Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2000;86:862-870

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Methods
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shimizu, S.
Right arrow Articles by Paul, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shimizu, S.
Right arrow Articles by Paul, R. J.
Related Collections
Right arrow Other Vascular biology
Right arrow Calcium cycling/excitation-contraction coupling
Right arrow Cell signalling/signal transduction
Right arrow Energy metabolism
Right arrow Ischemic biology - basic studies
(Circulation Research. 2000;86:862.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Effects of Hypoxia on Isometric Force, Intracellular Ca2+, pH, and Energetics in Porcine Coronary Artery

Shunichi Shimizu, Peggy Sue Bowman, George Thorne, III, Richard J. Paul

From the Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio.

Correspondence to Dr Richard Paul, Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, 231 Bethesda St, Cincinnati, OH 45267-0576. E-mail Richard.Paul{at}uc.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—When exposed to hypoxic conditions, coronary arteries dilate, which is an important protective response. Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we tested the major classes of hypotheses by measuring the effects of hypoxia on energetics, [Ca2+]i, K+ channel function, and pHi. Hypoxia relaxes porcine coronary arteries stimulated with either KCl or U46619. The extent of relaxation is dependent on both the degree and kind of stimulation. [Ca2+]i was measured in endothelium-denuded arteries using fura 2-AM and ratiometric fluorescent techniques. At lower stimulus levels, hypoxia decreased both force and [Ca2+]i. Inhibitor studies suggest that KCa and KATP channels are not involved in the hypoxic relaxation, whereas KV channels may play a minor role, if any. Despite the hypoxia-mediated decrease in force, [Ca2+]i was unchanged or increased at high levels of stimulation. Despite a marked increase in lactate content, pHi (measured with the ratiometric fluorescent dye BCECF) was also little affected by hypoxia. Measurement of the phosphagen and metabolite profile of freeze-clamped arteries with analytical isotachophoresis indicated that hypoxia increased lactate content by 4-fold and decreased phosphocreatine to 60% of control. However, neither ATP nor Pi was affected by hypoxia. Interestingly, additional stimulation under hypoxia increased force but not ATP utilization, as estimated from measurements of anaerobic lactate production. Thus, surprisingly, the economy of force maintenance is increased under hypoxia. In porcine coronary artery, both Ca2+-dependent and, importantly, Ca2+-independent mechanisms are involved in hypoxic vasodilatation. For the latter, mechanisms involving either ATP, [Ca2+]i, pHi, or Pi cannot be invoked. This novel oxygen sensing mechanism involves a decreased Ca2+ sensitivity.


Key Words: coronary arteries • hypoxia • pH • Ca2+ • metabolism


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The sensitivity of vascular contractility to PO2 has been an important subject among physiologists for more than 100 years.1 2 3 Although there is little question that PO2 affects vascular reactivity, the mechanisms have been the subject of much investigation. Given that pulmonary vessels contract, but most systemic vessels relax when exposed to hypoxia, the mechanisms are likely to be diverse. Hypoxia elicits a relaxation in coronary arteries that may be a physiologically protective response. Many theories have been put forth; however, they can be roughly divided into those involving energy limitation and those involving interruption of excitation-contraction coupling.

The first class involves some form of limitation on ATP synthesis. Relaxation is attributable to the inability of cellular ATP production under hypoxia to support the actin-myosin ATPase activity and hence contractile activity. This could be simply because of an inherently low glycolytic capacity for ATP synthesis under hypoxic conditions, or the capacity could be compromised; for example, acidic pH associated with hypoxia could inhibit glycolysis. It is also possible that regulatory sites for oxidative or anaerobic metabolism, such as phosphofructokinase, may be oxygen sensing modulatory elements.4 We have reported that an energy limitation mechanism is likely to underlie hypoxic relaxation observed in guinea pig taenia coli5 6 ; however, the available evidence does not support an energy limitation mechanism for the oxygen sensitivity of vascular smooth muscle. Pittman and Duling7 reported that contractility was depressed only when the smooth muscle cells in the core of a hypoxic artery became anoxic, suggesting that vascular sensitivity to PO2 could involve an energy limitation. However, because PO2 for arterioles would be unlikely to reach these low levels, a direct role for oxygen in situ could not be supported. There is also evidence that vascular reactivity of both large and small arteries is depressed for PO2s in the 20 to 100 torr range,2 8 which is higher than the range generally associated with inhibition of mitochondrial oxidative phosphorylation. Evidence for an oxygen sensing mechanism in vessels independent of mitochondrial metabolism was also provided by Coburn,9 who reported that even after inhibition of respiration by cyanide, contractility in rabbit aorta could still be depressed by hypoxia. More recent studies4 10 indicate that hypoxic relaxation is not simply a function of energy stores, and oxidative metabolism-contraction coupling is regulated by energy delivery to a reaction- or reactions-controlling muscle force.

An alternative class of hypotheses involves oxygen sensing mechanisms that would modulate the cell signaling pathways involved in excitation-contraction coupling, including mechanisms in which [Ca2+]i is the regulated parameter, for example by oxygen-dependent or ATP-dependent changes in Ca2+ permeability.11 12 A hypothesis of this class with considerable experimental support is that lowered [Ca2+]i concomitant with hyperpolarization is attributable to activation of K+ channels.13 14 Alternatively, evidence for signaling changes at constant [Ca2+]i is accumulating.3 5 Mechanisms involving altered Ca2+ sensitivity of the contractile apparatus, such as those potentially associated with pHi changes with hypoxia, would be in this category. These categories are not necessarily mutually exclusive.

Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we investigated the effects of hypoxia on energetics and [Ca2+]i, the central second messenger for control of vascular smooth muscle contractility. In addition, we studied the roles of K+ channels and pHi, major factors known to modulate contractility. In these coronary arteries, both Ca2+-dependent and -independent mechanisms underlie hypoxic vasodilatation.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Preparation of Arterial Rings
Porcine hearts obtained shortly after slaughter were rinsed of blood and placed in a cold (4°C) bicarbonate-buffered physiological salt solution (PSS). The distal portions of the left anterior descending coronary artery were dissected, cleaned of fat and connective tissue, and cut into 5-mm segments with a wall thickness between 300 and 500 µm. The segments were everted and deendothelialized by rubbing gently on filter paper.15

Isometric force measurements were previously described.16 17 Hypoxia was operationally defined as aeration with 95% N2/5% CO2, which decreases the bath PO2 to <1%, as we have reported.18

Intracellular Ca2+ measurements were made with the fluorescent probe fura 2-AM in an apparatus that permitted simultaneous measurements of force and [Ca2+]i, as previously described.19 20 In some experiments, the rings were mounted isometrically on a wire loop that was fitted into a spectrofluorometer cuvette21 ; no differences were noted in the [Ca2+]i values, and the data were averaged.

[Ca2+]i Calibration
The fluorescent intensity at 340-nm excitation was divided by fluorescent intensity 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 29 mmol/L KCl. This protocol was chosen as a general routine over absolute calibration of the fura 2 fluorescence because of the major uncertainties of absolute calibration.21 Using standard calibration procedures,22 we previously reported values of 50.4±17.2 nmol/L and 441±163 nmol/L for [Ca2+]i under baseline and KCl stimulation, respectively.20 21 In this limited range, the 340/380 ratio is nearly linearly dependent on [Ca2+]i, as reported by others.23

Intracellular pH measurements were made with the fluorescence pH indicator dye BCECF, as previously described,21 and calibrated using the high K+–nigericin technique.24 25 26

High-energy phosphagens, metabolites, and lactate production measurements have been previously reported in detail.27 Briefly, artery rings were frozen in liquid N2, pulverized, and mixed with frozen extraction solution (1 vol methanol and 1 vol 2 mmol/L EDTA) at liquid N2 temperature. An LKB analytical isotachophoresis apparatus was used to measure negatively charged metabolites in the thawed solution. Lactate production was measured using standard procedures previously reported in detail.15

Solutions
The PSS contained (in mmol/L) NaCl 122, KCl 4.73, NaHCO3 15, MgCl2 1.19, EDTA 0.02, KH2PO4 1.19, glucose 11.10, and CaCl2 2.50. When aerated with 95% O2/5% CO2, the pH of the PSS was 7.3 to 7.4 at 37°C.

Analysis of Data
The values given are mean±SEM. N values for sample size represent the number of hearts from which arteries were taken. Student’s t test for paired data and standard ANOVA with Bonferroni discrimination for multiple groups were used; a significance level of P<0.05 was chosen for rejection of the null hypothesis.

An expanded Materials and Methods section is available online at http://www.circresaha.org.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effects of Hypoxia on Isometric Force
Figure 1Down shows a typical isometric force response to hypoxia, defined here as aeration of the organ bath with 95% N2/5% CO2 instead of 95% O2/5% CO2. On switching to the N2 gas mixture, PO2 dropped rapidly to zero levels with a half-time of 1.7±0.2 minutes. Exact calibration at these low PO2 levels is difficult; however, at such low external PO2, the oxygen consumption of the artery itself would reduce the tissue level of oxygen to below that required for mitochondrial oxidative phosphorylation (1 to 2 torr) for the bulk of the artery. Isometric force, generated in response to 40 mmol/L KCl, was decreased by hypoxia to a steady state of {approx}50% of the initial value, as previously reported.17 The half-time of this decline of 4.8±0.6 minutes was considerably greater than that of the change in PO2. The response to hypoxia was reversible (Figure 2ADown), with no apparent loss of control-force responses to KCl over several hypoxia and reoxygenation cycles. In Figure 2BDown, the artery was subjected to hypoxia before KCl stimulation. There was a significant relaxation of basal tone, which has been reported in deendothelialized porcine coronary arteries to be {approx}20% of the maximum KCl contracture.15 16 28 Addition of 29 mmol/L KCl under hypoxia elicited a markedly diminished contraction, with maximum force similar to that obtained in the steady state after imposition of hypoxia on a control KCl contracture, as in Figure 2ADown.



View larger version (9K):
[in this window]
[in a new window]
  		Figure 1. Typical response of isometric force in porcine coronary artery to hypoxia; ie, changing the organ bath aeration from 95% O2/5% CO2 to 95% N2/5% CO2. PO2 refers to the output of an oxygen electrode in the bath and calibrated (right axis) as 100%=1 Atm O2. Contraction stimulated with 40 mmol/L KCl.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. Reversibility of the effects of hypoxia on isometric force in porcine coronary artery stimulated with KCl. A, Hypoxic relaxation of KCl contracture. B, Hypoxia decreases basal tone and inhibits force development in response to KCl.

Interestingly, increasing the level of stimulation can override the inhibitory effects of hypoxia. The dependence of the inhibitory effects of hypoxia on isometric force on the level of stimulus is shown in Figure 3Down. Cumulative concentration-response curves for KCl (Figure 3ADown) and receptor-mediated stimulation using U46619, a thromboxane A2 analog (Figure 3BDown), were generated under aerobic and hypoxic conditions. Both stimuli elicit similar levels of maximum isometric force; however, hypoxia clearly has a more pronounced inhibitory effect on the U46619 response. The extent of the inhibition by hypoxia was strongly dependent on the concentration of the stimuli used. For example, at 20 mmol/L KCl, which elicits {approx}80% of the maximum force, hypoxia reduces this response to only 20%. However, increasing KCl to 80 mmol/L increases isometric force under hypoxia to {approx}75% of that under oxygenated conditions. If metabolism were limited by hypoxia, this would be expected to set an absolute limit to force production independent of stimulus intensity. Thus, the effects of stimulus intensity on the inhibition of isometric force by hypoxia argue against an energy limitation hypothesis based on a limited anaerobic metabolic capacity.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Dependence of the inhibitory effects of hypoxia on isometric force on the level of stimulus. Cumulative concentration-response curves were generated with KCl (A) and U46619 (B) under aerobic ({circ}) and hypoxic conditions (•). Maximum isometric forces under control; oxygenated conditions were similar. Data are mean±SEM; n=8.

