Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture

George D. Thorne, Shunichi Shimizu, and Richard J. Paul

Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267 - 0576


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Hypoxia (95% N2-5% CO2) elicits an endothelium-independent relaxation (45-80%) in freshly dissected porcine coronary arteries. Paired artery rings cultured at 37°C in sterile DMEM (pH ~7.4) for 24 h contracted normally to KCl or 1 µM U-46619. However, relaxation in response to hypoxia was sharply attenuated compared with control (fresh arteries or those stored at 4°C for 24 h). Hypoxic vasorelaxation in organ cultured vessels was reduced at both high and low stimulation, indicating that both Ca2+-independent and Ca2+-dependent components are altered. In contrast, relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolin and isoproterenol) activation was not significantly affected by organ culture. Additionally, there was no difference in relaxation after washout of the stimulus, indicating that the inhibition is specific to acute hypoxia-induced relaxation. Simultaneous force and intracellular calcium concentration ([Ca2+]i) measurements indicate the reduction in [Ca2+]i concomitant with hypoxia at low stimulus levels in these tissue is abolished by culture. Our results indicate that organ culture at 37°C specifically attenuates hypoxic relaxation in vascular smooth muscle by altering dynamics of [Ca2+]i handling and decreasing a Ca2+-independent component of relaxation. Thus organ culture can be a novel tool for investigating the mechanisms of hypoxia-induced vasodilation.

hypoxia


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INTRODUCTION
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CHANGES IN OXYGEN TENSION (PO2) profoundly affect the normal physiology of an organism. Systemic vascular smooth muscle (VSM) relaxes in response to acute hypoxia or decrease in PO2 (6). Vasodilation to hypoxia in systemic VSM is likely a protective response to increase blood flow to areas of low PO2. A variety of theories have been proposed to explain the mechanism of hypoxia-induced vasodilation, such as release of vasoactive substances (25, 29), activation of ATP-sensitive K+ (K+ATP) channels (5, 18), and energy limitation due to a limited anaerobic metabolic capacity (4, 10, 16). The last is unlikely in light of evidence that suggests relaxation in response to hypoxia occurs without compromising energy metabolism (26). It has also been shown that hypoxic vasorelaxation is insensitive to various ion channel and metabolic antagonists (3, 26). Recently we showed that mechanisms involving reduction in ATP, decrease in pH, increased Pi, or activation of K+ATP or calcium-activated potassium (KCa) channels cannot explain hypoxic relaxation in pig coronary artery (26). Moreover, both Ca2+-dependent and Ca2+-independent mechanisms were required to account for hypoxia-induced vasodilation.

The role of intracellular calcium concentration ([Ca2+]i) handling in hypoxic sensitivity has been studied extensively. Hypoxia-induced pulmonary vasoconstriction has been linked to increases in [Ca2+]i attributed to release from intracellular stores (17) and inhibition of sarcolemmal K+ currents (15, 22), both of which activate Ca2+ channels. Ca2+ modulation in hypoxic vasodilation of systemic VSM is less clear. Although several studies have shown an inhibition of L-type Ca2+ channels by hypoxia (8, 13, 24), our studies (26) and other (1) indicate that hypoxia-induced relaxation can occur independently of the decreases in [Ca2+]i that are normally associated with smooth muscle vasodilation. Still others report dilation of arterial smooth muscle via KCa channel activation by release of Ca2+ from intracellular stores (21). Although Ca2+ modulation by hypoxia has been studied extensively, no consistent theory has emerged.

Hypoxia is known to cause an increase in coronary blood flow in vivo (7). Although many studies have demonstrated this effect of hypoxia on small coronary resistance vessels, hypoxia-induced vasodilation of larger coronary arteries has been shown both in vivo and in vitro (6, 6a, 7) and may be of physiological significance (30). This relaxation response to hypoxia can be demonstrated in isolated porcine coronary arterial rings (Fig. 1). Recently, we observed that organ culture severely inhibits coronary relaxation to hypoxia, whereas contractility appears normal (27). In this study, we characterize the effects of organ culture in detail to probe further the mechanisms of O2 sensing. We demonstrate that 1) the inhibition of relaxation is specific for hypoxia, 2) loss in ability to respond to hypoxia is linked to changes in [Ca2+]i handling properties after organ culture, and 3) Ca2+-independent components of vasorelaxation are also impaired. Thus organ culture may provide a novel way to investigate the mechanisms of hypoxic vasorelaxation.


