We (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24–C32, 2001) have recently shown that organ culture for 24 h specifically inhibits relaxation to acute hypoxia (95% N2-5% CO2) in the porcine coronary artery. Here we show similar results in the porcine carotid artery and the rat and mouse aorta. In the coronary artery, part of the inability to relax to hypoxia after organ culture is associated with a concomitant loss in ability to reduce intracellular Ca2+ concentration ([Ca2+]i) during hypoxia (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24–C32, 2001). To elucidate the mechanisms responsible for the loss of relaxation to hypoxia, we investigated changes in K+ and Ca2+ channel activity and gene expression that play key roles in [Ca2+]iregulation in vascular smooth muscle (VSM). Reduced mRNA expression of O2-sensitive K+ channels (Kv1.5 and Kv2.1) was shown by reverse transcriptase-polymerase chain reaction in the rat aorta. In contrast, no change in other expressed voltage-gated K+ channels (Kv1.2 and Kv1.3) or Ca2+ channel subtypes was found. Modified K+ channel expression is supported by functional evidence indicating a reduced response to general K+ channel activation, by pinacidil, and to specific voltage-dependent K+ (Kv) channel blockade by 4-aminopyridine. In conclusion, organ culture decreases expression of specific Kv channels. These changes are consistent with altered mechanisms of VSM contractility that may be involved in Ca2+-dependent pathways of hypoxia-induced vasodilation.
- potassium channel
- smooth muscle
the mechanism of hypoxia-induced vasodilation is not completely defined. However, it has in part been attributed to the possible involvement of various types of ion channels. K+ channel activation and inactivation has been suggested to contribute to hypoxic vasodilation and vasoconstriction, respectively, in different tissue varieties (13, 18, 28, 32). Modulation of Ca2+channel activity has also been demonstrated in a number of hypoxic responses, including smooth muscle relaxation (10, 17). We (26) have shown that at low levels of activation there is a reduction in intracellular Ca2+ concentration ([Ca2+]i) during acute hypoxia, suggesting the possible modulation of ion channel activity. We (29) recently demonstrated that the decrease in [Ca2+]i in response to hypoxia is abolished after organ culture.
Organ culture evokes a variety of cellular changes that alter normal physiological function. The physiological consequences of organ culture are often quite distinct from those of cell culture. Different components of the mechanism of hypoxia-induced vasodilation have been identified using organ culture or storage as a model (4,7). For instance, there is an endothelium-dependent relaxation after reoxygenation from hypoxia (4) that depends greatly on the duration of storage of the vessel at 4°C (25). We have shown that organ culture at 37°C for 24 h markedly reduces relaxation to hypoxia (29). It has also been shown that altered Ca2+ handling in organ cultured vascular smooth muscle (VSM) influences force development (14, 15). Recently, increases in L-type Ca2+ channel mRNA and the dihydropyridine receptor have been demonstrated in cardiac myocytes after culture (6). In addition, N-type Ca2+channels have been shown to appear in coronary myocytes (22). It is conceivable that changes in ion channel expression or function could lead to a change in Ca2+handling capabilities, especially whether the change occurs in those channels involved in the mechanism of hypoxic relaxation.
Considerable attention has been given to O2-sensitive Ca2+ and K+ channels. The effect of changes in Po 2 on these channels has been linked to hypoxic vasoconstriction and hypoxic vasodilation in pulmonary and systemic vessels, respectively. Hypoxia-induced decreases in arterial tone are correlated with an inhibition of Ca2+ channels (10) and activation of various K+ channel subtypes (19). Although the specific type of K+ channels may differ between tissues, systemic vascular O2 sensitivity is primarily thought to involve modulation of Ca2+- and ATP-dependent K+ channels. We (24) have shown that blocking Ca2+-sensitive K+ channels and ATP-sensitive K+ channels does not significantly affect relaxation to hypoxia. Recently, considerable attention has been given to voltage-dependent K+ (Kv) channels, such as Kv1.1–1.6, Kv2.1, and certain members of the Kv3 and Kv4 families, as O2 sensors (19). Our laboratory (24) has demonstrated a reduced magnitude of relaxation to hypoxia in the presence of the Kv channel blocker 4-aminopyridine (4-AP). Prolonged hypoxia changes the expression of many Kv channel subtypes (30) and hypoxia is known to activate K+ currents in arterial myocytes (28).
