ca2+-free medium. Values are means ± SE, n = 3-4, * P < 0.05, ** P < 0.01.

Metabolism of freshly dissected and cultured arterial rings. J_O2, measured under normoxic conditions, was greater in Ca²⁺-containing solution than in Ca²⁺-free normal solution in both fresh and cultured arterial rings, and higher still during depolarization and contraction in high-K⁺ solution (Fig. 2A). Under both basal and stimulated conditions, J_O2 values were lower in cultured than in fresh rings, and particularly the basal J_O2 was depressed after culture under hypoxia. However, the slope of J_O2 versus tension developed in response to high-K⁺ solution is unaltered by culture (freshly dissected 0.032 ± 0.003, culture normoxia 0.034 ± 0.004, and culture hypoxia 0.033 ± 0.003, n.s., n = 5 in all groups, Fig. 3A). Maximal mitochondrial capacity was determined using stimulation with the uncoupler DNP. JO₂ in the presence of DNP (30 µM) was similar after normoxic culture as in fresh tissue, but reduced after hypoxic culture (Fig. 3B).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Oxygen consumption (JO2) (A) and lactate production (Jlac) (B) in freshly dissected arterial rings and in rings cultured under normoxia or hypoxia. Effects of different extracellular Ca2+ concentrations and of depolarization in high-K+ solution are shown. Values are means ± SE, n = 5-6, * P < 0.05, ** P < 0.01, *** P < 0.001.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   A: JO2 vs. tension (energetic tension cost) in freshly dissected arterial rings and in rings cultured in normoxia or hypoxia under resting conditions and during stimulation with high-K+ solution. B: maximal JO2 after collapsing of the mitochondrial proton gradient with dinitrophenol (30 µM) in fresh and cultured tail artery rings. Values are means ± SE, n = 4-7. * P < 0.05.

Effects of acute hypoxia on Jlac. J_lac was determined under basal conditions and during stimulation with NE (Fig. 4). As in the experiments shown in Fig. 2, basal J_lac values were considerably greater in cultured than in fresh rings. In freshly dissected rings and in rings cultured under normoxia, J_lac increased only marginally during stimulation with NE (1 µM), but in rings cultured under hypoxia the increase was greater and statistically significant. When rings were stimulated with NE in hypoxic solution, J_lac increased in all groups of preparations, showing the presence of a Pasteur effect, i.e., increased glycolysis compensating for the lack of oxidative metabolism (Fig. 4).

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Lactate production in freshly dissected arterial rings and in rings cultured in normoxia or hypoxia. Responses to stimulation with NE in acute normoxia (O2) or hypoxia (N2) are shown. Values are means ± SE, n = 4-7, * P < 0.05, ** P < 0.01, *** P < 0.001.

Contractility during metabolic inhibition and glucose deprivation. Contractile properties of fresh and cultured preparations were determined under hypoxia and in the presence of several metabolic inhibitors. Hypoxia only marginally affected NE-induced tension development in the cultured rings, while in fresh preparations tension was reduced by 40% (Fig. 5). Mitochondrial inhibition by rotenone (10 µM) had a somewhat greater effect, and DNP (30 µM) was still more effective. Both inhibitors had significantly smaller effects on cultured than on fresh rings.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effects of inhibition of cellular metabolism by hypoxia (PO2 = 14 mmHg), rotenone (10 µM), and dinitrophenol (30 µM) on tension development in response to NE (1 µM) in freshly dissected arterial rings and in rings cultured in normoxia or hypoxia. Values are means ± SE, n = 4-7, * P < 0.05; ** P < 0.01.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   A: upregulation of glycogen stores after culture in hypoxia (n = 7 for all groups). B: effects of omission of glucose in bathing solution on tension development in response to NE (1 µM) is shown. The tension development is normalized to a contraction in the presence of glucose. The abscissa shows the number of contractions in glucose-free medium. Values are means ± SE, n = 4, * P < 0.05; ** P < 0.01; *** P < 0.001.

