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HIGHLIGHTED TOPICS
Oxygen Sensing in Health and Disease
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576
Submitted 1 August 2003 ; accepted in final form 9 October 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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cycloheximide; coronary arteries; hypoxia
In PCA, organ culture at 37°C for 24 h significantly alters expression of several genes. There is a downregulation of the voltage-dependent K+ channels, Kv1.5 and Kv2.1 (25), whereas the expression of ryanodine receptors, RYR2 and RYR3, increases after organ culture (27). This specific inhibition of hypoxic relaxation is also observed when vascular smooth muscle (VSM) is incubated with the protein synthesis inhibitor cycloheximide (26). Taken together, this suggests that protein synthesis is important for the maintenance of hypoxic relaxation.
Using proteomics, we investigated the changes in protein expression that occur after organ culture. We hypothesize that organ culture causes changes in VSM proteins important to vascular oxygen sensing. We demonstrate a downregulation of SM22, tropomyosin, and the small G protein Rho, novel candidates for the Ca2+-independent component of vascular oxygen-sensing mechanisms.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Organ bath studies. Organ bath experiments were performed as previously described (26). Briefly, arterial rings were manually deendothelialized by gentle rubbing of the lumen with a cotton-tipped applicator. One cultured ring and its paired control were placed into a bath containing PSS of the following composition (in mmol/l): 118.3 NaCl, 24.0 NaHCO3, 11.1 dextrose, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 0.026 EDTA, and 2.5 CaCl2. Bath pH is 7.4 when aerated with 95% O2-5% CO2 at 37°C. Tissues were allowed to equilibrate for 1 h. Tension was adjusted to 40 mN, 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 reference contraction was made with 0.1 µM U-46619 or 40 mM KCl, and relaxation to hypoxia was elicited. Hypoxia is defined as, after 95% N2-5% CO2 is bubbled through the baths for 
20 min, when the final oxygen tension of the bath solution, measured polarographically, is 1-2% (26). In some experiments, cumulative concentration-force relations were measured for U-46619 (1 nM to 1 µM). In separate experiments, rings were stimulated with 0.1 µM U-46619 and then concentration-relaxation relations to HA-1077 or Y-27632 (0.1 nM to 1 µM and 1 nM to 30 µM, respectively) were measured.
Relative receptor affinity. In separate experiments, concentration-force relations to U-46619 were constructed in the presence and absence of three different concentrations of SQ-29548. The average ED50 values at each concentration of SQ-29548 were used to construct a Schild plot. Briefly, the amount of SQ-29548 is plotted against the log of the ratio between the average ED50 for U-46619 in the absence of SQ-29548 and the ED50 in the presence of the particular concentration of the antagonist. The result is a line whose slope gives the relative affinity of receptors for the antagonist.
The hypoxic response was characterized in terms of the maximum hypoxic relaxation, expressed as a percentage of the initially developed isometric force, as were all other relaxation responses. All organ bath measurements were recorded with a digital data-acquisition system (Biopac). Force was normalized to cross-sectional area (change in force x circumference/2 x wet weight). Concentration-force relations are given in terms of percentage of the maximum force of the reference contraction to 0.1 µM U-46619 or percent inhibition of maximum force.
Protein isolation. Control and organ cultured arterial rings were frozen in liquid N2 and pulverized vigorously with a dental amalgamator. The resulting powder was dissolved in ice-cold homogenization buffer (0.15 M NaCl, 5 mM EDTA, 1 mM DTT, 20 mM sodium metabisulfite, 20 mM imidazole, and 1 mM PMSF). Homogenates were incubated on ice for 1 h and then centrifuged at 12,000 g, 4°C for 45 min. The supernatant was saved, and the pellet was homogenized a second time. The second pellet was discarded, and total protein was precipitated from the pooled supernatants with ice-cold acetone stored at -20°C for 3 h. Proteins were pelleted by centrifugation at 16,000 g, 4°C for 30 min and then resuspended in rehydration solution (8 M urea, 5% CHAPS, and 1 mM DTT). We determined total protein concentration from this sample with the Bradford assay. Samples were used either immediately for isoelectrical focusing or stored at -80°C.
Isoelectrical focusing. One-dimensional electrophoresis was performed according to a protocol compatible with immobilized pH gradient (IPG) dry strips (Amersham Biotech, Newark, NJ). Briefly, 20-40 µg of protein from control and cultured samples were mixed with isoelectrical focusing solution (rehydration solution plus trace amounts of bromophenol blue and 0.5% IPG buffer, pH range 3-10) to a final volume of 125 µl. Samples were loaded onto 7-cm IPG dry-strip holders. IPG dry strips were then laid into the holders on top of the samples. Holders were covered and placed on an IPGphor (Amersham Pharmacia Biotech) and rehydrated for 12 h. The focusing parameters are as follows: 500 V for 30 min, 1,000 V for 30 min, and then 8,000 V for 1 h.