Here we use the loss of O2 sensing in our organ culture model (29) to elucidate the Ca2+-dependent mechanism of hypoxia-induced relaxation. Using molecular, physiological, and pharmacological approaches, we show that1) organ culture results in a decreased expression of specific O2-sensitive Kv channels, 2) the effects of K+ channel activators and Kv channel inhibitors are reduced after organ culture, and 3) neither Ca2+ channel expression nor activity is altered by organ culture for 24 h. These findings provide new insight into the mechanisms of hypoxic relaxation and suggest that Kv channels play a more important role than Ca2+ channels in O2sensitivity of these tissues.
Artery culture conditions.
Organ culture was carried out as previously described (29). Briefly, left descending coronary arteries were dissected from adult porcine hearts obtained from the slaughterhouse on the day of death and two rings per artery were cleaned of adhering connective tissue. One ring was cultured in sterile Dulbecco's modified Eagle's medium + 1% antibiotic solution at 37°C. A paired ring (control) was stored in the same solution at 4°C. After 24 h, arterial rings were prepared for organ bath experiments. All organ culture preparations were performed under sterile conditions in a culture hood. Porcine carotid arteries were obtained from the slaughterhouse and similarly prepared.
Adult male Sprague-Dawley rats and adult male C57 Black Swiss mice were euthanized by CO2 asphyxiation. Small animal handling was done in accordance with, and with approval of, the local Institutional Animal Care and Use Committee. Aortas were dissected and rinsed with cold sterile culture media. Excess fat and connective tissue were removed and aortas were prepared for organ culture as described above.
Organ bath studies.
All tissue used for these experiments were mechanically deendothelialized by gentle rubbing of the luminal surface with a cotton-tipped applicator. Rings were mounted onto two wires; one of which was fixed and the other was connected to a force transducer. One cultured ring and its paired control were placed into a bath containing physiological saline solution of the following composition (in mmol/l): 118.3 NaCl, 25.0 NaHCO3, 11.1 dextrose, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 0.026 EDTA, and 2.5 CaCl2. Bath pH was 7.4 when aerated with 95% O2-5% CO2 at 37°C. Tissues were allowed to equilibrate for 1 h. Tension was adjusted to 40 mN for coronary and carotid arteries and 30 mN for mouse and rat aortas, which set each tissue length in the range for optimal force generation. At least two precontraction-relaxation cycles to 80 mM KCl (for coronary and carotid arteries) or 60 mM KCl (aortas) were performed until maximum reproducible muscle forces were observed. The absence of the endothelium was confirmed by lack of a response to substance P (10−8 M). A test contraction was elicited using 40 mM KCl for hog coronary and carotid arteries and 30 mM KCl for rat and mouse aortas. After steady force was obtained, hypoxia was obtained by bubbling the bath with 95% N2-5% CO2 for ≈20 min. The final O2 tension of the bath solution, measured polarographically, was ≈1–2%. We define these conditions as hypoxia. In some experiments, concentration-response curves to KCl were obtained in coronary artery. Rings were stimulated with increasing concentrations of KCl starting with 5 mM and ending with 55 mM in 5 mM increments.
In experiments with ion channel modulators, coronary rings were first tested for response to hypoxia as described above. Subsequently, arterial rings were stimulated with 1 μM U-46619. After stable forces were obtained, rings were treated with 4 mM pinacidil or 1 μM nifedipine. In separate experiments, rings were stimulated with 40 mM KCl for a reference contraction, rinsed with fresh physiological saline solution, and then treated with 1 mM 4-AP.
A = change in force × circumference/2 × wet weight). Concentration force relations are given in terms of percentage of the maximum force of the reference contraction.
Reverse transcriptase-plymerase chain reaction of K+ and Ca2+channels.
Total RNA from the rat aorta was isolated and used for reverse transcriptase-polymerase chain reaction (RT-PCR) according to the protocol of PE Life Sciences (Boston, MA). Briefly, an oligo-dT primer was used with the murine leukemia virus RT for first-strand synthesis. Amplification of desired K+ channel cDNA was performed with sense and antisense primers designed after the rat brain sequence at annealing temperatures previously described (2, 5, 20). The following K+ channels were investigated based on reported (19) voltage dependence and O2sensitivity: Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv2.1.