In addition to energy limitation or mechanisms involving the mitochondrial respiratory electron transport chain29 as the sensing element, a number of potential sites for oxygen sensing have been proposed. They include the Na+ pump,1 eicosanoid pathways,30 31 alterations in plasma membrane electrical properties,32 33 Ca2+ permeability,11 12 hydrogen peroxide formation,34 potentially oxygen free radicals,35 36 37 cGMP,38 and more recently, ATP-dependent K+ channels13 and the inositol phosphate pathway.29 To delimit the potential mechanisms of oxygen sensing in porcine coronary artery, we also screened a number of these potential pathways using similar pharmacological approaches. We have previously reported17 the effects of inhibitors, including ouabain, aminotriazole, superoxide dismutase, catalase, indomethacin, and propranolol, on the response to hypoxia. Although some minor modulatory effects were seen, as might be anticipated, on the whole these agonists had little effect on hypoxic relaxation.

To add to this database, we further tested the effects of chelerythrine (3 µmol/L), an inhibitor of the C-kinase pathway, and LY83583 (3 µmol/L), an inhibitor of the G-kinase pathway. Table 1Down shows that neither inhibitor had significant effects on the force generated by 29 mmol/L KCl or the relaxation to subsequent hypoxia. Thus, within the limitations of a pharmacological approach, the oxygen sensing pathway in porcine coronary artery does not seem to conform to any of the proposed hypotheses.


View this table:
[in this window]
[in a new window]
  		Table 1. Effects of Chelerythrine and LY83583 on Hypoxic Vasorelaxation in Porcine Coronary Artery

Effects of Hypoxia on [Ca2+]i
Central to most theories of hypoxic vasodilatation are mechanisms for decreasing [Ca2+]i. We studied the effects of hypoxia on [Ca2+]i using the ratiometric fluorescent dye fura 2. Figure 4Down shows representative recordings of the effects of hypoxia on [Ca2+]i at high (Figures 4ADown and 4CDown) and low (Figures 4BDown and 4DDown) stimulation intensity. At 40 mmol/L KCl, the 340/380 ratio rapidly increases and then is characterized by a small, slow decline. A decline in [Ca2+]i despite maintained force is characteristic of smooth muscle.39 40 After imposition of hypoxia, [Ca2+]i slightly increases (during which time isometric force is decreasing) and then resumes a slow decline. After 30 minutes of hypoxia, [Ca2+]i is indistinguishable from the level just before the hypoxia intervention. Reoxygenation elicits a small rapid decrease in [Ca2+]i followed by a return to the original small, slow decrease. The rise of [Ca2+]i is less steep with 20 mmol/L KCl than with 40 mmol/L KCl. The effects of hypoxia with 20 mmol/L KCl are considerably different than those with 40 mmol/L KCl. Imposition of hypoxia elicits a rapid and steady decline of {approx}25%, which is reversed on reoxygenation. Average values for these experiments are graphically summarized in Figure 5Down.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Effects of hypoxia on [Ca2+]i at high and low stimulus levels in representative experiments on porcine coronary artery. Ordinate: [Ca2+]i, as monitored by the 340/380 ratio of the absolute fluorescent intensities measured at 510 nm for fura 2-AM-loaded coronary artery excited at 340 and 380 nm. A and B, Stimulation by KCl depolarization. C and D, Stimulation using the receptor-mediated thromboxane A2 analog U46619. Note that hypoxia has relatively little effect on [Ca2+]i in arteries precontracted with high levels of KCl or U46619 (A and C), but hypoxia decreased [Ca2+]i at lower stimulus levels (B and D).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 5. Statistical summary of the effects of hypoxia on [Ca2+]i and isometric force at high and low stimulus levels for KCl (A) and U46619 (B). Data are mean±SEM; n values range from 4 to 13. *P<0.05 for differences between values in normoxia and hypoxia.

Ishida and Honda41 reported that [Ca2+]i was not affected by hypoxia when stimulated with KCl, whereas hypoxia decreased [Ca2+]i for receptor-mediated stimulation in guinea pig aorta. Thus, we measured the effects of hypoxia on [Ca2+]i for U46619 contractures. Typical experiments are shown in Figure 4Up, and the average values are graphically summarized in Figure 5Up. The general results are similar to those with KCl stimulation. At 1 µmol/L U46619, hypoxia increased [Ca2+]i by 23% but force decreased by 50%. At 0.1 µmol/L U46619, hypoxia decreased both force ({approx}80%) and [Ca2+]i ({approx}50%).