Two-dimensional SDS-PAGE. After isoelectrical focusing was completed, we prepared strips for SDS-PAGE using 10-20% Trisglycine precast gradient gels (Invitrogen, Carlsbad, CA). Focused IPG strips were incubated in equilibration solution (50 mM Tris·HCl, 6 M urea, 30% glycerol, 2% SDS, and trace amounts of bromophenol blue) for 15 min with gentle shaking. Strips were then rinsed with electrophoresis running buffer (25 mM Tris-base, 192 mM glycine, and 0.1% SDS) and then laid into the two-dimensional well of the gel. Strips were covered with warm SDS agarose, and the gel box apparatus was filled with running buffer. Gels were run at 125 V for 1.5 h.
Silver staining. Visualization of proteins after SDS-PAGE was done by mass spectroscopy-compatible silver staining. Briefly, gels were fixed in a mixture of methanol, acetic acid, and distilled H2O (dH2O) (45:5:50 vol/vol/vol) for 90 min. Gels were washed twice in dH2O for 20 min each. Gels were then washed with 0.2% sodium thiosulfate for 3 min; this was followed by two rinses in dH2O for 30 s each. The gels were next incubated in 0.1% silver nitrate for 30 min and rinsed once with dH2O for 30 s. Next, incubation in developing solution (2.5% sodium carbonate-0.02% formaldehyde) was performed until protein spots were visible (5 min). Gels were then rinsed with 1% acetic acid for 10 min and dH2O twice for 20 min each. All steps were performed at room temperature with gentle shaking.
In-gel extraction, digestion, purification, and protein identification. Mass spectrometric identification of proteins was done according to strategies previously described (10, 21). Briefly, the protein bands were excised from one- or two-dimensional silver-stained polyacrylamide gels (as shown in Fig. 1). After reduction and alkylation, proteins were digested in the gel with an excess of sequencing-grade trypsin (Promega, Madison, WI). The digestions were carried out for 12 h at 37°C. The resulting peptides were extracted, and samples for MALDI analysis were prepared by using the "fast evaporation" method (28). Mass spectra were recorded on a Bruker Biflex III MALDI reflecting time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany). Matrix-related ions and trypsin autolysis products were used for internal spectra calibration. Delayed ion extraction resulted in peptide masses with better than 50 ppm mass accuracy on average, limited mainly by ion statistics in the smaller peaks. Locally installed PROWL software (Proteometrics) was used to search current nonredundant protein sequence databases (National Center for Biotechnology Information) with a list of peptide masses.
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Statistical analysis. We analyzed data using the t-test for paired two-sample means or two-way repeated-measures ANOVA with one-factor balance design. Statistical significance was accepted at P < 0.05. Values are expressed as means ± SE; n values represent the number of hearts from which arteries were isolated.
Chemicals. U-46619, anti-tropomyosin, anti-Rho, and anti-calponin antibodies were from Sigma-Aldrich Chemical (St. Louis, MO). HA-1077 was from Calbiochem (Pasadena, CA). Y-27632 was a gift from the Welfide (Osaka, Japan). SQ-29548 was a gift from Dr. Robert Rapoport (Univ. of Cincinnati). All two-dimensional gel reagents were from Amersham Biotech or from Invitrogen. Antibodies to SM22 were a gift from Dr. Julian Solway (Univ. of Chicago). Antibodies to tropomyosin, calponin, and Rho were from Sigma-Aldrich Chemical.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-actin, was readily identifiable by comparison with two-dimensional gel databases. This actin band and three reproducible spots (see Fig. 1), representing specific proteins, were used for orientation of all subsequent gels from control and cultured extracts.
A comparison of typical gels from control and cultured arteries is shown in Fig. 2. Figure 2, top, is a magnification of the higher molecular mass portion of the gels (30-200 kDa), whereas Fig. 2, bottom, shows the lower molecular mass proteins (6-30 kDa). Notice that organ culture causes a change in expression of several proteins. This may be related to the significant change in mRNA expression observed in previous studies (27). Many proteins exhibited a decrease in expression after organ culture, but increased expression was also observed. Two proteins in particular exhibited decreased expression after organ culture and were most consistent compared with all others (labeled A and B in Fig. 2). Protein A is a relatively small protein of 20 kDa and an isoelectric point of 8. Protein B is more acidic (isoelectric point of 4), of higher molecular mass (35 kDa), and ran close to the distinct actin smear. These candidate proteins were the main focus of subsequent experiments and ultimate identification.
