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1 Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
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Abstract |
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S (1 mM), or in the absence of Ca2+ after treatment with ATPS (1 mM), were of similar magnitude. Hypoxia also relaxed GTPS contractures but importantly, arteries could not be relaxed after treatment with ATPS. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATPS suggests that this Ca2+-independent mechanism(s) may operate through alteration of MRLC-Pi or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 µM) relaxed GTPS and Ca2+ contractures; but the latter required a higher concentration (10 µM) for consistent relaxation. Relaxations to N2 and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca2+-independent O2-sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca2+ is clamped at a constant value.
(Received 11 August 2004;
accepted after revision 28 November 2004;
first published online 25 November 2004)
Corresponding author R. J. Paul: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576, USA. Email: richard.paul{at}uc.edu
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Mechanisms underlying Ca2+-independent hypoxic relaxation have been less extensively investigated. There is now compelling evidence for Ca2+-independent oxygen- sensing in coronary artery (Shimizu et al. 2000) and in other vascular smooth muscles (VSM) (Coburn et al. 1992; Sward et al. 1993 Aalkjaer & Lombard, 1995), as well as other smooth muscle tissues (Obara et al. 1997; Taggart & Wray, 1998). The results of these studies are in general based on ‘global’ measurements of [Ca2+]i, averaging over a large number of cells in whole smooth muscle tissue. An alternative hypothesis is that local changes in Ca2+ not detected by global Ca2+ measurements may underlie this O2 sensing, Recently, much attention has been given to the significance of Ca2+ waves occurring at the single cell level (Ruehlmann et al. 2000). Relaxation due to inhibition of oxidative metabolism, often used as a model for hypoxia-induced vasorelaxation, may result from changes in ‘local’ Ca2+ wave dynamics. These waves could be altered in a manner which may not change the measured global [Ca2+]i and thus account for these apparent Ca2+-independent phenomena (Sward et al. 2002).
Our hypothesis is that a decrease in Ca2+ sensitivity for activation of the contractile apparatus, rather than modulation of local [Ca2+]i, underlies the globally measured, Ca2+-independent component of hypoxic vasorelaxation. There is a growing body of literature indicating that modulation of force at a fixed [Ca2+]i is a common aspect of smooth muscle regulatory mechanisms (Pfitzer, 2001; Somlyo & Somlyo, 2003). Much attention is currently being given to the small G-protein, RhoA, for its association with Ca2+ sensitization of force via activation of Rho kinase in VSM (Somlyo & Somlyo, 2000; Brozovich, 2002; Somlyo & Somlyo, 2003). We have shown that the receptor-mediated contraction to the thromboxane A2 mimetic, U46619, nearly exclusively involves an increase in Ca2+ sensitivity mediated by the RhoA/Rho kinase pathways in porcine coronary artery (Nobe & Paul, 2001). Since this pathway has been implicated in the hypoxia-induced vasoconstriction in rat pulmonary artery (Robertson et al. 2000; Wang et al. 2001), it may well be a potential site for coronary oxygen sensing.
In this investigation we utilize a permeabilized coronary artery preparation to study hypoxic relaxation with fixed global and local Ca2+ concentrations. We demonstrate that relaxation to hypoxia can occur at fixed local Ca2+ concentrations and that hypoxia can reduce the force of GTPS-mediated but not ATPS-mediated activation.
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Methods |
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Adult porcine hearts were obtained immediately after killing from a local slaughterhouse and placed in a cold (4°C) physiological salt solution (PSS). The left anterior descending coronary artery was dissected and cleaned of fat and connective tissue. The artery was cut into 1-mm-wide rings and had a circumference between 6 mm and 10 mm. The rings were mechanically de-endothelialized by gentle rubbing between the thumb and index finger. The rings were mounted on wire hooks with one hook fixed and the other connected to a Harvard Apparatus Capacitance force transducer and placed into a tissue bath (0.5 ml) at 37°C containing MOPS-PSS (pH 7.4) of the following composition (mM): 140 NaCl, 4.7 KCl, 1.2 NaH2PO4, 20 MOPS, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2, 11.0 glucose. The bathing solution was aerated with hydrated air or N2. The aerating gases were hydrated by aerating through a water trap, which also removed any soluble impurities in the gases. Aeration was achieved by directing a stream of gas over the surface of the bath, which was covered by a plastic shield, which reduced evaporation and aided equilibration with the aerating gas.