Role of K+ Channels in the Ca2+-Dependent Hypoxic Vasodilatation
Activation of K+ channels is one of the more extensively studied hypotheses for hypoxic vasodilatation. This hypothesis involves K+ channel activation, hyperpolarization, and consequent closing of voltage-dependent Ca2+ channels.13 Because [Ca2+]i was decreased by hypoxia at low levels of stimulation, the potential for K+ channel involvement was suggested. We used a pharmacological approach to identify whether K+ channels were involved in the low-stimulus hypoxic vasodilatation. Arteries were contracted with U46619 (0.1 µmol/L) and treated with K+ channel inhibitors before exposure to hypoxia. As with any pharmacological approach, the specificity of K+ channel inhibitors is open to interpretation. We made the following operational assignments in our investigation based on the literature42 and our recent studies on K+ channels in porcine coronary artery43 : KCa channel inhibition, tetraethylammonium (TEA) (1 mmol/L), apamin (1 µmol/L), and charybdotoxin (0.1 µmol/L); KV channel inhibition, 4-aminopyridine (4-AP) (1 mmol/L); KATP channel inhibition, glibenclamide (10 µmol/L); KCa and KV, TEA (10 mmol/L), KIR and KATP, BaCl2 (10 µmol/L); non-specific, tetrabutylammonium (TBA) (5 mmol/L). The effects of these K+ channel inhibitors on isometric force in response to U46619 and subsequent hypoxia are summarized in Table 2Down. Inhibition of KATP or KCa channels was associated with only small effects. This is somewhat surprising because the literature13 suggests that KATP channels could be an important mediator of hypoxic vasodilatation. However, our results are consistent with a recent study from our laboratory indicating that KV channels are the predominant K+ channels in porcine coronary artery.43 TBA was associated with a significant reduction (60%) of the response to hypoxia. More specific inhibition of KV channels with 4-AP was associated with a small (<20%) but significant increase in force and a moderate (15%) reduction in the relaxation to hypoxia.


View this table:
[in this window]
[in a new window]
  		Table 2. Effects of K+ Channel Inhibitors on Hypoxic Vasorelaxation in U46619 Contractures in Porcine Coronary Artery

Mechanisms of Ca2+-Independent Hypoxic Vasodilatation
The data in Figure 5Up demonstrate that at high levels of stimulation, hypoxia decreases force with a little change or increase in [Ca2+]i. To further demonstrate a Ca2+-independent relaxation, we attempted to maximize [Ca2+]i by exposure of the arteries to KCl (40 mmol/L), added ionomycin (1 µmol/L), a Ca2+ ionophore, and finally added U46619 (1 µmol/L) to promote sarcoplasmic reticulum Ca2+ release. Data from these experiments, graphically summarized in Figure 6Down, show that isometric force under normoxic conditions increased to 150% of a KCl contracture by the cumulative treatment with ionomycin and U46619. Even under these conditions to fix [Ca2+]i at maximal levels, hypoxia was still able to elicit a substantial relaxation. In light of this Ca2+-independent relaxation to hypoxia, we investigated pHi and energy metabolism, 2 alternative mechanisms proposed for hypoxic vasodilatation.5 6



View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. Effects of hypoxia on isometric force under stimulus conditions designed to maximize [Ca2+]i by cumulative treatment of 40 mmol/L KCl contracture with ionomycin (1 µmol/L), then U46619 (1 µmol/L). A relaxation to hypoxia was observed in all cases (P<0.05) implicating Ca2+-independent mechanisms of hypoxic vasodilatation. Note that rightmost 3 bars are the decrease in force with hypoxia, whereas the others are absolute force values. Data are mean±SEM; n=4.

Effects of Hypoxia on pHi
Hypoxia is known to increase both lactate content (see below) and lactate production in porcine coronary artery.44 Moreover, we have reported that acidic pHi is associated with an inhibition of force in coronary arteries.21 Thus, it is plausible that the inhibition of force by hypoxia could be attributed to an acidification of pHi. We measured the effects of hypoxia on pHi using the ratiometric fluorescent dye BCECF, shown in Figure 7Down. As previously reported,21 KCl addition had only minor effects on pHi despite the known increase in lactate production.45 Similarly, imposition of hypoxia did not acidify, as might be anticipated, because of the significant increase in lactate production (see below). The trend with hypoxia is alkalinization, although the differences were not statistically significant. Because alkalinization was shown to increase force in previous studies,21 the effects of hypoxia on pHi are unlikely to play any role in the observed Ca2+-independent relaxation.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 7. Effects of hypoxia on pHi for KCl contracture in porcine coronary artery. pHi was measured using the dye BCECF and ratiometric fluorometry. Dark line represents the average for 3 arteries; gray areas represent SEM.

Effects of Hypoxia on Phosphagen Content and Metabolite Profile
To gain more insight into the mechanisms of hypoxic vasorelaxation, we measured the phosphagen content and metabolite profile in freeze-clamped coronary arteries using isotachophoresis.27 The results are graphically summarized in Figure 8Down. As previously reported for carotid artery, stimulation of maximum isometric force with KCl was associated with little change in high-energy phosphagens or other metabolites. Hypoxia, as might be expected, was associated with a 3-fold increase in lactate content. The only other measured change was a decrease of {approx}50% in phosphocreatine (PCr) content. Significantly, there was no change in ATP or Pi.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 8. Effects of hypoxia on high-energy phosphagens (ATP and PCr), Pi, and lactate of porcine coronary artery. Bar graph shows mean±SEM; n=12. Open bars, Unstimulated, oxygenated conditions. Shaded bars, KCl stimulation, oxygenated conditions. Filled bars, KCl stimulation, hypoxia. *P<0.001.

As shown in Figure 9ADown, addition of U46619 (1 µmol/L) reverses the hypoxic relaxation of a KCl (29 mmol/L) contracture. This large increase in force under hypoxic conditions was not associated with a proportional increase in [Ca2+]i (Figure 9BDown). The order of stimulation was not important, because adding KCl reversed the effects of hypoxia on a U46619 contracture (88.7±5.5% of normoxic force, n=4, not significant). These data would again support the hypothesis that metabolic ATP production is not limiting, for presumably there is additional ATP mobilized to support the higher level of isometric force. In general, ATP utilization is proportional to the level of isometric force.46



View larger version (19K):
[in this window]
[in a new window]
  		Figure 9. Effects of addition of U46619 (1.0 µmol/L) to a KCl (29 mmol/L) contracture under hypoxia for a representative artery, isometric force (A), and [Ca2+]i (B). Under hypoxia, addition of U46619 to a KCl contracture was associated with a significant increase in isometric force, without a proportionate increase in [Ca2+]i.