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Fig. 1.   Typical recording of a freshly isolated porcine coronary arterial ring in response to KCl stimulation (40 mM) and hypoxia (95% N2-5% CO2). The right axis bar shows %O2 as determined by the output of an O2 electrode in the bath and calibrated as 100% = 760 mmHg.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Coronary Artery Culture Conditions

Adult pig hearts were obtained from a slaughterhouse immediately after death. Left descending coronary arteries were dissected, and two rings per artery were cleaned of adhering connective tissue. One ring was cultured in sterile DMEM + 1% antibiotic solution at 37°C. A paired ring (control) was stored in the same solution at 4°C. In a subset of these experiments, arteries were mounted isometrically on wires set to a diameter slightly greater than the resting diameter. After 24 h, rings were prepared for organ bath studies. All organ culture preparations were performed under sterile conditions in a culture hood. Arterial rings were prepared immediately after dissection for organ bath studies, and these results are designated as the fresh artery response. In a separate group of experiments, several rings from the same artery were cultured/stored under the conditions described above for 6, 12, 18, 24, and 30 h and then prepared for organ bath studies. In another group of experiments, paired rings were cultured for 24 h with 10 µM cycloheximide, a protein synthesis inhibitor in the media.

Organ Bath Studies

All tissue used for these experiments were mechanically de-endothelialized 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 salt solution (PSS) of the following composition (in mmol/l): 118.3 NaCl, 15.0 NaHCO3, 11.1 dextrose, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 0.026 EDTA, and 2.5 CaCl2. In experiments using cycloheximide, the 10 µM concentration was maintained in the baths of those rings cultured with the inhibitor. pH was 7.4 when the bath was aerated with 95% O2-5% CO2 at 37°C. Tissues were allowed to equilibrate for 1 h. Tension was adjusted to 40 mN, which sets the tissue length in the range for optimal force generation. At least two contraction/relaxation cycles to 80 mM KCl 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 0.1 µM U-46619 or 40 mM KCl. After steady force was generated, hypoxia was obtained by bubbling 95% N2-5% CO2 through the baths for approx 20 min. The final oxygen tension of the bath solution, measured polarographically, was approx 1-2%. We define these conditions as hypoxia. Upon steady contracture (40 mM KCl), rings were also challenged cumulatively with increasing concentrations (10-9-10-5 M) of one of three vasodilators [sodium nitroprusside (SNP), forskolin, or isoproterenol].

The hypoxic response was characterized in terms of the maximum hypoxic relaxation, expressed as a percentage of the initial isometric force, and the time course, quantified by time to half-maximal relaxation (t1/2). All organ bath measurements were recorded using a digital data acquisition system (Biopac). Force was normalized to cross-sectional area [F/A = (change in force × circumference)/(2 × wet weight)].

Intracellular Calcium Imaging

A Photon Technology International spectrofluorimeter adapted for force acquisition permitted simultaneous force and [Ca2+]i measurements as previously described (2, 20). Briefly, rings were mounted onto a force transducer and submersed in a cuvette containing a solution for loading the fluorescent probe fura 2-AM (MOPS-buffered PSS, BSA, 0.2% Pluronic acid, and 5 µM fura 2-AM) for 3 h at room temperature. After a steady baseline tension was attained, rings were rinsed and constantly perfused (20 ml/min) with 95% O2-5% CO2-bubbled PSS at 37°C. Tissues were stimulated by adding agonists to the perfusate PSS. Hypoxia was obtained by perfusing with the same solution but aerated with 95% N2-5% CO2.