Ca2+ channel cDNA amplification was performed using the following primers and conditions: L-type sense TGC TCT GCC TGA CTC TGA AG, antisense GAG TGC CTT CAC ATC GAA TC, annealed at 46°C for 38 cycles; T-type sense GCC GTC TCA GCC GCG GCC TTT CT, antisense CAA AGG TGA GTG TAT CCT CAG GC, annealed at 50°C for 38 cycles; and P/Q-type sense CCA GTC TGT GGA GAT GAG AGA AAT GGG, antisense TTT GGA GGG CAG GTC ACC CGA TTG, annealed at 52°C for 38 cycles. PCR products were analyzed on 1.2% DNA agarose gels containing ethidium bromide (3 μg/μl) for visualization.
RT-PCR using all K+ and Ca2+ channel primers was also attempted in porcine coronary artery. None of these primers recognized porcine coronary artery cDNA. Therefore, the rat aorta was used for these experiments and subsequent Western blot analysis.
Western blot analysis.
Control and organ cultured rat aortic rings were frozen in liquid N2 and pulverized vigorously in a dental amalgamator. The resulting powder was dissolved in ice-cold homogenization buffer (0.15 M NaCl, 5 mM EDTA, 1 mM dithiothreitol, 20 mM sodium metabisulfite, 20 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride). Homogenates were incubated on ice for 1 h and then centrifuged at 16,000g and 4°C for 45 min. The supernatant was saved and total protein concentration was determined from this sample by using the Bradford assay. Samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 1.5 h. Proteins were transferred to nitrocellulose membranes for 1 h using the Semi-Dry Transfer apparatus (Bio-Rad; Hercules, CA). After transfer, membranes were blocked with 3% nonfat dry milk for 1 h and then incubated with primary antibody at 4°C for 16–18 h. Membranes were then washed four times for 20 min each with phosphate-buffered saline, followed by 1-h incubation at room temperature in secondary antibody (horseradish peroxidase-conjugated anti-rabbit). Membranes were washed again four times in phosphate-buffered saline, subjected to enhanced chemiluminescence detection for 1 min, and then exposed to film.
Data were analyzed using the t-test for paired two-sample means or two-way repeated-measures analysis of variance with one-factor balance design. Statistical significance was accepted forP < 0.05. Values are expressed as means ± SE.n values represent the number of hearts from which arteries were isolated.
U-46619, pinacidil, 4-AP, and nifedipine were from Sigma-Aldrich (St. Louis, MO). RT-PCR reagents were from Applied Biosystems (Atlanta, GA). Antibodies for K+ channels (Kv1.5 and Kv2.1) were from Upstate Biotechnology (Lake Placid, NY) and for Kv1.3, Kv1.2, and L-type Ca2+ from Alomone Labs (Jerusalem, Israel). Antibodies to calponin were from Sigma-Aldrich.
Effects of organ culture on hypoxic relaxation in porcine coronary and carotid artery and rat and mouse aorta.
We (29) investigated whether the marked reduction in hypoxia-induced relaxation after organ culture was specific to coronary arteries. Figure 1 shows relaxation to hypoxia for four different control and organ-cultured systemic vessels. The reduced relaxation to hypoxia in the cultured porcine coronary artery (29) is similar to that of the rat and mouse aorta. Both show a 50–60% reduction in hypoxic relaxation. Porcine carotid artery exhibited a greater level of inhibition at ∼80%. Whereas control carotids had a significantly higher magnitude of hypoxic relaxation compared with the other tissues, cultured carotid arteries relaxed even less compared with the controls. These results indicate that inhibition of hypoxic relaxation after organ culture occurs in many vascular tissue types.
Effects of organ culture on depolarization-contraction coupling, K+ and Ca2+channel function.
We reported that organ culture causes specific inhibition of hypoxic relaxation in porcine coronary artery without significantly effecting maximum force development or relaxation via A- or G-kinase pathways (29). Intracellular Ca2+ handling during hypoxia was also altered after organ culture. One possible explanation for these changes in contractility and Ca2+ handling is a change in depolarization-contraction coupling. Figure2 shows KCl concentration-force relations for both control and organ-cultured coronary arteries. There is a significant rightward shift in the developed force to KCl after organ culture. The ED50 for control and cultured arteries are 14.8 ± 0.5 and 22.3 ± 0.7 mM KCl, respectively. These data suggest a decrease in sensitivity to KCl depolarization without adversely affecting maximum isometric force (Fig. 2, inset) after organ culture. An alteration in ion channel function could result in compromised Ca2+ handling leading to reduced relaxation to hypoxia. It is conceivable that a loss in K+ or Ca2+ vital for the reduction in [Ca2+]i during hypoxia could manifest as an inability to relax to hypoxia.