The rings were equilibrated for 45–60 min. Baseline tension during this period was adjusted to 8 mN, which sets the tissue to the optimal length range for maximum isometric force generation. Following equilibration, rings were stimulated with 80 mM KCl for 10 min for at least two contraction–relaxation cycles until reproducible forces were generated. Test contractions were induced by 30 mM KCl and force was allowed to plateau. After attainment of a steady-state force (at 
15 min post-stimulation), the aeration of the bathing solution was switched from air to 100% N2. The PO2 at which relaxation, occurred previously measured polarographically, was 1–2% (Close et al. 1994; Shimizu et al. 2000). We defined these conditions as ‘hypoxia’. Isometric force was recorded by AcqKnowledge software, a digital data acquisition system (Biopac). The force was normalized to cross-sectional area: F/A
= (change in force x circumference)/(2 x wet weight). The decrease in force in response to hypoxia was characterized in terms of the maximum hypoxic relaxation (at 20 min), expressed as percentage of the isometric force immediately preceding aeration with N2. Similar normalization for maximum force and hypoxic relaxation were used for the permeabilized preparations.
Tissue permeabilization
After validating a hypoxic relaxation for an intact coronary artery, the bath temperature was lowered to 23°C and the artery was permeabilized. The arteries were first calcium depleted in a solution containing (mM) 5 EGTA, 20 imidazole, 50 KCl and 150 sucrose, pH 7.4 for 10 min at room temperature. Then ß-escin (500 µM) or Triton X-100 (5% v/v) was added to the bath and the arteries were incubated for a further 20 min for the former and up to 60 min for the latter. The rings were then equilibrated in a relaxing solution (see below).
Solutions
‘Relaxing solution’ for skinned fibres consisted of (mM): 10 MgCl2, 7.5 Na2ATP, 4 EGTA, 20 imidazole (pH 6.7), 10 phosphocreatine. This nominally Ca2+-free solution had a calculated Ca2+ concentration of <10 nM
‘Ca2+ contracting solution’ for skinned fibres was similar to relaxing solution but also contained (mM): 4.0 CaCl2 with 1.94 free Mg2+, 7.2 MgATP, an ionic strength of 110, and a free Ca2+ of 6.6 µM calculated using a multiple ionic equilibrium program (Godt & Maughan, 1988). ‘Rigor solution’ contained (mM): 2 MgCl2, 4 EGTA, 20 imidazole (pH of 6.7), 50 KCl with an ionic strength of 110. ‘Thiophosphorylating (ATPS) solution’ was similar to contracting solution but excluded ATP and phosphocreatine and included (mM): 1 Na2ATPS and 20 KCl for an ionic strength of 110.
Thiophosphorylating protocol
Permeabilized arteries were subjected to a control contraction–relaxation cycle. This was followed by two short 3 min rinses in rigor solution. They were then incubated for at least 10 min in ATPS solution, rinsed for 2 min in rigor solution, and finally transferred back to relaxing solution.
Myosin regulatory light chain phosphorylation
Four rings from each coronary artery were mounted isometrically, one for force measurements to assess viability, and the other three for myosin regulatory light chain phosphorylation (MRLC-Pi) measurements. At defined times and under specified conditions, tissues were placed in 5% trichloroacetic acid (TCA), 10 mM beta-mercaptoethanol (BME), 95% acetone cooled on dry ice and incubated for 20 min. The frozen fibres were then washed in dry-ice-cooled 5 mM BME–acetone twice, each for 20 min followed by a third wash in the same solution overnight. The segments were minced with fine scissors in extracting solution containing 10 M urea, 2 mM EDTA, 5 mM dithiothreitol, 0.01% bromophenol blue and 20 mM Tris-HCl (pH 7.4). Isoelectric focusing polyacrylamide, glycerol urea gel electrophoresis (IEF-PAGE) using pH 4–6 ampholyte was conducted to separate phosphorylated and unphosphorylated forms of MRLC (Obara et al. 1997). IEF gels were electrophoresed overnight at 300 V followed by 1 h at 500 V at 4°C. The proteins were electrotransferred from the gel to a nitrocellulose membrane in 20% methanol containing 25 mM Tris and 192 mM glycine for 150 min at 120 mA. After transfer, the nitrocellulose membranes were incubated in Tris buffered saline (TBS, composition: 150 mM NaCl, 20 mM Tris, pH 7.5) containing 5% fat-free dried milk for 1 h followed by incubation with 1 µg ml–1 MRLC-specific mouse monoclonal antibodies (Sigma, St Louis, MO, USA) in TBS overnight at 4°C. The membranes were washed in TBS and incubated with peroxidase-conjugated goat antimouse IgG antibodies (1 : 4000 Sigma A90D44) in TBS containing 3% horse serum for 7 h. Signals were detected by using enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Biosciences) and film. The fraction of phosphorylated MRLC was quantified from densitometric scans of ECL film as: (MRLC-P1 + MRLC-P2)/(MRLC + MRLC-P1 + MRLC-P2). MRLC-P1 and MRLC-P2, mono- and di-phosphorylated MRLC, respectively.
