Ascorbate transport in pig coronary artery smooth muscle: Na(+) removal and oxidative stress increase loss of accumulated cellular ascorbate.

Pig deendothelialized coronary artery rings and smooth muscle cells cultured from them accumulated ascorbate from medium containing Na(+). The accumulated material was determined to be ascorbate using high-performance liquid chromatography. We further characterized ascorbate uptake in the cultured cells. The data fitted best with a Hill coefficient of 1 for ascorbate (K(asc) = 22 +/- 2 microM) and 2 for Na(+) (K(Na) = 84 +/- 10 mM). The anion transport inhibitors sulfinpyrazone and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibited the uptake. Transferring cultured cells loaded with (14)C-ascorbate into an ascorbate-free solution resulted in a biphasic loss of radioactivity - an initial sulfinpyrazone-insensitive faster phase and a late sulfinpyrazone-sensitive slower phase. Transferring loaded cells into a Na(+)-free medium increased the loss in the initial phase in a sulfinpyrazone-sensitive manner, suggesting that the ascorbate transporter is bidirectional. Including peroxide or superoxide in the solution increased the loss of radioactivity. Thus, ascorbate accumulated in coronary artery smooth muscle cells by a Na(+)-dependent transporter was lost in an ascorbate-free solution, and the loss was increased by removing Na(+) from the medium or by oxidative stress.

Effects of hydrogen peroxide on pig coronary artery endothelium.

Peroxides and other reactive oxygen species damage arteries during ischemia-reperfusion. Here, we report on the effects of H(2)O(2) on contractility of pig coronary artery. We either treated 3-mm coronary artery rings with 0 to 0.5 mM H(2)O(2) in organ baths or we perfused the arteries with H(2)O(2) and then cut them into rings. In each instance, we monitored the force of contraction of 3-mm rings in H(2)O(2)-free solution with 30 mM KCl and then we determined the A23187 induced endothelium dependent relaxation as a percent of this contraction. Treatment with H(2)O(2) in the organ bath caused a decrease in the contraction but it did not affect the percent relaxation. Treating arteries with H(2)O(2) by luminal perfusion did not affect the contraction but it decreased the percent relaxation. Perfusion alone decreased the amount of endothelium remaining in the arteries and perfusing with H(2)O(2) decreased it further. The percent relaxation with A23187 correlated well with the endothelium remaining in the arteries. We propose that H(2)O(2) and shear stress can cause a loss of endothelium and that endothelium can also protect the underlying smooth muscle against luminal H(2)O(2).

On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells.

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca(2+)-dependent and Ca(2+)-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca(2+) with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe(2+) to NTA with and without Ca(2+). We observed Ca(2+) activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe(2+) to NTA with or without Ca(2+). However, Ca(2+) did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca(2+) and Fe(2+) by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe(2+), and total Ca(2+) concentrations). In solutions based on these constants and taking Ca(2+) chelation into account, Ca(2+) did not affect NTBI uptake over a range of free Fe(2+) concentrations. Thus, the Ca(2+) activation of NTBI uptake observed using the constant total Fe(2+) to NTA ratio was because of Ca(2+)-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca(2+) dependence of NTBI uptake be re-evaluated.

Sarco/endoplasmic reticulum Ca2+-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b.

Endo/sarcoplasmic reticulum (ER) Ca2+-pumps are important for cell survival and communication but they are inactivated by reactive oxygen species (ROS). We have previously reported that the Ca2+-pump isoform SERCA3a is more resistant than SERCA2b to damage by peroxide. Since peroxide and superoxide differ in their redox potentials, we now report the effects of superoxide on the two Ca2+-pump isoforms. We isolated microsomes from HEK293 cells transiently transfected with SERCA2b or SERCA3a cDNA. We exposed these microsomes to superoxide which was generated using xanthine plus xanthine oxidase and catalase to prevent accumulation of peroxide due to superoxide dismutation. Superoxide damaged the Ca2+-transport activity of both isoforms but SERCA3a was damaged at higher concentrations of superoxide and upon longer periods of exposures than was SERCA2b. Thus the SERCA3a isoform is more resistant than SERCA2b to inactivation by both superoxide and peroxide.

Catalase activity in coronary artery endothelium protects smooth muscle against peroxide damage.