[Ca2+]i calibration. The fluorescent emission at 510 nm for intensity at 340 nm excitation was divided by that measured at 380 nm, and this ratio was used as an index of [Ca2+]i. For statistical analysis, the ratio was assigned values of 0% for resting muscle and 100% for tissue stimulated with 40 mM KCl. This protocol was chosen as a general routine over absolute calibration of the fura 2 fluorescence in lieu of the major uncertainties in terms of absolute calibration (2). Using standard calibration procedures (9), we previously reported values of 50.4 ± 17.2 nM and 441 ± 163 nM for [Ca2+]i under baseline and KCl simulation, respectively (2, 20). In this limited range, the 340/380 ratio is nearly linearly dependent on [Ca2+]i as reported by others (19).

Statistical Analysis

Data were analyzed using the t-test for paired two-sample means or two-way repeated ANOVA with one-factor balance design. Statistical significance was accepted for P < 0.05. Values are expressed as the mean ± SE. Values of n represent the number of hearts from which arteries were isolated.


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Hypoxia-induced Vasodilation in Fresh, Cold, Stored, and Cultured Arteries

Fresh coronary artery relaxes upon exposure to hypoxia (PO2 < 10 mmHg; Fig. 1). The magnitude of relaxation depended on the type of stimulus used for the precontraction. Fresh arterial rings relaxed up to 45 and 80% of maximum force when stimulated with KCl or the thromboxane A2 analog U-46619, respectively. Similar responses were seen in arteries stored at 4°C for 24 h (Fig. 2, top; Fig. 3, B and D). The paired arteries that were stored at 4°C were subsequently used as the control for cultured arteries. Figure 2 illustrates a typical response of control (stored at 4°C for 24 h) and organ-cultured (37°C for 24 h) arterial rings to 40 mM KCl or 1 µM U-46619 followed by hypoxia. Organ culture results in a marked attenuation in magnitude of relaxation to hypoxia. Data from these types of experiments for U-46619 and KCl are summarized in Fig. 3. Similar experiments were performed on control and cultured arterial rings maintained isometrically on wires so that they were under tension for 24 h. As with the untethered conditions, hypoxic relaxation was reduced in the organ cultured arteries compared with their paired controls (n = 4; data not shown).


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Fig. 2.   Effect of differential organ culture on porcine coronary artery. Cultured rings (bottom) have an attenuated hypoxia-induced relaxation relative to control (top, n = 24). Tracings show relaxation to hypoxia during 40 mM KCl (left) and 1 µM U-46619 (right) activation.



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Fig. 3.   A summary of the effects of organ culture on maximum isometric force generation and hypoxia-induced vasodilation for 40 mM KCl (top) and 1 µM U-46619 (bottom) contractions. B and D: maximum relaxation in response to acute hypoxia (15-20 min) is attenuated by 40-60% after organ culture at 37°C. A and C: the ability to generate isometric force is not significantly altered by this treatment. Results represent the means ± SE for 12 experiments. *P < 0.05 for differences between organ cultured and control.

The reduction in hypoxic relaxation after organ culture occurred without any significant change in maximum force developed in response to either KCl or U-46619 (Fig. 3, A and C). Figure 4 shows the average time course of isometric force after bubbling with N2 from experiments such as that shown in Fig. 1. Organ culture can be seen to result in a significant decrease in the rate of hypoxia-induced vasodilation and increased t1/2 compared with control.


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Fig. 4.   Average (n = 12) time course of isometric force after bubbling with N2 from experiments such as that shown in Fig. 1. The ordinate represents the amount of force remaining after imposition of hypoxia. Organ culture can be seen to result in a significant decrease in the rate of hypoxia-induced vasodilation and to increase the half-maximal time (t1/2) compared with control. Time course for KCl (A) and U-46619 (B) activation are both slowed by about 50% (n = 12). C: average t1/2 for hypoxic relaxation in control and cultured arteries for 40 mM KCl and 1 µM U-46619 activation. Bar graph shows the means ± SE for 8 (KCl) and 6 (U-46619) experiments. *Differences were significant at P < 0.05.

Figure 5 shows the effects of the duration of time under control or culture conditions on the hypoxic relaxation. Isometric force generating capability with depolarization or receptor-mediated activation in either group was not affected until 30 h of storage (Fig. 5, A and C). In contrast, reduced relaxation to hypoxia occurred as early as 6 h with KCl stimulation and 12 h with U-46619 stimulation in cultured rings. Control rings exhibited a small attenuation in relaxation to hypoxia at 30 h compared with shorter durations of storage when stimulated with 40 mM KCl (Fig. 5B). There was no loss of relaxation to hypoxia with U-46619 stimulation in control rings (Fig. 5D).