Intracellular Ca²+ waves. We tested whether temporal Ca²⁺ coding in the form of waves was affected by culture, and whether this would correlate with the altered responsiveness to metabolic inhibition. With the use of confocal microscopy, intracellular Ca²⁺ waves were detected in cells within intact rings loaded with Fluo-4. While wave activity was low under resting conditions, it increased during stimulation by NE. To avoid confluence of individual waves and thus allow calculation of wave frequency, an intermediate concentration of NE (0.1 µM) was used, representing close to EC₅₀ for cultured preparations (see above) but clearly below EC₅₀ for freshly prepared rings (0.6 µM), in which the sensitivity to exogenous NE is influenced by prejunctional uptake (16, 19). Despite the relatively greater level of stimulation by NE compared with fresh tissue, the number of cells with wave activity in cultured preparations and also the frequency of waves in individual cells, was lower by ~50% (Fig. 7). Culture under hypoxia further decreased both parameters of wave generation.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Intracellular Ca2+ concentration waves detected in single cells in the intact arterial wall. Rings were loaded with Fluo-4-acetoxymethyl ester, and wave activity after stimulation with NE (0.1 µM) was detected in all discernible cells by confocal microscopy. A: percentage of cells exhibiting wave activity; B: frequency of waves in these cells. Values are means ± SE, n = 4, ** P < 0.01, *** P < 0.001.

Protein synthesis in cultured arterial rings. Protein synthesis in arterial rings cultured for 4 days was studied as an incorporation of L-[4,5-³H]leucine in normoxic medium. Rings cultured under hypoxia showed a lower incorporation of leucine compared with rings cultured under normoxic conditions, which in turn showed lower incorporation than freshly dissected rings (Fig. 8).

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Incorporation of L-[4,5-3H]leucine under normoxic conditions in freshly dissected arterial rings and in rings cultured under normoxia and hypoxia, respectively. Rings were transferred to normoxic conditions 1 h before incubation with the isotope. Values are means ± SE, n = 6-10, * P < 0.05, ** P < 0.01.

	DISCUSSION

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Exposure of vascular smooth muscle tissue to maintained hypoxia for several days was found to cause reduced basal J_ATP and rate of protein synthesis. Under our hypoxic conditions (14 mmHg), and assuming a PO₂ of 7 mmHg at the surface of the vessel, we calculate that all cells are near or above the mitochondrial limiting PO₂ of 1-2 mmHg. Although this neither supports nor excludes a potential limitation on mitochondrial O₂ supply, other mechanisms may be involved. Whereas basal J_ATP had decreased by 40% when measured in oxygenated medium following hypoxic culture, it was the same as in fresh tissue after normoxic culture. However, in common with hypoxic culture, the regulation of glycolysis was altered, with an increased rate of glycolytic relative to oxidative metabolism.

Expression of glucose transporters and glycolytic enzymes is under the control of hypoxia-inducible factor 1, which regulates a multitude of genes by a molecular- sensing mechanism that is rapidly becoming understood (reviewed in Ref. 26). It thus appears that conditions during normoxic culture are sufficient to activate this program to some extent, although responses to hypoxic culture are more pronounced. The diminished relaxation response to hypoxia seen after both normoxic and hypoxic culture may reflect the shift from oxidative to glycolytic metabolism. Thorne et al. (31) recently reported a diminished relaxation response to hypoxia after 24-h normoxic culture of porcine coronary artery.

Total protein contents were better preserved during hypoxic than during normoxic culture. It is notable that environmental stress, such as hypoxia, generally causes decreased protein turnover, especially evident as a slowing of protein degradation (9). However, both during hypoxic and normoxic culture conditions protein was lost, indicating that degradation is faster than synthesis, although contractility is preserved over the presently used time span. Because no growth promoter (i.e., fetal calf serum) was added, protein synthesis was expected to be slow. Furthermore, we applied no mechanical tension, a factor that has been shown to increase the rate of protein synthesis and to promote growth during culture of vascular tissue (2, 34). Electron microscopy of tail arterial rings cultured under the normoxic conditions used here shows essentially normal morphology (16), and thus the loss of protein is not associated with a change in cellularity of the tissue, as also suggested by the maintained basal J_ATP shown here. The energetic tension cost, evaluated as JO₂ relative to developed force for high-K⁺ stimulation, was closely similar among all three experimental groups, suggesting that the basic mechanisms of force generation had not been affected by culture, either hypoxic or normoxic. JO₂ has been shown to correlate with force development in vascular tissue, whereas J_lac primarily reflects energy turnover associated with ion pumping (23), which may have been affected by culture (see below).