Cyclopiazonic acid contracts pig coronary artery de-endothelialized rings, and pretreating the rings with hydrogen peroxide (H(2)O(2)) inhibits this contraction (IC(50)=0.097+/-0.013 mM). We used the cyclopiazonic acid contraction to test the novel hypothesis that endothelium can protect underlying smooth muscle against luminal H(2)O(2). We perfused the arteries with Krebs' solution containing 0. 3 or 1 mM H(2)O(2), removing endothelium from the arteries either before or after the perfusion. We then cut rings from them to monitor their contraction to 10 microM cyclopiazonic acid in a H(2)O(2)-free solution. The inhibition of the cyclopiazonic acid contraction by perfusion with H(2)O(2) was significantly less when endothelium was removed after the perfusion than when it was removed before it. The specific activity of catalase in post-nuclear supernatants from freshly isolated endothelium (14.1+/-2.7 micromol/min/mg protein) was 17+/-3-fold greater than in those from smooth muscle (0.83+/-0.22 micromol/min/mg protein). Thus endothelium contained high catalase activity and protected the underlying smooth muscle against luminal peroxide.

Effects of peroxide on contractility of coronary artery rings of different sizes.

Reactive oxygen species (ROS, free radicals) produced during cardiac ischemia and reperfusion can damage the contractile functions of arteries. The sarcoplasmic reticulum (SR) Ca2+ pump in coronary artery smooth muscle is very sensitive to ROS. Here we show that contractions of de-endothelialized rings from porcine left coronary artery produced by the hormone Angiotensin II and by the SR Ca2+ pump inhibitors cyclopiazonic acid and thapsigargin correlate negatively with the tissue weight. In contrast, the contractions due to membrane depolarization by high KCl correlate positively. Peroxide also produces a small contraction which correlates negatively with the tissue weight. When artery rings are treated with peroxide and washed, their ability to contract with Angiotensin II, cyclopiazonic acid and thapsigargin decreases. Thus, the SR Ca2+ pump may play a more important role in the contractility of the smaller segments of the coronary artery than in the larger segments. These results are consistent with the hypothesis that ROS which damage the SR Ca2+ pump affect the contractile function of the distal segments more adversely than of the proximal segments.

Effects of peroxide on endothelial nitric oxide synthase in coronary arteries.

Reactive oxygen species (ROS) are produced in ischemia and reperfusion. Since endothelial nitric oxide synthase (eNOS) is key to the endothelium-dependent vasodilation, we examined the effects of peroxide on this enzyme. We treated cells cultured from pig coronary artery endothelium with different concentrations of hydrogen peroxide, washed them, solubilized them and measured NOS activity by arginine to citrulline conversion. Hydrogen peroxide inhibited the eNOS activity with an IC50 value of 0.85 +/- 0.39 mM. In another experiment, we perfused arteries with solutions containing 0 or 1 mM hydrogen peroxide, washed them, removed the endothelium using a cotton swab, centrifuged and solubilized the endothelium and monitored its NOS activity. Hydrogen peroxide (1 mM) did not affect the NOS activity significantly (p > 0.05) in this assay. We conclude that the inactivation of eNOS by hydrogen peroxide does not play a major role in the ischemia-reperfusion damage because the peroxide concentrations attained during ischemia-perfusion are much lower than those affecting the eNOS activity.

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function.

We examined the effects of peroxide on the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump in pig coronary artery endothelium and smooth muscle at three organizational levels: Ca2+ transport in permeabilized cells, cytosolic Ca2+ concentration in intact cells, and contractile function of artery rings. We monitored the ATP-dependent, azide-insensitive, oxalate-stimulated 45Ca2+ uptake by saponin-permeabilized cultured cells. Low concentrations of peroxide inhibited the uptake less effectively in endothelium than in smooth muscle whether we added the peroxide directly to the Ca2+ uptake solution or treated intact cells with peroxide and washed them before the permeabilization. An acylphosphate formation assay confirmed the greater resistance of the SERCA pump in endothelial cells than in smooth muscle cells. Pretreating smooth muscle cells with 300 microM peroxide inhibited (by 77 +/- 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic Ca2+ concentration in a Ca2+-free solution, but it did not affect the endothelial cells. Peroxide pretreatment inhibited the CPA-induced contraction in deendothelialized arteries with a 50% inhibitory concentration of 97 +/- 13 microM, but up to 500 microM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 microM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 +/- 2%, but it inhibited the bradykinin-induced, endothelium-dependent relaxation by only 40 +/- 13%. The greater resistance of the endothelium to reactive oxygen may be important during ischemia-reperfusion or in the postinfection immune response.