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Our laboratory has previously shown that inhibition of hypoxic relaxation by organ culture can be mimicked by incubation of control arteries with the protein synthesis inhibitor cycloheximide (26). We compared the two-dimensional gels from organ cultured and cycloheximide-treated coronary arteries in Fig. 3. Those spots, used as orientation markers, were similar in gels that used protein extracts from cycloheximide-treated arteries. The change in expression of the two candidate proteins was also consistent (labeled A and B in Fig. 3, top).
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We obtained tryptic maps for both candidate proteins (A and B from Fig. 2) using solid-phase matrix-assisted ionization and time-of-flight analysis (MALDI TOF). The resulting peptide masses were used in a National Center for Biotechnology Information database query for comparison with known proteins. Table 1 gives identities of the candidate proteins and relative significance values according to the database search. Protein A had a tryptic map most comparable to the smooth muscle-specific contractile protein, SM22, with 65% coverage of the tryptic peptides (matching 65% of all of the measured to theoretical peptide masses) and an average error of 22 ppm. Protein B was most comparable to tropomyosin, with 55% coverage and an average error of 12 ppm.
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We used Western blots to validate protein identification and expression pattern from two-dimensional gels using antibodies specific to the two candidate proteins (Fig. 4A). The average data are expressed in Fig. 4B. Both SM22 and tropomyosin show about a 50% reduction in expression in organ-cultured arteries compared with control arteries. Expression of the smooth muscle contractile protein, calponin, was also measured and showed no change.
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Smooth muscle protein actin and SM22 promoters contain elements whose activity is regulated by the small G protein Rho (11). To see whether Rho expression correlated with the change in SM22 expression, we performed Western blots using specific anti-Rho antibodies. Figure 5A shows results of Western blot experiments performed with an antibody recognizing all three Rho isoforms (A, B, and C). Again, a 50% decrease in expression was observed (Fig. 5B).
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Response to receptor-mediated agonist, antagonist, and Rho kinase inhibitors. To indirectly assess the functional consequence of decreased Rho expression, organ bath experiments were performed. Receptor-mediated contraction to the thromboxane analog, U-46619, is largely controlled by the activation of the Rho/Rho kinase pathway (15). Concentration-response curves to U-46619 for control and organ-cultured coronary arteries are shown in Fig. 6. There is a rightward shift in sensitivity to U-46619 after organ culture. ED50 values were 12 ± 0.2 nM and 36 ± 5.7 nM for control and organ-cultured arteries, respectively. These results correlate with the loss in Rho protein.
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Concentration-relaxation relations for the Rho kinase inhibitors Y-27632 and HA-1077 are shown in Fig. 7. There is no significant difference in the percent inhibition for either antagonist, suggesting that the pathway from Rho kinase downstream is intact after organ culture. Figure 8 is a Schild plot representing the affinity of thromboxane receptors for the antagonist SQ-29548 in control and cultured arteries. This method compares the control concentration-force relations for U-46619 with those in the presence of varying concentrations of SQ-29548. The slope of the line represents the level of competitive antagonism between agonist and antagonist, thus providing an estimate of relative receptor density. There was no significant difference between the slopes for control and cultured tissue, suggesting that the receptors mediating U-46619 activation have not been significantly altered by organ culture.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In our organ culture system, we demonstrated a decreased expression of VSM proteins associated with the regulation of contractile function. SM22 and tropomyosin are contractile apparatus-associated proteins whose functions are not clear but may involve inhibition of actin-myosin interaction (6, 9, 12, 13). The Rho/Rho kinase pathway has been implicated in Ca2+ sensitization of isometric force in VSM (1, 15, 24). The specific decrease in expression of these proteins may contribute to the inhibition of relaxation to hypoxia after organ culture. Although the exact function of these proteins remains unclear, their preferential downregulation over other known contractile proteins, such as calponin, correlate with decreased expression of the small G protein Rho, altered sensitivity to U-46619, and specific inhibition of hypoxia-induced relaxation. These proteins are therefore novel candidates for the investigation of vascular oxygen sensing in PCA.