It is possible that the increase in force with additional stimulation under hypoxia could be accomplished by increasing the force per ATP utilization (economy) rather than increasing ATP utilization at a constant economy. Thus, from an energetics viewpoint, it is of particular interest to know what, if any, changes in metabolism were associated with the increase in force observed when U46619 was added to the KCl contracture under hypoxic conditions (Figure 10Down).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 10. Statistical summary of the effects on isometric force, rate of lactate production (Jlac), and high-energy phosphagens (ATP and PCr) of addition of U46619 (0.1 µmol/L) to a KCl (29 mmol/L) contracture under hypoxia. Light shaded bars, Mean values with KCl stimulation. Dark shaded bars, Mean values with KCl+U46619. Bar graph shows mean±SEM; all paired differences were significant at P<0.5; n=8 to 11. Despite the maintenance of higher forces with addition of U46619 than with KCl alone, Jlac was lower, indicating an increase in economy under hypoxic conditions.

Under these hypoxic conditions ({approx}1% O2), anaerobic glycolysis is virtually the only source for production of ATP. The rate of lactate production (Jlac) was equal to that measured in the bath, and no significant differences in tissue lactate content were found. Under hypoxia with KCl stimulation (Figure 10Up), Jlac was {approx}4 times the rate of oxygen consumption.47 This would suggest that the capacity for anaerobic glycolysis is adequate to stoichiometrically replace the ATP attributable to oxidative metabolism. After the addition of U46619 under hypoxia, force increased and remained elevated for the entire 60-minute measurement period at levels similar to the original force in the presence of KCl under aerobic conditions. Jlac did not increase with U46619 treatment, but in fact was somewhat lower, although not significantly (P=0.08), than that observed in KCl alone under hypoxia. This was surprising, because an increase in glycolytic ATP production would have been predicted to match the increase in force elicited by U46619. The ATP and PCr content measured in freeze-clamped arteries was reduced by the end of the measurement (P<0.05). However, the decrease of ATP was inconsequential when compared with the ATP produced over 60 minutes by aerobic glycolysis ({approx}60x Jlac). Thus, it would seem that hypoxia not only decreases the Ca2+ sensitivity (ie, less force at similar [Ca2+]i concentrations) but also can alter the relation between force production and ATP utilization (ie, less ATP utilization per unit force maintained).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Hypoxia inhibits force development and relaxes force in precontracted porcine coronary artery. This is likely a physiologically protective response to increase blood flow under stress conditions. Our understanding of the mechanisms is complicated by our incomplete knowledge of the regulation of smooth muscle.48 49 It is generally accepted that [Ca2+]i is the major second messenger, acting via binding to calmodulin and subsequent activation of myosin light chain kinase. There is evidence suggesting a role for modulatory mechanisms, including those linked to the thin filament, such as caldesmon50 and calponin.51 In addition, there is a growing body of evidence suggesting that the Ca2+ sensitivity of the contractile system is also a parameter subject to modulation.20 49 Metabolism is also known to be closely linked to contractile function in smooth muscle46 ; however, its role as a regulatory mechanism is less well-defined.

A most striking observation in these studies is that the relaxation or inhibition of force associated with hypoxia could be ascribed to at least two different mechanisms. At low stimulus levels, the inhibition of force was accompanied by a decrease in [Ca2+]i. At high stimulus levels, hypoxia decreased force in the presence of constant or increasing [Ca2+]i. In our conditions, hypoxia (bubbling with N2) was such that bath PO2 was <1%, and the tissue PO2 was likely below that for mitochondrial oxidative phosphorylation throughout. In physiological terms, this is severe hypoxia, but it does enable one to set defined conditions for the tissue as a whole, which is useful to separate Ca2+-dependent and -independent mechanisms. The dependence of these mechanisms on the PO2 is currently under investigation and will be important to understanding tissue oxygen sensing, which can be observed at considerably higher PO2.52

The decrease in [Ca2+]i at low stimulus levels was not affected by inhibitors of KATP or KCa channels. There was a moderate inhibition ({approx}15%) of the hypoxic relaxation associated with inhibition of KV channels. These results are consistent with a recent study from our laboratory indicating that KV channels are the predominant K+ channels in porcine coronary artery.43 Interpretation of these data, however, is not straightforward, because the specificity of KV channel inhibitors is not absolute. In addition, 4-AP and TEA (but not TBA) also increased the force (<20%) before exposure to hypoxia, which could also account for some of the reduction in the relaxation to hypoxia.

A direct effect of hypoxia on Ca2+ channels11 12 or Ca2+ uptake or extrusion systems, such as enhanced Na+-Ca2+ exchange,1 cannot be ruled out. These arteries are able to relax to hypoxia after inhibition of the sarcoplasmic reticulum Ca2+ pump with cyclopiazonic acid (G.T. and R.J.P., unpublished observations, 1999). Thus, a major role for the sarcoplasmic reticulum in hypoxic relaxation is unlikely in these arteries.

The Ca2+-independent relaxation to hypoxia was observed for both receptor-mediated stimulation and depolarization, as long as the intensity was high. A Ca2+-independent pathway has been suggested for several smooth muscles.3 The Ca2+-independent effects of hypoxia are by definition a decrease in Ca2+ sensitivity; ie, a lower force maintained under hypoxic conditions was not associated with a change in [Ca2+]i.

A change in pHi would be the most obvious choice as an effector of Ca2+ sensitivity. It is known that pHi can modify, although not dramatically, the Ca2+ sensitivity in permeabilized preparations.53 Our studies on the effects of pHi on intact coronary artery21 in fact show a larger dependence of force on pHi than would be predicted from studies on permeabilized smooth muscle. One might predict an inhibition of force caused by a reduction of pHi under hypoxic conditions, based on the increases in lactate production previously reported44 and specifically shown here (Figures 8Up and 10Up). This would be consistent with the inhibition of force seen in response to the acidification elicited by removal of NH4Cl.21 However, our studies indicate that pHi is not affected by hypoxia. This finding, although initially surprising, is consistent with other studies on smooth muscle showing that hypoxia and cyanide do not affect pHi measured with nuclear magnetic resonance methodology54 or weak acid distribution.55 The increased acid load may be partially offset by the decrease in PCr, which would exert an alkalinizing effect. Although it is an attractive hypothesis, our results indicate that the hypoxic relaxation and the change in Ca2+ sensitivity observed here for porcine coronary artery cannot be attributed to alterations in pHi.