Sarco(endo)plasmic reticulum Ca2+ pump isoform SERCA3 is more resistant than SERCA2b to peroxide.

Sarco(endo)plasmic reticulum Ca2+ pumps are encoded by genes SERCA1, SERCA2, and SERCA3. Most tissues express SERCA2 Ca2+ pumps (splice SERCA2b) which are inactivated by reactive oxygen. In contrast, SERCA3 is expressed in tissues such as tracheal epithelium, mast cells, lymphoid cells, and aortic endothelium, which are frequently exposed to oxidative stress. Therefore, we compared SERCA3 and SERCA2b proteins for their sensitivity to oxidation. We isolated microsomes from HEK-293 cells overexpressing SERCA3 or SERCA2b. We incubated the microsomes with different concentrations of hydrogen peroxide and then determined Ca2+ pump activities in them in the following three assay systems: ATP-dependent oxalate-stimulated azide-insensitive 45Ca2+ uptake by the microsomal vesicles, Ca(2+)-Mg(2+)-ATPase, and Ca(2+)-dependent acylphosphate formation. Peroxide inhibited the pump activities in microsomes with half-maximal inhibitory concentration (IC50) values of 69 +/- 14, 66 +/- 13, and 84 +/- 15 microM for the 45Ca2+ uptake, Ca(2+)-Mg(2+)-ATPase, and the acylphosphate formation reactions, respectively. However, for microsomes from SERCA3-expressing cells, the corresponding values of IC50 for peroxide were 274 +/- 47, 857 +/- 110, and 746 +/- 40 microM. Thus, in each assay system, the resistance to inactivation by peroxide was significantly (P < 0.05) higher for the SERCA3 protein than for SERCA2b. The SERCA3 resistance to oxidants may aid the cells expressing it to function during exposure to oxidative stress.

SR Ca2+ pump heterogeneity in coronary artery: free radicals and IP3-sensitive and -insensitive pools.

Reactive oxygen species are known to decrease the action of agents that mobilize Ca2+ from sarcoplasmic reticulum (SR) in pig coronary artery smooth muscle. Potentially, this may be due to damage to the SR Ca2+ pump or to the myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release channels. Here we report on the effects of peroxide and superoxide on the SR Ca2+ pump and the subsequent IP3-induced Ca2+ release. Smooth muscle cells cultured from pig left coronary arteries were permeabilized using saponin and then loaded with 45Ca2+ in the presence of an ATP-regenerating system and the mitochondrial Ca2+ uptake inhibitor sodium azide. IP3 caused a release of up to 65% of the loaded 45Ca2+, whereas the Ca2+ ionophore A-23187 caused a release of > 95%. The nature of the IP3-insensitive component of the Ca2+ uptake is not known. The IP3-induced Ca2+ release occurred at 0 or 37 degrees C and was complete in < 30 s. The 50% effective concentration for IP3 was 2.7 +/- 1.0 microM at pH 6.8 and 37 degrees C. At pH 7.4 the IP3-induced Ca2+ release was slightly lower than at pH 6.4-6.8. The IP3-induced release was also inhibited by Ca2+ concentration in the release medium. To investigate the effects of peroxide or superoxide, the cells were treated with these agents, washed, skinned, and then used to examine the IP3-sensitive and -insensitive Ca2+ pools under the conditions in which the IP3-sensitive pool was 60-65% of the total. Peroxide pretreatment was equipotent in inhibiting loading into the IP3-sensitive and -insensitive Ca2+ pools. In contrast, superoxide pretreatment inhibited loading into the IP3-sensitive pool but not into the IP3-insensitive pool. These data are consistent with a model in which the SR Ca2+ pumps are heterogeneous: those required to pump Ca2+ into the IP3-sensitive pool are inhibited by peroxide and superoxide, but those loading the IP3-insensitive pool are inhibited by peroxide only.

Sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle is regulated by a novel pathway.