Statistical analysis
Data were analysed using the t test for paired two-sample means or one-way ANOVA, using appropriate post hoc tests to determine significance of paired differences. Statistical significance was accepted for P < 0.05. Values are expressed as the mean ± S.E.M. n values represent the number of hearts with one coronary ring used per heart for force, or per treatment for MRLC-Pi measurements.
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Results |
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Hypoxia decreased force in ß-escin permeabilized preparations by 32.2% ± 3.3% (n = 5), which was similar to that for the intact preparation 43.0% ± 5.6%. We also studied a more severe permeabilization using Triton X-100 (Ruegg & Paul, 1982). Force in Ca2+ contraction solution was similar to that after permeabilization with ß-escin (Fig. 4). However, the hypoxic relaxation was decreased to 16.8% ± 1.7% (n = 6) after 40 min and was abolished by a 60 min permeabilization in the Triton X-100-containing solution. This suggested that the integrity of the sarcolemma was critical to preservation of key signalling components of the Ca2+-independent hypoxic relaxation.
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S, a nonhydrolysable form of GTP known to activate the small G-protein RhoA and downstream effectors like Rho kinase (Somlyo & Somlyo, 2003). A typical isometric myogram from these types of experiments is shown in Fig. 2A. The permeabilized coronary artery was initially placed in an EGTA-buffered submaximal Ca2+-containing contraction solution ([Ca2+]
= 0.17 µM), which elicited a small contraction (7.8%
± 2.6%, n
= 5). Then GTPS (1 mM) was added and substantial force developed, typical of the increased Ca2+ sensitivity upon activation of RhoA. Maximum force generation to GTPS was similar to that developed in the Ca2+ contraction solution; a summary of data from these experiments is shown in Fig. 4. Hypoxia caused a rapid and significant reduction of the GTPS-activated force (Fig. 2A), which was of similar magnitude to the relaxation observed in the Ca2+ contraction solution (Fig. 4B). To validate that the contracture elicited by GTPS was mediated by Rho kinase, we used the Rho kinase inhibitor Y27632. As shown in Fig. 2B, Y27632 (1 µM) caused a substantial relaxation.
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S is a substrate for myosin light chain kinase (MLCK), but a poor substrate for the phosphatase (MLCP) leading to near irreversible thiophosphorylated MRLC (Cassidy et al. 1979). We used a thiophosphorylation protocol that involves a Ca2+ contraction solution in which ATPS has been substituted for ATP. After thiophosphorylation, placing the arteries now in a Ca2+-free, ATP-containing solution elicits a contraction as shown in Fig. 3. The maximum force developed was similar to that generated in either 6.6 µM Ca2+ contraction solution or in response to 1 mM GTPS (Fig. 4A). ATPS contractures achieved a peak value in approximately 15 min then declined at a rate of 0.55%
± 0.33% min–1 (n
= 5). In contrast to the GTPS contractures, there was virtually no decrease in force to hypoxia with ATPS thiophosphorylated fibres, with total relaxation at the end of 20 min of 8.3 ± 2.3% (n
= 5). Moreover the contracture elicited by ATPS was not affected by Rho kinase inhibition with 10 µM Y27632, as shown in Fig. 3B.
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S-activated contractures to this end, is that both N2 and Y27632 are inhibitory, such that using 10 µM Y27632 can nearly completely abolish the contracture, making estimates of the effects of N2 difficult to quantify. However, there is a fair amount of evidence suggesting that Rho kinase can and does play a role in Ca2+ contractures (Urban et al. 2003). In about half of our preparations, 1 µM Y27632 elicited relaxation of 6.6 µM Ca2+ contractures (see Fig. 5A). With 10 µM Y27632, all rings relaxed (Fig. 5B; 30%
± 5%, n
= 4).
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S contractures suggested the MRLC-Pi may be an effector site. To further investigate the mechanism(s) underlying hypoxic relaxation, we measured MRLC-Pi in permeabilized coronary arteries. A typical film from our IEF gel/Western blot is shown in Fig. 7A. In the EGTA relaxing solution, baseline MRLC-Pi was 0.18 ± 0.02 MRLC-Pi/total MRLC (n
= 18). This is similar to that recently reported (0.15) for baseline MRLC-Pi in intact hog carotid artery (Rembold et al. 2004). As shown in Fig. 7B, after 15 min in 6.6 µM Ca2+ contracting solution, MRLC-Pi increased to 0.35 ± 0.02 (n
= 18). After exposure to N2 for 20 min, MRLC-Pi decreased to 0.28 ± 0.02 (n
= 20). Both the Ca2+-induced increase and the hypoxia-induced decrease were statistically significant (P < 0.05). Figure 7B also shows the isometric force data whose changes parallel those of MRLC-Pi.