K+ and Ca2+ channels directly regulate [Ca2+]i in response to depolarization-mediated activation in VSM. We measured force production in response to general K+ channel activation (pinacidil), Kv channel blockade (4-AP), and L-type Ca2+ channel blockade (nifedipine) to determine whether changes in these ion channel activities underlie changes in Ca2+ handling and depolarization-contraction coupling. After organ culture of coronary arteries, activation of K+ channels by pinacidil is markedly reduced (Fig. 3 A). Both the magnitude and rate of relaxation of coronary rings after 4 μM pinacidil are decreased. In a previous report (26), we demonstrated a major role of Kv channels in modulating porcine coronary contractility. Approximately 40% of the pinacidil vasodilation is inhibited by 1 mM 4-AP (Fig. 3 C). In addition, there is evidence implicating a role for Kv channels in O2-sensing mechanisms (1, 26, 28). Organ culture results in a decreased force response to Kv channel blockade (Fig. 3 B). These data are summarized in Fig.4. Both relaxation after K+activation and the increase in force after Kv channel blockade are inhibited by organ culture. The degree of inhibition of relaxation after K+ activation is similar to that observed for relaxation to hypoxia. Relaxation after L-type Ca2+ channel blockade was not significantly affected by organ culture. These results indicate a reduction in K+ channel function, specifically Kv channels after organ culture that correlates with a loss in hypoxic relaxation.
Expression of O2-sensitive Kv channels and Ca2+ channel subtypes after organ culture.
The activity of certain Kv channel subtypes and Ca2+channels has been shown to be O2 sensitive (1, 9,10). We investigated the expression of a subset of these channels that have been previously reported (19, 31) to be involved in hypoxic vasoconstriction in pulmonary arteries. Whereas our functional database is most extensive for hog coronary artery, many Kv channel molecular tools are not available. RT-PCR was performed using specific sense and antisense primers designed to amplify rat cDNA fragments of the following ion channels: Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, L-type Ca2+, T-type Ca2+, and P/Q-type Ca2+. Because organ culture also inhibited hypoxic relaxation in rat aorta, this vessel was used in these experiments and for Western blot analysis. The results are shown in Fig. 5. Only Kv1.2, Kv1.3, Kv1.5, Kv2.1, and the three Ca2+ channels subtypes were present in this tissue. RT-PCR was performed on rat brain RNA as a control to confirm that the primers were amplifying the cDNA fragment of the correct size (data not shown). Whereas there was no change in Kv1.2 and Kv1.3 expression, there was a marked downregulation of Kv1.5 and Kv2.1. There was also no change in Ca2+ channel expression after organ culture. To confirm decreased expression of Kv1.5 and Kv2.1 at the protein level, Western blots were made using antibodies specific to each channel. Figure 6 shows that there is a loss in both Kv1.5 and Kv2.1 protein after organ culture. These results suggest that organ culture inhibits the expression of Kv1.5 and Kv2.1 without affecting that of other Kv or Ca2+ channels present.
Recently, we (29) have shown that organ culture specifically reduces relaxation to hypoxia in the porcine coronary artery. In this study, we demonstrate that this is also observed for the porcine carotid artery and rat and mouse aorta. We have shown that inhibition of relaxation to hypoxia after organ culture involves changes in [Ca2+]i handling (29). Others have shown that organ culture alters overall Ca2+ handling in VSM (15). Figure 2 shows that the KCl-force relation is shifted rightward in cultured arteries, suggesting altered Ca2+ handling. Stimulation by depolarization-mediated agonist involves reduced K+ efflux through K+ channels and activation of voltage-gated Ca2+ channels. Our data indicate that there is a change in the excitation-contraction coupling pathway for KCl after organ culture (Fig. 2) that is likely due to reduced Ca2+ influx through voltage-dependent Ca2+ channels. We have previously shown that there is a reduction in [Ca2+]i during hypoxic relaxation under low levels of activation (24). In a recent study, we demonstrated that organ culture for 24 h at 37°C abolishes not only relaxation to hypoxia but also the concomitant decrease in [Ca2+]i(29). An inability to reduce [Ca2+]i may reflect altered function or expression of ion channels, presumably K+ channels and Ca2+, that are responsible for regulating Ca2+homeostasis specifically during hypoxia.