Coronary artery smooth muscle expresses an alternative splice (SERCA2b) of the sarcoplasmic reticulum (SR) Ca2+ pump gene SERCA2, which is also expressed in cardiac muscle (SERCA2a), but how the activity of this transporter is regulated in the coronary artery is not known. SERCA2a in the cardiac muscle can be regulated via phospholamban or, as recently reported, by a direct phosphorylation of this protein by calmodulin kinase (Xu, A., C. Hawkins, and N. Narayanan. J.Biol. Chem. 268:8394-8397, 1993). Because both SERCA2a and SERCA2b contain this calmodulin kinase phosphorylation site, we examined the effect of endogenous calmodulin kinase phosphorylation of the SR Ca2+ pump in the coronary artery. SR-enriched membranes were isolated from coronary artery smooth muscle and washed in ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to remove bound calmodulin. When these membranes were incubated with MgATP2- in the presence of Ca2+/calmodulin, a 115-kDa protein was phosphorylated. This phosphorylated 115-kDa protein was identified as SERCA2b in Western blots and by immunoprecipitation using a SERCA2-selective antibody. Preincubating the membranes in MgATP2- in the presence of Ca2+/calmodulin stimulated the subsequent Ca2+ uptake in the presence of oxalate plus MgATP2- and azide. The stimulation of Ca2+ uptake was inhibited by including the SR Ca2+ pump inhibitors thapsigargin and cyclopiazonic acid in the Ca2+ uptake medium or by including the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or the calmodulin kinase II peptide fragment 290-309 in the phosphorylation solution. Thus an endogenous calmodulin-dependent kinase phosphorylated SERCA2b and activated it. Phospholamban could not be detected in these membranes in Western blots. Therefore, the regulation of the SR Ca2+ pump activity in coronary artery smooth muscle may involve a direct phosphorylation of the pump protein by an endogenous calmodulin-dependent kinase.

Sarcoplasmic reticulum Ca(2+)-pump density is higher in distal than in proximal segments of porcine left coronary artery.

Densities of sarcoplasmic reticulum (SR) Ca(2+)-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca(2+)-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca(2+)-pump acylphosphate was 7.1 +/- 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca(2+)-pump antibody was 5.3 +/- 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca(2+)-pump inhibitors Cyclopiazonic acid (CPA, EC50 = 0.19 +/- 0.06 microM) and thapsigargin (EC50 = 0.0095 +/- 0.0035 microM). With 10 microM CPA, the force of contraction per tissue wet weight was 4.2 +/- 0.5 -fold greater in distal than in proximal rings, and with 1 microM thapsigargin it was 4.0 +/- 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca(2+)-pump than the proximal segments.

Properties of the sarcoplasmic reticulum Ca(2+)-pump in coronary artery skinned smooth muscle.

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca(2+)-uptake which increased linearly with time for > 1 h. The Ca(2+)-uptake occurred with Km values of 0.20 +/- 0.03 microM for Ca2+ and 400 +/- 34 microM for MgATP2-. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13 +/- 0.02 and 0.56 +/- 0.04 microM, respectively. These properties of SR Ca(2+)-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4-6.8).

Effects of peroxide and superoxide on coronary artery: ANG II response and sarcoplasmic reticulum Ca2+ pump.

The sarcoplasmic reticulum (SR) Ca2+ pump in membranes isolated from arterial smooth muscle is damaged by reactive oxygen species (ROS). Because angiotensin II (ANG II) contracts arterial smooth muscle by mobilizing intracellular Ca2+ concentrations ([Ca2+])i, we determined the effects of ROS pretreatment on ANG II-induced contractions in coronary artery rings and [Ca2+]i transients in smooth muscle cells (SMC) cultured from them. This experimental design eliminates direct ROS interference in assay solutions, thus monitoring only the tissue damage. Pretreating the arteries with peroxide inhibited the ANG II contractions with the concentration for half-maximal activation (K0.5) = 74 +/- 5 microM. Peroxide (250 microM) inhibited the contractions to ANG II and cyclopiazonic acid (CPA, SR Ca(2+)-pump inhibitor) by 78.3 +/- 5.1 and 67.4 +/- 6.3%, respectively, but did not significantly affect the contractions by 60 mM KCl. Pretreating SMC with peroxide inhibited the ANG II-induced increase in [Ca2+]i with K0.5 = 24 +/- 3 microM for peroxide. Peroxide (100 microM) inhibited the increase in [Ca2+]i in response to ANG II and CPA by 78.9 +/- 5.1 and 38.3 +/- 4.9%, respectively. The SR Ca(2+)-pump activity was also measured as the Ca(2+)-dependent formation of 115-kDa acylphosphate. Pretreating SMC with 100 microM peroxide inhibited the acylphosphate levels by 36.3 +/- 3.2%. Peroxide (100 microM) pretreatment of SMC did not significantly affect their ANG II binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II contractions in coronary artery. Nature of receptors and calcium pools.