To further circumscribe the mechanisms of the inhibition of force by hypoxia, we measured its effects on the high-energy phosphagen and metabolite profile. Hypoxia was characterized by a significant decrease in PCr to {approx}60% of control; ATP, however, was not altered. These values are similar to those reported56 for porcine femoral artery, measured during 6 hours of hypoxia using nuclear magnetic resonance techniques. The only other significant change in metabolite content seemed to be an increase in lactate content, as expected. Mechanisms that are dependent on decreasing ATP content as the signal for vasorelaxation, such as those involving ATP-dependent K+ channels, would seem to be ruled out by these measurements. Alternatively, measurement of bulk ATP may not reflect changes in the localized compartments involved, such as a purported subsarcolemmal region that may be distinct from the cytosol. Similar comments would apply to Pi, which has been associated with the effects of hypoxia on striated muscle57 but did not change with hypoxia in our experiments (Figure 8Up).

In a related set of experiments, we observed that although hypoxia inhibited force, increasing stimulation could override this inhibition. As shown in Figure 9Up, addition of U46619 to KCl-stimulated arteries under hypoxia (or vice versa) restored force to near initial levels. No major changes in high-energy phosphagens from those of hypoxia alone were measured (Figure 10Up). Thus, changes in ATP levels do not seem to be a major signaling pathway.

To further investigate the effects of hypoxia on energetics, we measured the rate of ATP production by arterial lactate production in parallel experiments of similar design. Because changes in high-energy phosphagen were negligible, the ATP production is equivalent to utilization. Interestingly, whereas U46619 increased force under hypoxia, there was no parallel increase in Jlac (Figure 10Up). Thus, hypoxia seems to affect the coupling between force production and ATP utilization. This may simply mean that ATP was diverted to the myosin ATPase from other processes. It is also possible that the crossbridge cycle might be modified under hypoxic conditions. Because PCr was decreased by hypoxia, the calculated free ADP levels would increase, assuming that the creatine kinase reaction was in equilibrium.58 Thus, it is plausible that changes in [ADP] may underlie hypoxic relaxation. One might anticipate that increasing ADP could affect the crossbridge detachment rate, which may also underlie the change in energetics. Changes in [ADP] are known to alter the crossbridge cycle rate in smooth muscle, but the effective concentration ranges seem to be too high59 or too low60 based on the calculated [ADP] in vascular smooth muscle.

It is also plausible that changes in myosin regulatory light chain phosphorylation (MLC-Pi) may be the regulated step for hypoxic relaxation. Coburn et al4 reported that hypoxia decreased MLC-Pi in receptor-mediated contractures but was less affected in KCl contractures in rabbit aorta. Because ATP was not unaffected, presumably myosin light chain kinase activity would not be the site for the Ca2+-independent oxygen sensing. There is evidence that myosin phosphatase may be a site for MLC-Pi regulation,61 which may involve a G protein and C kinase. Within the scope of limited pharmacological data, our negative results with chelerythrine lessen the likelihood for the involvement of C kinase in the mechanism of hypoxic relaxation. However, oxygen sensing at the level of MLC-Pi remains to be tested.

In conclusion, hypoxic vasorelaxation in porcine coronary arteries involves both Ca2+-dependent and -independent mechanisms. The hypoxic vasodilatation cannot be attributed to changes in pHi, Pi, or ATP. Under hypoxia, Ca2+ sensitivity is decreased but the force per ATP seems to be increased. Large coronary arteries can play a significant role in the regulation of coronary circulation.62 The relaxation induced by hypoxia in coronary arteries likely plays a protective role in coronary ischemia. Our results rule out many of the current theories for hypoxic relaxation, but the sites for this important oxygen sensing mechanism remain to be elucidated.


*    Acknowledgments
 
This work was supported in part by the National Institutes of Heath, National Heart, Lung, and Blood Institute (HL23240), and the American Heart Association (OH-SW-91-13; National 92007130).