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Discussion |
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We previously showed that an acidification of intracellular pH (pHi) can relax porcine coronary artery (Nagesetty & Paul, 1994) with little change in [Ca2+]i. It is possible that with the milder permeabilization by ß-escin, some metabolic component, either glycolytic, or mitochondrial, might lead to lowering of pHi and thus force during hypoxia. This is unlikely to be a cause of the relaxation, given the buffer capacity of 20 mM imidazole. In control experiments, the contracting solution pH measured with a pH electrode was unaffected to within 0.01 pH unit by repeated switches for 20 min each between aeration with air and N2. Moreover, hypoxia had little effect in an ATPS contracture which would be anticipated to be similarly affected by local changes in pHi. Similar control experiments using Fura-2 to monitor solution [Ca2+], also showed that it was not affected by the aerating gas.
MRLC-Pi depends on the relative levels of MLC kinase and phosphatase activities, the latter of which can be regulated independently of [Ca2+]i (Somlyo & Somlyo, 2000). MLC phosphatase activity can be decreased by activation of the small G-protein, RhoA, and subsequent activation of Rho kinase which, via phosphorylation of the phosphatase myosin binding subunit, inhibits the phosphatase (Brozovich, 2002). We show that hypoxia can inhibit a GTPS-mediated contraction (Fig. 2), indicating that the relaxation mechanism(s) can override maximum RhoA activation, and of course, any other GTPases that may also be activated by GTPS. Importantly, treatment with ATPS inhibited the relaxation to hypoxia. Logical targets for near irreversible thiophosphorylation by ATPS are MLC and the myosin-binding subunit of MLC phosphatase (MYPT1). Thus reasonable candidates for the Ca2+-independent O2-sensing mechanism would be pathways leading to modulation of the level of MRLC-Pi or MYPT1. Since we had shown that Rho kinase is a major modulatory factor in porcine coronary artery (Nobe & Paul, 2001), the RhoA/Rho-kinase pathway was a logical site to investigate. Loss of sarcolemmal integrity with prolonged Triton X-100 permeabilization, which would also lead to loss of RhoA function, abolished the hypoxic response. Treatment with the Rho kinase inhibitor, Y27632, relaxed force in contractures elicited by RhoA activated by GTPS. Somewhat surprisingly, Y27632 also relaxed Ca2+ contractures, though at higher concentrations than those enhanced by GTPS. The reported involvement of Rho kinase in KCl contractures in intact arteries (Urban et al. 2003) suggests that this also occurs under physiologically relevant conditions. Importantly, Y27632 did not inhibit ATPS contractures, indicating that the effects of this compound were probably mediated through effects on MRLC-Pi. It is also of interest that the effects of Y27632 and N2 were not additive (Fig. 6), supporting the notion that both may operate through a common end effector. These data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca2+-independent O2-sensing mechanism. Clearly identification of the protein or proteins targeted by the withdrawal of oxygen in permeabilized arteries will be critical to understanding the mechanisms of the non-Ca2+-dependent O2-sensing pathway in vascular smooth muscle. This may also be potentially relevant to a wide variety of cell types.
For porcine coronary artery under physiological conditions, the relative significance of oxygen-sensing mechanisms that lead to a decrease in the Ca2+ sensitivity of force, and oxygen-sensing mechanisms that lead to a decrease in [Ca2+]i is unknown. We are aware of one report in which MRLC-Pi was measured under hypoxic conditions in intact arteries. Coburn et al. (1992) showed for rabbit aorta that MRLC-Pi decreased with hypoxia for noradrenaline (norepinephrine) stimulation, but did not differ for KCl contractures relative to MRLC-Pi when oxygenated. This would be consistent with our findings that the Ca2+-independent hypoxic relaxation could be clearly discerned at high levels of stimulation (Shimizu et al. 2000). Hai et al. (1993) also reported no steady-state differences in MRLC-Pi between N2 aerating and air, for KCl stimulation in the trachea. Our laboratory reported similar results for taenia coli (Obara et al. 1997), but the relevance to vascular tissue is open to question. Thus the relevance of Ca2+-independent hypoxic relaxation under physiological conditions remains the subject for future experimentation. However our data and previous studies on intact coronary arteries provide strong evidence for the existence of Ca2+-independent O2-sensing mechanisms.
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Footnotes |
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