Sue E. Samson

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-stimulated45Ca2+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 μM 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 μM, but up to 500 μM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 μM 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.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/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 inactivatedby 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. (Mol Cell Biochem 000: 000-000, 1999)

Benefits of antioxidant supplements for knee osteoarthritis: rationale and reality

Arthritis causes disability due to pain and inflammation in joints. There are many forms of arthritis, one of which is osteoarthritis whose prevalence increases with age. It occurs in various joints including hip, knee and hand with knee osteoarthritis being more prevalent. There is no cure for it. The management strategies include exercise, glucosamine plus chondroitin sulfate and NSAIDs. In vitro and animal studies provide a rationale for the use of antioxidant supplements for its management. This review assesses the reality of the benefits of antioxidant supplements in the management of knee osteoarthritis. Several difficulties were encountered in examining this issue: poorly conducted studies, a lack of uniformity in disease definition and diagnosis, and muddling of conclusions from attempts to isolate the efficacious molecules. The antioxidant supplements with most evidence for benefit for pain relief and function in knee osteoarthritis were based on curcumin and avocado-soya bean unsaponifiables. Boswellia and some herbs used in Ayurvedic and Chinese medicine may also be useful. The benefits of cuisines with the appropriate antioxidants should be assessed because they may be more economical and easier to incorporate into the lifestyle.

Ca2+‐pumps and Na+–Ca2+‐exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco‐/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+–Ca2+‐exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT‐PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT‐PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.

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).

Ca2+-mediated ascorbate release from coronary artery endothelial cells

The addition of Ca2+ ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca2+‐mediated release. An increase in Ca2+‐mediated Asc release was observed from PCEC preincubated with Asc, Asc‐2‐phosphate or dehydroascorbic acid (DHAA). The effects of various ATP analogs and inhibition by suramin were consistent with the ATP‐induced release being mediated by P2Y2‐like receptors. ATP‐stimulated Asc release was Ca2+‐mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca2+], (b) Ca2+ ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca2+ and chelating intracellular Ca2+inhibited the ATP‐induced release, and (d) inositol‐selective phospholipase C inhibitor U73122 also inhibited this release. Accumulation of Asc by PCEC was examined at Asc concentrations of 10 μM (Na+‐Asc symporter not saturated) and 5 mM (Na+‐Asc symporter saturated). At 10 μM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. PCEC but not pig coronary artery smooth muscle cells show a Ca2+‐ mediated Asc release pathway that may be activated by agents such as ATP.

Functional linkage of Na+-Ca2+-exchanger to sarco/endoplasmic reticulum Ca2+ pump in coronary artery: comparison of smooth muscle and endothelial cells.

An increase in cytosolic Ca2+ concentration in coronary artery smooth muscle causes a contraction but in endothelium it causes relaxation. Na+‐Ca2+‐exchanger (NCX) may play a role in Ca2+ dynamics in both the cell types. Here, the NCX‐mediated 45Ca2+ uptake was compared in Na+‐loaded pig coronary artery smooth muscle and endothelial cells. In both the cell types, this uptake was inhibited by KB‐R7943, SEA 0400 and by monensin, but not by cariporide. Prior loading of the cells with the Ca2+ chelator BAPTA increased the NCX‐mediated 45Ca2+ uptake in smooth muscle but not in endothelial cells. In the presence or absence of BAPTA loading, the Na+‐mediated 45Ca2+ uptake was greater in endothelial than in smooth muscle cells. In smooth muscle cells without BAPTA loading, thapsigargin diminished the NCX‐mediated 45Ca2+ entry. This effect was not observed in endothelial cells or in either cell type after BAPTA loading. The results in the smooth muscle cells are consistent with a limited diffusional space model in which the NCX‐mediated 45Ca2+ uptake was enhanced by chelation of cytosolic Ca2+ or by its sequestration by the sarco/endoplasmic reticulum Ca2+ pump (SERCA). They suggest a functional linkage between NCX and SERCA in the smooth muscle but not in the endothelial cells. The concept of a linkage between NCX and SERCA in smooth muscle was also confirmed by similar distribution of NCX and SERCA2 proteins when detergent‐treated microsomes were fractionated by flotation on sucrose density gradients. Thus, the coronary artery smooth muscle and endothelial cells differ not only in the relative activities of NCX but also in its functional linkage to SERCA.

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 (Kasc = 22 ± 2 μM) and 2 for Na+ (KNa = 84 ± 10 mM). The anion transport inhibitors sulfinpyrazone and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) inhibited the uptake. Transferring cultured cells loaded with 14C-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.

Ascorbate uptake in pig coronary artery endothelial cells

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When 14C-ascorbate uptake in endothelial cells was examined as 14C or by HPLC, the two values did not differ from each other. 14C-ascorbate uptake was Na+-dependent, stereoselective for l-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: Km = 27± 3 μM (Hill coefficient = 1) for ascorbate and Km = 73± 14 mM (Hill coefficient = 2) for Na+. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect. (Mol Cell Biochem 271: 43–49, 2005)

PROPERTIES OF THE SARCOPLASMIC RETICULUM CA2+-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 Ca2+-uptake which increased linearly with time for >1 h. The Ca2+-uptake occurred with Km values of 0.20±0.03 μM for Ca2+ and 400±34 μM for MgATP2−. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13±0.02 and 0.56±0.04 μM, respectively. These properties of SR Ca2+-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).