Received August 31, 1999; accepted February 29, 2000.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Detar R. Mechanism of physiological hypoxia-induced depression of vascular smooth muscle contraction. Am J Physiol. 1980;238:H761–H769.[Free Full Text]
  2. Jackson WF. Arteriolar oxygen reactivity: where is the sensor? Am J Physiol. 1987;253:H1120–H1126.[Abstract/Free Full Text]
  3. Taggart MJ, Wray S. Hypoxia and smooth muscle function: key regulatory events during metabolic stress. J Physiol (Lond). 1998;509:315–325.[Abstract/Free Full Text]
  4. Coburn RF, Moreland S, Moreland RS, Baron CB. Rate-limiting energy-dependent steps controlling oxidative metabolism-contraction coupling in rabbit aorta. J Physiol (Lond). 1992;448:473–492.[Abstract/Free Full Text]
  5. Ishida Y, Paul RJ. Effects of hypoxia on high-energy phosphagen content, energy metabolism and isometric force in guinea-pig taenia caeci. J Physiol (Lond). 1990;424:41–56.[Abstract/Free Full Text]
  6. Obara K, Bowman PS, Ishida Y, Paul RJ. Effects of hypoxia on [Ca2+]i, pHi and myosin light chain phosphorylation in guinea-pig taenia caeci. J Physiol (Lond). 1997;503:427–433.[Medline] [Order article via Infotrieve]
  7. Pittman RN, Duling BR. Oxygen sensitivity of vascular smooth muscle, I: in vitro studies. Microvasc Res. 1973;6:202–211.[Medline] [Order article via Infotrieve]
  8. Chang AE, Detar R. Oxygen and vascular smooth muscle contraction revisited. Am J Physiol. 1980;238:H716–H728.[Abstract/Free Full Text]
  9. Coburn RF. Oxygen tension sensors in vascular smooth muscle. Adv Exp Med Biol. 1977;78:101–115.[Medline] [Order article via Infotrieve]
  10. Scott DP, Davidheiser S, Coburn RF. Effects of elevation of phosphocreatine on force and metabolism in rabbit aorta. Am J Physiol. 1987;253:H461–H465.[Abstract/Free Full Text]
  11. Franco-Obregon A, Urena J, Lopez-Barneo J. Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation. Proc Natl Acad Sci U S A. 1995;92:4715–4719.[Abstract/Free Full Text]
  12. Lorenz JN, Paul RJ. Dependence of Ca2+ channel currents on endogenous and exogenous sources of ATP in portal vein smooth muscle. Am J Physiol. 1997;272:H987–H994.[Abstract/Free Full Text]
  13. Daut J, Maier-Rudolph W, von Beckerath N, Mehrke G, Gunther K, Goedel-Meinen L. Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels. Science. 1990;247:1341–1344.[Abstract/Free Full Text]
  14. Yuan XJ, Goldman WF, Tod ML, Rubin LJ, Blaustein MP. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol. 1993;264:L116–L123.[Abstract/Free Full Text]
  15. Ngai JH, Roth PS, Paul RJ. Effects of endothelium on basal tone and agonist and O2 sensitivity in porcine coronary artery. J Pharmacol Exp Ther. 1990;252:1053–1059.[Abstract/Free Full Text]
  16. Rubanyi G, Paul RJ. Two distinct effects of oxygen on vascular tone in isolated porcine coronary arteries. Circ Res. 1985;56:1–10.[Abstract/Free Full Text]
  17. Close LA, Bowman PS, Paul RJ. Reoxygenation-induced relaxation of coronary arteries: a novel endothelium-dependent mechanism. Circ Res. 1994;74:870–881.[Abstract/Free Full Text]
  18. Hashimoto M, Close LA, Ishida Y, Paul RJ. Dependence of endothelium-mediated relaxation on oxygen and metabolism in porcine coronary arteries. Am J Physiol. 1993;265:H299–H306.[Abstract/Free Full Text]
  19. Tosun M, Paul RJ, Rapoport RM. Coupling of store-operated Ca++ entry to contraction in rat aorta. J Pharmacol Exp Ther. 1998;285:759–766.[Abstract/Free Full Text]
  20. Bowman P, Haikala H, Paul RJ. Levosimendan, a calcium sensitizer in cardiac muscle, induces relaxation in coronary smooth muscle through calcium desensitization. J Pharmacol Exp Ther. 1999;288:316–325.[Abstract/Free Full Text]
  21. Nagesetty R, Paul RJ. Effects of pHi on isometric force and [Ca2+]i in porcine coronary artery smooth muscle. Circ Res. 1994;75:990–998.[Abstract/Free Full Text]
  22. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450.[Abstract/Free Full Text]
  23. Mitsui M, Nakao K, Inukai T, Karaki H. Inhibitory effects of cadralazine and its metabolite, ISF-2405, on contractions and the level of cytosolic Ca2+ in vascular smooth muscle. Eur J Pharmacol. 1990;178:171–177.[Medline] [Order article via Infotrieve]
  24. Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979;18:2210–2218.[Medline] [Order article via Infotrieve]
  25. Krampetz IK, Rhoades RA. Intracellular pH: effect on pulmonary arterial smooth muscle. Am J Physiol. 1991;260:L516–L521.[Abstract/Free Full Text]
  26. Motley ED, Paul RJ, Matlib MA. Role of Na+-Ca2+ exchange in the regulation of vascular smooth muscle tension. Am J Physiol. 1993;264:H1028–H1040.[Abstract/Free Full Text]
  27. Krisanda JM, Paul RJ. Phosphagen and metabolite content during contraction in porcine carotid artery. Am J Physiol. 1983;244:C385–C390.[Abstract/Free Full Text]
  28. Cohen RA, Zitnay KM, Weisbrod RM, Tesfamariam B. Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine. J Pharmacol Exp Ther. 1988;244:550–555.[Abstract/Free Full Text]
  29. Coburn RF, Fillers WS. Oxidative metabolism-contraction coupling in smooth muscle. Prog Clin Biol Res. 1989;315:391–404.[Medline] [Order article via Infotrieve]
  30. Kalsner S. Intrinsic prostaglandin release. A mediator of anoxia-induced relaxation in an isolated coronary artery preparation. Blood Vessels. 1976;13:155–166.[Medline] [Order article via Infotrieve]
  31. Rubanyi G, Paul RJ. O2-sensitivity of ß-adrenergic responsiveness in isolated bovine and porcine coronary arteries. J Pharmacol Exp Ther. 1984;230:692–8.[Abstract/Free Full Text]
  32. Hellstrand P, Johansson B, Norberg K. Mechanical, electrical, and biochemical effects of hypoxia and substrate removal on spontaneously active vascular smooth muscle. Acta Physiol Scand. 1977;100:69–83.[Medline] [Order article via Infotrieve]