Maximum contractile responses to NE were unaffected after hypoxic culture, whereas responses to high-K⁺ depolarization were reduced. This pattern is also seen when preparations cultured under normoxia are compared with freshly prepared tissue (Ref. 15 and this study). Culture of arterial tissue downregulates the expression of voltage-activated Ca²⁺ channels (6), which may explain the decreased high-K⁺-induced force. This would not affect NE-induced force to the same extent because NE causes sensitization to Ca²⁺ (7). Although the effect of chronic hypoxia on the expression of voltage-dependent Ca²⁺ channels has not been examined in systemic vascular smooth muscle cells, it is interesting that a decreased Ca²⁺ transient was found after hypoxic culture of cardiac myocytes and suggested to be part of a protective response against Ca²⁺ overload (27).

NE stimulates glycogenolysis and thus mobilizes energy from glycolysis, evident as an increased J_lac. Rings cultured under normoxia lost force faster than fresh rings when repeatedly stimulated with NE in glucose-free medium, consistent with the greater reliance on glycolytic metabolism after culture. In contrast, rings cultured under hypoxia were markedly more resistant to glucose-free medium than freshly dissected rings, correlating with the dramatically increased glycogen stores in this tissue. A similar phenomenon has been described in cardiac myocytes cultured under hypoxia and subsequently reoxygenated (27), although the increase in glycogen stores was only about twofold, in contrast to the 40-fold increase seen here in the vascular rings. Overall, the metabolic reactions to chronic hypoxia in cultured vessels resemble alterations occurring in atherosclerosis (3).

We (7) have previously shown that intracellular Ca²⁺ stores are upregulated during culture of the tail artery. The present results suggest that culture under hypoxia further augments these stores, because it increased the magnitude of caffeine-induced contractions, dependent on Ca²⁺ release from intracellular stores via ryanodine receptors. This suggests that the uptake of Ca²⁺ into the sarcoplasmic reticulum is increased, implying increased demand of energy for pump activity. This is consistent with the increase in glycolytic rate in cultured vascular preparations. Mitochondrial uncoupling produced maximal rates of JO₂, which were essentially unaffected by culture under normoxia, but significantly decreased after hypoxic culture. All experimental groups also exhibited Ca²⁺-dependent control of mitochondrial function (21).

Inhibition of mitochondrial oxidative phosphorylation by rotenone and uncoupling of ATP production from oxidative phosphorylation using DNP produced similar responses as hypoxia in fresh and cultured preparations, although the loss of contractile force was greater, particularly with DNP. Adrenergic stimulation promotes generation of intracellular Ca²⁺ transients in the form of recurrent waves, as demonstrated both in arterial (13) and venous (24) smooth muscle. In Xenopus laevis oocytes, this kind of temporal Ca²⁺ coding appears to be regulated by mitochondrial metabolism (14), and in the tail artery we have demonstrated that intracellular Ca²⁺ wave activity is altered by rotenone, such that a pattern of low-frequency, high-amplitude waves is shifted to lower amplitude and higher frequency (29). In contrast, DNP eliminates all wave activity. The present results show that following organ culture, the number of cells with wave activity is decreased, as well as the frequency of waves in individual cells. These alterations were enhanced after hypoxic culture, suggesting that they are related to mitochondrial inhibition. The implications of this finding need to be explored further, but may be considered to include a role of wave activity in regulating several cellular processes. In addition to a possible role of intracellular Ca²⁺ waves in force production (13, 24), Ca²⁺ waves may influence protein synthesis because intermittent Ca²⁺ transients have been shown to be more efficient than sustained elevations in activating Ca²⁺-dependent transcription factors in cultured cells (4) and native smooth muscle (28).

The present study shows that exposure of vascular smooth muscle to chronic hypoxia shifts cellular metabolism toward glycolytic energy production, increases glycogen stores, and confers resistance to acute hypoxia. These effects are accompanied by altered cellular Ca²⁺ handling and decreased protein synthesis, whereas the energetic cost of contraction is unaffected. Because this study was performed on intact differentiated vascular tissue, its results point to deviations from normal function that may be early responses to chronic hypoxia of the vascular wall, a known risk factor for vascular disease.