K+ channels play a major role in modulating force and ultimately Ca2+ homeostasis in vascular smooth muscle. After organ culture there is a reduction in the vasodilator response to pinacidil, a general K+ channel activator (Figs.3 A and 4). This suggests that K+ channels are not functional or that there is a decrease in the number of K+ channels. The specific type of K+ channel activity that is altered cannot be inferred from these experiments because pinacidil has been shown to activate several different K+ channel subtypes (3). However, 40% of the pinacidil response is 4-AP sensitive suggesting a major Kv channel contribution (Fig. 3 C). Shimizu et al. (26) showed that 4-AP-sensitive Kv channels were the most significant K+ channels in terms of modulating force and [Ca2+]i in porcine coronary artery. There is evidence that hypoxia relaxation of arteries can involve hyperpolarization mediated through ATP-sensitive K+channels and Ca2+-sensitive K+ channels (12, 16, 19). However, using a variety of K+channel inhibitors, only 4-AP treatment resulted in a significant decrease in the hypoxic relaxation (24). However, because 4-AP pretreatment also increased the prehypoxic force, interpretation of the attenuated hypoxic relaxation in terms of Kv channel blockade is not straightforward.
Data in Fig. 3 B (and summarized in Fig. 4) show a significantly reduced reactivity to 4-AP blockade after organ culture. The blunted force increase to 4-AP may indicate Kv channels not affected by organ culture that contribute to the small remaining hypoxic relaxation observed (Fig. 1, lane 1). The reduction in the hypoxic relaxation after organ culture also paralleled the decreased relaxation to pinacidil (Fig. 4). The loss of Kv channel function was also paralleled by the inability of hypoxia to reduce [Ca2+]i. The inability to reduce [Ca2+]i during hypoxia could be explained by altered Ca2+ channel function as well. However, our results indicate that voltage-dependent Ca2+ channel function was not altered by organ culture (Fig. 4).
Our conclusions based on functional evidence are strongly supported by our biochemical results. Organ culture is known to alter the expression of several ion channel subtypes. It has been reported (21) that chronic hypoxia decreases the expression of Kv1.5 and Kv2.1 in pulmonary myocytes (5). We investigated the effect of organ culture on Kv channels known to be O2 sensitive. Our results indicate that expression of Kv1.5 and Kv2.1 is decreased in the rat aorta after organ culture. Functional results with K+channel modulators in both the hog coronary and rat artery support these findings (Fig. 4). Expression of other Kv channel subtypes did not change (Fig. 5). This is consistent with the ability to contract to depolarization. The lack of change in expression of Ca2+channel subtypes not only supports the maintenance of L-type Ca2+ channel function, but also suggests that inhibition of relaxation to hypoxia after organ culture primarily involves altered Kv channel expression. The functional and molecular data presented here suggest that at least Kv1.5 and Kv2.1 are important for hypoxic-vasorelaxation after organ culture.
The mechanism(s) by which organ culture results in altered ion-channel expression is unknown. VSM cell culture is associated with differentiation and progression from a contractile to a proliferative phenotype (23). Organ culture is also known to alter both smooth muscle-specific and ubiquitously expressed genes (27). These changes could manifest into altered ion channel expression and/or function by several mechanisms including inhibition of gene transcription, decrease in mRNA or protein expression, or modulation of transcription factors that regulate ion channel expression. A change in transcription factors and promoter elements necessary for regulation of Kv channel expression (8), for example, could occur during organ culture and cause the downregulation of Kv1.5 and Kv2.1 that we observe. The exact mechanism may actually be a combination of several and warrants continued investigation.
Our results provide compelling evidence for the involvement of Kv channels in the hypoxic relaxation. However, it is possible that mechanisms not involving Kv channels may be involved. We have championed the hypothesis that a Ca2+-independent mechanism of hypoxic relaxation also exists (24). Moreover, hypoxic vasodilation of some vessels is in fact, not associated with hyperpolarization (11). In this investigation, we demonstrate that organ culture inhibits hypoxia-induced relaxation in four different vascular smooth muscle tissues. We previously showed that altered Ca2+ handling contributes to this attenuated relaxation response. Our current data show that K+channels, specifically Kv1.5 and Kv2.1, but not Ca2+channels are decreased in parallel to the hypoxic relaxation by organ culture. Further investigation is needed to unambiguously link these Kv channels to the hypoxic relaxation.
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-54829 and American Heart Association Grant 0030091N.
Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (E-mail:).
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- Copyright © 2002 the American Physiological Society