  33. Roulet MJ, Coburn RF. Oxygen-induced contraction in the guinea pig neonatal ductus arteriosus. Circ Res. 1981;49:997–1002.[Abstract/Free Full Text]
  34. Burke TM, Wolin MS. Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol. 1987;252:H721–H732.[Abstract/Free Full Text]
  35. Kontos HA. Oxygen radicals from arachidonate metabolism in abnormal vascular responses. Am Rev Respir Dis. 1987;136:474–477.[Medline] [Order article via Infotrieve]
  36. Pieper GM, Gross GJ. Oxygen free radicals abolish endothelium-dependent relaxation in diabetic rat aorta. Am J Physiol. 1988;255:H825–H833.[Abstract/Free Full Text]
  37. Rubanyi GM, Vanhoutte PM. Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle. Am J Physiol. 1986;250:H815–H821.
  38. Coburn RF, Eppinger R, Scott DP. Oxygen-dependent tension in vascular smooth muscle: does the endothelium play a role? Circ Res. 1986;58:341–347.[Abstract/Free Full Text]
  39. Murphy RA. Contraction in smooth muscle cells. Annu Rev Physiol. 1989;51:275–283.[Medline] [Order article via Infotrieve]
  40. Morgan KG, Bradley A, DeFeo TT. Calcium transients in smooth muscle. Ann N Y Acad Sci. 1988;522:328–337.[Medline] [Order article via Infotrieve]
  41. Ishida Y, Honda H. Underlying mechanisms for hypoxia-induced relaxation of the guinea-pig isolated aorta. Jpn J Pharmacol. 1992;58(suppl 2):307P.
  42. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol. 1995;268:C799–C822.[Abstract/Free Full Text]
  43. Shimizu S, Yokoshiki H, Sperelakis N, Paul RJ. Role of voltage-dependent and Ca2+-activated K+ channels on the regulation of isometric force in porcine coronary artery. J Vasc Res. 2000;37:16–25.[Medline] [Order article via Infotrieve]
  44. Ishida Y, Hashimoto H, Paul RJ. Does a limitation of energy supply to the contractile apparatus underlie the relaxation induced by hypoxia in smooth muscle. Prog Clin Biol Res. 1987;245:463–464.
  45. Paul RJ, Bauer M, Pease W. Vascular smooth muscle: aerobic glycolysis linked to sodium and potassium transport processes. Science. 1979;206:1414–1416.[Abstract/Free Full Text]
  46. Paul RJ. The chemical energetics of vascular smooth muscle: intermediary metabolism and its relation to contractility. In: Bohr DF, Somlyo AP, Sparks HV, eds. Handbook of Physiology. Bethesda, Md: American Physiological Society; 1980:201–235.
  47. Close LA, Bowman PS, Paul RJ. Effects of endothelium on metabolic and mechanical parameters in porcine coronary artery. Endothelium. 1994;1:317–323.
  48. de Lanerolle P, Paul RJ. Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am J Physiol. 1991;261:L1–L14.[Abstract/Free Full Text]
  49. Horowitz A, Menice CB, Laporte R, Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev. 1996;76:967–1003.[Abstract/Free Full Text]
  50. Sobue K, Muramoto Y, Fujita M, Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981;78:5652–5655.[Abstract/Free Full Text]
  51. Winder SJ, Walsh MP. Smooth muscle calponin: inhibition of actomyosin MgATPase and regulation by phosphorylation. J Biol Chem. 1990;265:10148–10155.[Abstract/Free Full Text]
  52. Fredricks KT, Liu Y, Rusch NJ, Lombard JH. Role of endothelium and arterial K+ channels in mediating hypoxic dilation of middle cerebral arteries. Am J Physiol. 1994;267:H580–H586.[Abstract/Free Full Text]
  53. Hellstrand P, Arner A. Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pig Taenia coli. Pflugers Arch. 1985;405:323–328.[Medline] [Order article via Infotrieve]
  54. Hellstrand P, Vogel HJ. Phosphagens and intracellular pH in intact rabbit smooth muscle studied by 31P-NMR. Am J Physiol. 1985;248:C320–C329.[Abstract/Free Full Text]
  55. Stephens NL, Kroeger EA, Loh W. Intracellular pH in hypoxic smooth muscle. Am J Physiol. 1977;232:E330–E335.
  56. Leach RM, Sheehan DW, Chacko VP, Sylvester JT. Effects of hypoxia on energy state and pH in resting pulmonary and femoral arterial smooth muscles. Am J Physiol. 1998;275:L1051–L1060.[Abstract/Free Full Text]
  57. Godt RE, Nosek TM. Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. J Physiol (Lond). 1989;412:155–180.[Abstract/Free Full Text]
  58. Clark JF, Dillon PF. Phosphocreatine and creatine kinase in energetic metabolism of the porcine carotid artery. J Vasc Res. 1995;32:24–30.[Medline] [Order article via Infotrieve]
  59. Kerrick WG, Hoar PE. Non-Ca2+-activated contraction in smooth muscle. Prog Clin Biol Res. 1987;245:437–448.[Medline] [Order article via Infotrieve]
  60. Nishiye E, Somlyo AV, Torok K, Somlyo AP. The effects of MgADP on cross-bridge kinetics: a laser flash photolysis study of guinea-pig smooth muscle. J Physiol (Lond). 1993;460:247–271.[Abstract/Free Full Text]
  61. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature. 1994;372:231–236.[Medline] [Order article via Infotrieve]
  62. Young MA, Vatner SF. Regulation of large coronary arteries. Circ Res. 1986;59:579–596.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. L. Wardle, M. Gu, Y. Ishida, and R. J. Paul
Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H23 - H29.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
S. Wray
Insights into the uterus
Exp Physiol, July 1, 2007; 92(4): 621 - 631.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
L. A. Shimoda, T. Luke, J. T. Sylvester, H.-W. Shih, A. Jain, and E. R. Swenson
Inhibition of hypoxia-induced calcium responses in pulmonary arterial smooth muscle by acetazolamide is independent of carbonic anhydrase inhibition
Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L1002 - L1012.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
K. R. Olson, R. A. Dombkowski, M. J. Russell, M. M. Doellman, S. K. Head, N. L. Whitfield, and J. A. Madden
Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation
J. Exp. Biol., October 15, 2006; 209(20): 4011 - 4023.
[Abstract] [Full Text] [PDF]