The role of 3'-untranslated region (3'-UTR) mediated mRNA stability in cardiovascular pathophysiology.

Knowledge of transcription and translation has advanced our understanding of cardiac diseases. Here, we present the hypothesis that the stability of mRNA mediated by the 3'-untranslated region (3'-UTR) plays a role in changing gene expression in cardiovascular pathophysiology. Several proteins that bind to sequences in the 3'-UTR of mRNA of cardiovascular targets have been identified. The affected mRNAs include those encoding beta-adrenergic receptors, angiotensin II receptors, endothelial and inducible nitric oxide synthases, cyclooxygenase, endothelial growth factor, tissue necrosis factor (TNF-alpha), globin, elastin, proteins involved in cell cycle regulation, oncogenes, cytokines and lymphokines. We discuss: (a) the types of 3'-UTR sequences involved in mRNA stability, (b) AUF1, HuR and other proteins that bind to these sequences to either stabilize or destabilize the target mRNAs, and (c) the potential role of the 3'-UTR mediated mRNA stability in heart failure, myocardial infarction and hypertension. We hope that these concepts will aid in better understanding cardiovascular diseases and in developing new therapies.

SERCA pump isoform expression in endothelium of veins and arteries: every endothelium is not the same.

Endothelium from rat aorta expresses sarco/endoplasmic reticulum Ca2+(SERCA) pump gene SERCA3 where as the smooth muscle expresses SERCA2. This has led to the postulate that vascular endothelium expresses SERCA3. To test this postulate, we examined the SERCA2 and SERCA3 mRNA expression in endothelium and smooth muscle dissected from coronary artery, coronary vein, aorta and vena cava of pig. Smooth muscle from all arteries and veins expressed only the SERCA2 mRNA. Endothelium from coronary artery, coronary vein and aorta expressed both SERCA2 and SERCA3 mRNA but the endothelium from vena cava did not express SERCA3 mRNA although it expressed SERCA2. These observations support the postulate that vascular endothelium expresses SERCA3 but the affirmation is equivocal because vena cava endothelium does not express SERCA3.

Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves.

Mobilization of endoplasmic reticulum Ca2+ is pivotal to the ability of a cell to send or respond to stimuli. Ca(2+)-Mg(2+)-ATPases, termed SERCA pumps, sequester Ca2+ into the sarco/endoplasmic reticulum. There are several SERCA protein isoforms encoded by three genes. This paper summarizes the structure, function, tissue and subcellular distribution, and regulation of various SERCA isoforms. Then it attempts to link divergence in the signal transduction processes of cells to the types and levels of SERCA proteins they express and to how the cells regulate their SERCA pump activity. The paper examines possible linkages between SERCA pumps and receptor-activated Ca2+ entry, SERCA isoform localization and Ca(2+)-waves, and the role of SERCA pumps in nuclear Ca2+ in cell proliferation and apoptosis. Then it uses available information on cardiac function and chronic stimulation of the fast-twitch muscle to answer a series of basic questions on the regulation of SERCA activity and expression and their linkage to signal transduction. Finally, it discusses the possibility that neurons exhibit complex Ca(2+)-waves whose interactions have the potential to explain the operational basis of neural networks. A series of unanswered questions emerge based on this synthesis, including the unsettling issue of whether all the isoforms are needed to achieve the divergence in signal transduction or if there is a degree of redundancy in the system.

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.

Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle.

Cardiac muscle expresses sarcoplasmic/endoplasmic-reticulum Ca2+ pump isoform SERCA2a; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100-200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (mean+/-S.E.M.) were (29+/-4)-fold higher in the heart. In both tissues the SERCA2 mRNA was associated with polyribosomes. In a sucrose-density-gradient sedimentation velocity experiment on polyribosomes, there was no difference in the sedimentation pattern of SERCA2 mRNA between the two tissues, suggesting that the translation efficiency of SERCA2 RNA in the two tissues is quite similar. Thus the main difference in the control of SERCA2 expression in the two tissues is post-transcriptional and pretranslational.

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.

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.

Involvement of cyclooxygenase 2 (COX-2) in intrinsic tone of isolated guinea pig trachea.

Indomethacin and related nonsteroidal anti-inflammatory drugs relax prostanoid-dependent intrinsic tone of isolated guinea pig trachea by inhibiting cyclooxygenase (COX). Recently, a second isoform of COX (COX-2) was discovered, which differed from COX-1 with respect to protein structure, transcriptional regulation, and susceptibility to inhibition by pharmacological agents. It is now known that indomethacin nonselectively inhibits COX-1 and COX-2, whereas NS-398 is a selective inhibitor of COX-2. In the present study we compared the activity of a selective (NS-398) and nonselective (indomethacin) COX-2 inhibitor on intrinsic tone of isolated guinea pig trachea. NS-398 > or = indomethacin produced a reversal of intrinsic tone with a similar concentration-dependent (10 nM to 1 microM) time course (Tmax approximately 20-45 min), potency (EC50 1.7 and 5.6 nM, respectively), and maximal response. Contractions to cholinergic nerve stimulation (45 V, 0.5 ms, 0.1-32 Hz) and histamine were similarly modulated in tissues relaxed with the selective or nonselective COX-2 inhibitors. Immunoblot analyses showed that COX-2 protein synthesis was induced in both the cartilage and smooth muscle portions of the trachea during changes in intrinsic tone. These findings are consistent with pharmacological results and provide the first demonstration that prostanoid tone in isolated guinea pig trachea is dependent on COX-2 activity. The results also suggest that the activity of indomethacin in this preparation is likely related to COX-2 inhibition.