Pig coronary artery rings denuded of endothelium contract to the vasoactive hormone angiotensin II (Ang II). The nature of Ang II receptors and their Ca(2+)-pool utilization were examined for contraction of the artery rings and for increase in ultracellular [Ca2+] ([Ca2+]i) in smooth muscle cells cultured from them. Ang II contracted the arteries (EC50 = 7 +/- 4 nM) but with a lower maximal force (1.4 +/- 0.25 N/g tissue) than the contraction with 60 mM K+ (6.11 +/- 0.63 N/g tissue). In the cultured cells it caused a transient increase in [Ca2+]i with an EC50 value of 11 +/- 4 nM. The cells bound Ang II with a dissociation constant (Kd) of 7 +/- 2 nM. Based on the effects of the Ang II antagonists saralasin, DuPont 753, dithiothreitol and PD123319, the Ang II receptors responsible for contraction, increase in [Ca2+]i and Ang II binding to coronary artery smooth muscle were of type AT1. The contraction to Ang II was abolished by EGTA but not by nitrendipine. The sarcoplasmic Ca2+ pump inhibitors cyclopiazonic acid (10 microM CPA) and thapsigargin (1 microM) produced contractions of 4.35 +/- 0.73 and 2.07 +/- 0.54 N/g, respectively. Ang II contractions in the control arteries were nearly abolished upon pretreatment with CPA and thapsigargin. CPA and thapsigargin induced contractions were abolished by exposure to EGTA for 1 h but short exposure of the cells to EGTA only modulated the CPA or thapsigargin induced increase in [Ca2+]i; Ang II induced increase in [Ca2+]i was not inhibited by 1 microM nitrendipine but was reduced significantly by a 30-60 sec exposure to EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary artery contractility, Na(+)-pump and oxygen radicals.

Oxygen radicals accumulated during ischemia and reperfusion may affect coronary contractility by endothelium dependent and independent pathways one of which may involve Na(+)-pump. Here we report a contractility assay for Na(+)-pump in pig coronary artery and use it to examine the effects of hydrogen peroxide and superoxide. Coronary artery rings contracted in a K(+)-free Krebs solution and relaxed upon subsequent exposure to K+. The relaxation approximated a single exponential decay whose rate constant depended on [K+]2. This K(+)-induced relaxation was abolished by ouabain and was attributed to Na(+)-pump. In tissues pretreated with peroxide, the rate of relaxation of the K(+)-free contracted arteries decreased with an IC50 = 1.6 +/- 0.6 mmol/l for peroxide. Another set of tissues was pretreated with the superoxide generating system containing 0.3 mmol/l xanthine + varying concentrations of xanthine oxidase (XO) and precontracted in K(+)-free Krebs solution. The rate of the K(+)-induced relaxation decreased with IC50 = 24 +/- 8 mU/ml for XO. Thus, using the relaxation assay we conclude that exposing coronary arteries to oxygen radicals can damage Na(+)-pumps.

Free radicals uncouple the sodium pump in pig coronary artery.

Free radicals may impair vital functions of several types of tissues including coronary artery smooth muscle. Because the Na+ pump plays a key role in maintaining coronary tone, the effects of superoxide and peroxide on this protein were examined. Ouabain-sensitive Rb+ uptake by denuded coronary artery rings was used in lieu of K+ transport by this pump. It was inhibited by exposing the rings for 90 min either to peroxide [50% inhibitory concentration (IC50) = 0.56 +/- 0.18 mM] or to superoxide generated by xanthine oxidase (XO; 0.3 mM xanthine and xanthine oxidase, IC50 = 0.08 +/- 02 mU/ml). The effect of peroxide was not overcome by superoxide dismutase and that of superoxide was not prevented by catalase. K(+)-activated ouabain-sensitive hydrolysis of p-nitrophenyl phosphate in the plasma membrane-enriched fraction isolated from the coronary artery smooth muscle was monitored as the hydrolytic activity of the Na+ pump. It was inhibited by exposing the membranes only to very high concentrations of peroxide (IC50 = 9.85 +/- 3.5 mM) or XO (IC50 = 5 +/- 2 mU/ml). The exposure to 2.5 mM H2O2 or 0.5 mU/ml XO reduced the Na(+)-dependent acylphosphate levels only by 41 +/- 3 and 30 +/- 4%, respectively even though either inhibited the Rb+ uptake by > 80%. Thus superoxide and peroxide uncoupled the hydrolytic activity of the Na+ pump from Rb+ uptake. We speculate that such an uncoupling in ischemia and reperfusion would result in dual damage: ion imbalance and continuous hydrolysis of ATP in the cells that are already starved.