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Fig. 5.   Effects of time in storage on control and cultured coronary arterial rings. The ability to generate isometric force was not significantly altered in response to 40 mM KCl (A) and 1 µM U-46619 (C) stimulation. Only after 30 h did control or cultured rings exhibit a decline in force. Relaxation response to hypoxia was significantly different between control and culture groups as early as 12 h (B and D). Bar graph shows the means ± SE for 8 (KCl) and 6 (U-46619) experiments. *Differences were significant at P < 0.05.

We investigated whether organ culture impaired relaxation in general by studying the response to various smooth muscle vasodilators. Figure 6 shows the average force remaining after increasing concentrations of SNP, isoproterenol, and forskolin. Sensitivity to increasing concentrations of the nitric oxide donor SNP was not significantly different between experimental groups (Fig. 6C). Similarly, organ culture did not significantly affect the sensitivity of coronary rings to receptor-mediated (isoproterenol) or direct (forskolin) activation of adenylate cyclase (Fig. 6, A and C). Thus the ability of porcine coronary arterial rings to relax to G- or A-kinase mediated pathways was not affected by organ culture at 37°C for 24 h. Analysis of the decrease in isometric force upon washout of agonist (Fig. 7) indicates that organ culture also does not alter this mechanism of relaxation as well.


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Fig. 6.   Concentration-response curves for sodium nitroprusside (A), forskolin (B), and isoproterenol (C). The ordinate represents the average force remaining after treatment with increasing concentrations of each drug. Results indicate no change in G- or A-kinase-mediated smooth muscle relaxation pathways after organ culture at 37°C. There was no significant difference in sensitivity to these vasodilators at any concentration used (n = 8).



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Fig. 7.   A: typical recording of a control and cultured arterial ring stimulated with 40 mM KCl and rinsed with fresh physiological salt solution under normoxic conditions. B: summary of t1/2 to relaxation for 8 experiments. The bar graph shows the means ± SE.

We recently showed that with high stimulus levels hypoxic vasorelaxation occurred without a decrease in [Ca2+]i (26). The response to hypoxia under high and low stimulus conditions thus gives an index of the relaxation's dependence on [Ca2+]i. Figure 8 illustrates that in all cases (low and high activation, U-46619 and KCl), organ-cultured arteries had a significantly reduced relaxation in response to hypoxia. The largest difference was seen with 20 mM KCl stimulation (lane 4), a low-level depolarization, where cultured arteries relaxed 60% less than control. Interestingly, the smallest difference between control and cultured arteries was observed during low-level receptor-mediated stimulation, 0.1 mM U-46619 (lane 3). These data suggest that both Ca2+-dependent and -independent components of hypoxia-induced relaxation are affected by organ culture.


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Fig. 8.   Hypoxia-induced relaxation during high (1 µM U-46619 and 40 mM KCl) and low (0.1 µM U-46619 and 20 mM KCl) activation. Organ culture at 37°C for 24 h significantly reduces relaxation response to hypoxia regardless of stimulation type or level. Bar graph shows means ± SE; n = 8-12. *Differences were significant at P < 0.05.

Effects of Protein Synthesis Inhibition on Relaxation in Response to Hypoxia

To investigate whether an enhancement or inhibition of protein synthesis underlies the loss of relaxation response to hypoxia, we incubated both control and cultured coronary rings with 10 µM cycloheximide, a protein synthesis inhibitor. Figure 9 shows that after 24 h of incubation with cycloheximide, control rings exhibit a significant reduction in relaxation in response to hypoxia compared with control rings not incubated with the inhibitor (Fig. 9B). This was true for hypoxia during both KCl and U-46619 stimulation. Organ cultured rings incubated with cycloheximide showed no change in their inhibited relaxation response to hypoxia. Similar to organ culture, relaxation to SNP and forskolin were unaffected by cyclohexamide. In all cases, isometric force generation was not significantly affected by incubation of these rings with cycloheximide (Fig. 9A).