Results from two-dimensional gel electrophoresis and mass spectroscopy suggested the downregulation of SM22 and tropomyosin (Figs. 2 and 4). These results were further confirmed by Western blot analysis (Fig. 5). Our laboratory has previously shown that treatment of coronary arteries with cycloheximide is similar to culturing the tissue in terms of specific inhibition of relaxation to hypoxia (26). Our two-dimensional gel results also indicated changes in the same protein species with either cycloheximide-treated or organ-cultured VSM. Although the exact function of these proteins remains unclear, each is a well-known contractile-associated protein possibly acting as inhibitors of actin myosin interaction (9). Tropomyosin is associated with the actin thin filament and plays a role in regulation of regulation of skeletal muscle, although its role in smooth muscle is unknown (2). In this context, the maximum force-generating capacity is not affected by organ culture, but sensitivity to agonists is decreased. SM22 is reported to be specifically associated with the smooth muscle-specific protein calponin and has been proposed as a potential regulatory protein (13, 20). Interestingly, expression of calponin is not changed by organ culture (Fig. 5), suggesting the preferential inhibition of certain contractile proteins over others. This differs from smooth muscle cell culture, in which SM22 and calponin decrease in parallel (8). SM22 expression may be regulated by a Rho-mediated pathway (11). Our results indicate a downregulation of Rho (Fig. 6) after organ culture.
Response to agonists and antagonists. In an attempt to functionally evaluate the consequence of decreased Rho (and indirectly SM22), we analyzed the sensitivity of the arteries to U-46619 and inhibitors of the Rho/Rho kinase pathway in control and organ-cultured coronary preparations. Receptor-mediated activation in PCA is in part regulated by activation of Rho and Rho kinase (15). Our results show a rightward shift in the U-46619 concentration-force relation after organ culture (Fig. 7). This suggests that there is a loss within the coupling of the U-46619 signal to force development, possibly associated with the decrease in Rho expression. The lack of change in sensitivity to the Rho kinase inhibitors HA-1077 and Y-27632 indicates that the decrease in sensitivity to U-46619 is likely at the level of the G protein, Rho. Decreased sensitivity to U-46619 could also be explained by a change in thromboxane A2 receptor function. However, our results and Schild plot analysis (Fig. 8) suggest that the capacity of these receptors to respond is not affected by organ culture. Although only a correlation, these results support the hypothesis that a downregulation of Rho may manifest as altered downstream pathways potentially important for oxygen sensing.
Relevance to hypoxic relaxation. All three of the proteins identified here are connected with the modulation of contractile force in VSM. There is either a direct interaction with contractile proteins, as is probably true for SM22 and tropomyosin, or there is a modulation of Ca2+ sensitivity, as is the case with the Rho/Rho kinase pathway. A change in the expression of these proteins could lead to altered physiological responses, such as in hypoxia, crucial for maintenance of normal VSM function. Several mechanisms and the possible involvement in modulation oxygen sensitivity are readily proposed. First, the loss of inhibitors of actin-myosin interaction could be manifest as an inability to decrease force via this inhibition pathway. This might be anticipated to inhibit many different modes of relaxation following organ culture. However, we have demonstrated that organ culture exclusively inhibits hypoxic relaxation, suggesting a possible role for SM22 or tropomyosin in modulation of this response specifically. Similar correlated decreases in hypoxic relaxation and protein expression are also observed with cycloheximide treatment, further supporting this hypothesis.
Chronic hypoxia has been shown to increase expression of a 22-kDa protein highly homologous to SM22 (17). However, to affect function, this kind of regulation would be more long term, as would the hypoxia-induced synthesis of tropomyosin observed in pulmonary arterial myocytes (18). Further investigation is necessary to determine the exact role of these proteins in hypoxic relaxation or in maintenance of hypoxic relaxation after organ culture.
The Rho pathway is more defined, and its influence of smooth muscle contractility has been investigated extensively (14, 16, 23). It is the major pathway underlying the high Ca2+ sensitivity of force production associated with receptor-mediated stimulation (15). Furthermore, the Rho kinase pathway has been implicated in hypoxia-induced vasoconstriction in pulmonary VSM (19). If hypoxia induces the inactivation of Rho in systemic VSM, causing a decrease in force, then downregulation of Rho could result in a decreased responsiveness to hypoxia in organ-cultured VSM. Although a direct effect of hypoxia on the Rho/Rho kinase pathway remains to be evaluated, such changes after organ culture may explain the loss of hypoxic relaxation. The modulation of hypoxic relaxation through regulation of Rho-mediated SM22 expression is also possible. In this case, the organ culture-induced change in Rho expression would lead to a downregulation of SM22, which in turn contributes to a loss in hypoxic relaxation.
The combination of organ culture and application of proteomics has produced unique and novel candidates for oxygen sensing in PCA. In this investigation, SM22, tropomyosin, and Rho decreased after organ culture. Decreased expression of these proteins correlates with an inability to relax to hypoxia after organ culture. Functional studies and evidence from other investigations suggest that these are indeed plausible candidates for the mechanism of vascular oxygen sensing. Further investigation is needed to directly link these proteins to the mechanism of hypoxic relaxation.
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FOOTNOTES |
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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.
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REFERENCES |
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