Coronary artery acidosis: pH and calcium pump stability.

To study the effects of prolonged exposure to different pH, pig coronary artery smooth muscle subcellular fraction F3 enriched in sarcoplasmic reticulum was preincubated at 0 or 37 degrees C and pH 6.4-7.8 and then used for monitoring the rate of the oxalate-stimulated component of the ATP-dependent azide-insensitive Ca2+ uptake at constant pH (6.8) and temperature (37 degrees C). Ca2+ uptake by F3 decreased with the increasing preincubation pH. The loss of Ca2+ uptake was more rapid upon preincubation at 37 degrees C than at 0 degrees C. Dithiothreitol (DTT), when included in the preincubation solution, protected against the loss. Sucrose, KCl, ATP, and ATP plus CaCl2 protected slightly, and glutathione, catalase, superoxide dismutase, azide, ascorbate, CaCl2, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, mannitol, mercaptopropionylglycine, and FeSO4 had only marginal or no effects. Efflux of accumulated Ca2+ was more rapid from membranes that had been preincubated at 37 degrees C than from those preincubated at 0 degrees C, but it was not affected by the preincubation pH or by DTT. The loss of Ca2+ uptake due to incubation at pH 7.8 accompanied a decrease in the 115-kDa Ca(2+)-dependent acylphosphate formation due to the Ca2+ pump. The presence of DTT in the preincubation mixture increased the acylphosphate level in the control and protected against its loss at the preincubation pH 6.8 or 7.8. Thus, in the membranes isolated from coronary artery, acidosis may protect against damage to the sarcoplasmic reticulum Ca2+ pump by protonation of a key sulfhydryl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pH on stability of sarcoplasmic reticulum calcium pump in rabbit heart.

The sarcoplasmic reticulum (SR) membranes isolated from rabbit heart were preincubated at pH 6.8 or 7.8 and their Ca2+ pump properties were compared at pH 6.8. The ATP-dependent azide insensitive oxalate-stimulated Ca2+ uptake was reduced more rapidly from the membranes preincubated at 37 degrees C at pH 7.8 than from those preincubated at pH 6.8. The Ca(2+)-Mg(2+)-ATPase, and the Ca(2+)-dependent formation of 110 kDa acylphosphate were also inhibited by the preincubation at the higher pH. Including 1 mM DTT in the preincubation medium reduced the inactivation. The preincubation at 37 degrees C in the presence or absence of DTT caused membranes to become more leaky as the loss of Ca2+ uptake was more rapid than that of ATPase or the acylphosphate formation. The loss of these activities was not accompanied by a breakdown of the protein as monitored in Western blots. It is hypothesized that the SR Ca2+ pump inactivation involves a key-SH group and that the lower pH provides a compensatory protective mechanism for the SR during acidosis.

Expression of the internal calcium pump in pregnant rat uterus.

Uterine contraction to agents such as oxytocin during labour may utilize Ca2+ sequestered by Ca2+ pump into the sarcoplasmic reticulum (SR). Uterus expresses the SR Ca2+ pump gene SERCA2 as the mRNA splice which encodes the protein SERCA2b. The expression of SERCA2 mRNA and protein was monitored in uteri of day 15 pregnant and delivering rats. The ratio of SERCA2 mRNA to 28S RNA increased by 20% from day 15 to delivery. SERCA2 protein examined by two antibodies increased by 54-55%. Ca2+ dependent acylphosphate intermediate of 115 kD corresponding to SERCA2 also increased 72% during this period. Thus, there is an increase in the SERCA2 expression from day 15 to delivery.