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Fig. 9.   The effects of 10 µM cycloheximide (chx) incubation on force (A) and hypoxic relaxation (B) for control and cultured porcine coronary arteries. Inhibition of protein synthesis causes an attenuated relaxation to hypoxia in control arterial rings but no further changes in cultured rings. Bar graph shows means ± SE; n = 8. Differences were significant at P < 0.05 (*control vs. cultured; §control + chx vs. control - chx).

Intracellular Calcium Measurements

Organ culture has been reported to alter [Ca2+]i handling through modification of Ca2+ channel expression and intracellular Ca2+ store function (11, 17, 17a). We thus measured [Ca2+]i and force generation during hypoxic challenge to determine if Ca2+ handling was affected by organ culture. Figure 10 shows typical [Ca2+]i response to hypoxia with low (20 mM) and high (40 mM) KCl stimulation in fresh coronary artery. A significant decrease in [Ca2+]i during hypoxia with 20 mM KCl was observed, but little change with 40 mM KCl, as previously reported (26). The decrease in [Ca2+]i concomitant with hypoxia during low stimulation was also observed in control arteries (Fig. 11B). In contrast, in organ cultured arteries [Ca2+]i does not decrease in response to hypoxia (Fig. 11A). Data from these types of experiments are summarized in Table 1. These data suggest an impaired [Ca2+]i handling capacity during hypoxia after organ culture.


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Fig. 10.   Calcium tracings during hypoxia on 40 (A) and 20 (B) mM KCl contractures for a fresh coronary ring. Although hypoxia decreases force in either case (not shown in this figure), intracellular calcium concentration ([Ca2+]i) is decreased only at 20 mM (B).



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Fig. 11.   A: simultaneous force and Ca2+ measurements showing a typical tracing of a coronary artery after organ culture that has lost the ability to reduce [Ca2+]i during hypoxia under low stimulus levels (20 mM KCl). B: consistent with freshly isolated vessels, there is a concomitant decrease in [Ca2+]i during hypoxia with low activation in control vessels.


                              
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Table 1.   Decrease in [Ca2+]i during hypoxia-induced vasorelaxation under low-level activation in porcine coronary artery


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In this investigation we show that organ culture at 37°C for 24 h significantly reduces the relaxation response to hypoxia in porcine coronary artery. The ability to generate control levels of isometric force after organ culture suggests that significant smooth muscle cell alteration does not occur. Moreover, the ability to respond to both depolarization (KCl) and receptor-mediated (U-46619) contractile stimuli suggests that the machinery necessary for these two different excitation pathways is not altered by organ culture. These results are quite different from those of cultured smooth muscle cells where both contractility and activation pathways can be significantly altered (8, 13, 24).

The attenuation of the hypoxic relaxation was dependent on the duration of organ culture (Fig. 5), with a reduction compared with control seen as early as 6 h. In control rings, a significant reduction in the hypoxic relaxation did not occur until 30 h. The importance of this observation is less clear, as there was also a significant drop in force generation at 30 h as well, possibly simply reflecting tissue degradation. Although control rings showed some minor loss in relaxation in response to hypoxia at various time points compared with earlier ones, they always relaxed significantly more than cultured rings. These results suggest that the specific attenuation of hypoxic vasorelaxation depends both on time and storage condition. The most consistent results in terms of a maximal loss of relaxation response to hypoxia without a concomitant loss of force production were obtained with a duration of 24 h, which is when all other experiments were performed.

Importantly, relaxation in response to activation of cGMP and cAMP-mediated vasodilatory pathways was not affected by organ culture. Sensitivity to these pathways activated directly (SNP, forskolin) or through receptor stimulation (isoproterenol) was not significantly different (Fig. 6). This evidence implies that organ culture does not impair the innate ability of coronary arteries to respond to the major smooth muscle pathways of relaxation. Therefore, the loss in hypoxic relaxation in these tissues cannot be attributed to a generalized loss in inherent relaxation capability. Moreover, this also suggests that O2 sensing does not involve the effector pathways downstream from A- or G-kinase activation. This is consistent with previous studies based on pharmacological approaches (3, 26). The idea that organ culture specifically reduces hypoxia-induced vasodilation is strengthened further by the fact that cultured vessels will relax when the stimulus is removed (Fig. 7).

We previously identified both Ca2+-independent and -dependent components of the mechanism(s) underlying hypoxic relaxation in coronary arteries (24, 26). The Ca2+-dependent component is visible at lower K+ stimulus levels such as 20 mM KCl (Figs. 8 and 9) or at low-level receptor-mediated stimulation with 0.1 µM U-46619 (Fig. 8). In response to hypoxia, both freshly dissected coronary artery and those stored at 4°C (control) decrease [Ca2+]i when activated at low levels (Fig. 10). However, after 24 h in organ culture, both the decrease in [Ca2+]i (Table 1) and the decrease in force (Fig. 8) in response to hypoxia are lost. A reduction in the capacity to lower [Ca2+]i could directly translate into a reduced ability to relax in response to hypoxia in cultured arteries. The fact that relaxation in response to washout of agonist is not affected by organ culture (Fig. 7) suggests that this impairment of Ca2+ handling appears to be specific to hypoxia-induced vasodilation. Our results indicate that both Ca2+-independent and -dependent mechanisms are markedly reduced after organ culture (Fig. 8).

At high stimulus intensities, 40 mM KCl or 1 µM U-46619, little change in [Ca2+]i occurs during acute hypoxic challenge in fresh or control arteries despite a significant decrease in force (Fig. 8, lanes 1 and 2). In cultured arteries under these high-intensity stimulus conditions, the relaxation response to hypoxia is severely inhibited (Fig. 8). Our results thus indicate that both Ca2+-independent and -dependent mechanisms of the relaxation response to hypoxia are markedly reduced after organ culture (Fig. 8).

Organ culture elicits a very specific reduction in the relaxation response to hypoxia, which may help delimit the underlying O2-sensing mechanisms. As the mechanisms regulating smooth muscle contraction are complex, mechanisms of hypoxic relaxation would be expected to be dependent on the nature of the stimulus. It is of interest to note that with KCl stimulation, the hypoxia relaxation in control arteries is attenuated at high stimulus levels (40 mM) relative to low (20 mM). This suggests that K+ channels, some classes of which are known to be sensitive to oxygen tension (15, 22), may be involved, because their effects would be expected to decrease with increasing KCl depolarization. The loss of the hypoxia-induced decrease in [Ca2+]i could reflect a loss of K+ channels or Ca2+ channels, which have also been shown to be oxygen sensitive (8a). This would be consistent with the reduction of the hypoxic relaxation after cycloheximide treatment in control arteries (Fig. 9).

With U-46619 stimulation, one might anticipate that both Ca2+-dependent mechanisms, as do K+ channel activation and Ca2+-independent mechanisms, could play a role in hypoxic relaxation. This would be expected particularly at low intensity (0.1 µM U-46619), for which decreases in [Ca2+]i with hypoxia have been observed. But the magnitude of hypoxic relaxation at 0.1 and 1 µM was similar (Fig. 8), thus the effects are not additive in any simple fashion. This is not surprising, because relaxation is complex, particularly with U-46619, when Ca2+ sensitization of the contractile elements is likely (14).

There is also a change in the Ca2+-independent component of relaxation in response to hypoxia. Again, this could be due to an decrease or increase in some factor(s) not involved in Ca2+ handling, but the evidence here suggests the loss of function by degradation. Many genes and proteins change during 24 h of organ culture of VSM (28). Presumably an alteration in contractile proteins, heat shock proteins, or redox proteins could make the tissue less responsive to hypoxia, directly or indirectly. With the recent development of molecular biology techniques such as subtraction cDNA libraries and proteomics for identification of differential mRNA or protein changes, the specific loss of hypoxic relaxation may provide a new approach to elucidating the mechanism(s) of hypoxic vasorelaxation.

In this investigation summary, our results show that hypoxic vasorelaxation is lost after 24 h in organ culture at 37°C, while contractility and other relaxation pathways are unaffected. Thus organ culture may provide a tool for identifying new targets for understanding the elusive mechanisms underlying this important physiological response.


    FOOTNOTES

Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0576.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 June 2000; accepted in final form 12 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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