Beta blockers and improved progression-free survival in patients with advanced HER2 negative breast cancer: a retrospective analysis of the ROSE/TRIO-012 study.

BACKGROUND: Recent retrospective studies suggest that beta-adrenergic blocking drugs (BB) are associated with improved outcomes in patients with a range of cancers. Although limited and discordant data suggest that BB may increase overall survival (OS) in localized breast cancer (BC), there is no information on the effects of BB in women with advanced BC. PATIENTS AND METHODS: To explore the association between BB use and BC outcomes, we retrospectively reviewed ROSE/TRIO-012, a double-blinded, multinational phase III trial that randomized 1144 patients with HER2-negative advanced BC to first-line docetaxel in combination with ramucirumab or placebo. We compared progression-free survival (PFS), OS, overall response rate, and clinical benefit rate in patients who received BB to those who did not. RESULTS: 153/1144 (13%) patients received BB; 62% prior to enrolment and 38% began after enrolment. Median PFS in BB treated patients was longer than in patients who did not receive them (10.3 versus 8.3 months; HR 0.81; 95% CI 0.66-0.99; P = 0.038). Patients treated with BB only after enrolment had even higher median PFS (15.5 versus 8.3 months, P < 0.001). In the TNBC subset, median PFS was 13.0 months with BB, compared to 5.2 months without BB (HR 0.52; 95% CI 0.34-0.79; P = 0.002). The benefit of BB intake in PFS was independent of treatment-emergent hypertension (P = 0.476) but associated with treatment arm (P = 0.037). The test for interactions between BB and treatment arm was not significant (P = 0.276). No differences were seen in OS, overall response rate, or clinical benefit rate. A validation dataset analysis had consistent but less substantial improved outcomes for women with node positive operable breast cancer receiving BB in the BCIRG-005 trial. CONCLUSIONS: In this exploratory analysis, BB intake was associated with significant improvement in PFS, particularly in patients with TNBC and patients not previously exposed to BB. CLINICAL TRIAL NUMBER: NCT00703326.

Cardiac-specific adipose triglyceride lipase overexpression protects from cardiac steatosis and dilated cardiomyopathy following diet-induced obesity.

BACKGROUND: Although obesity increases the risk of developing cardiomyopathy, the mechanisms underlying the development of this cardiomyopathy are incompletely understood. As obesity is also associated with increased intramyocardial triacylglycerol (TAG) deposition, also referred to as cardiac steatosis, we hypothesized that alterations in myocardial TAG metabolism and excess TAG accumulation contribute to obesity-induced cardiomyopathy. OBJECTIVE AND DESIGN: To test if increased TAG catabolism could ameliorate obesity-induced cardiac steatosis and dysfunction, we utilized wild-type (WT) mice and mice with cardiomyocyte-specific overexpression of adipose triglyceride lipase (MHC-ATGL mice), which regulates cardiac TAG hydrolysis. WT and MHC-ATGL mice were fed either regular chow (13.5 kcal% fat) or high fat-high sucrose (HFHS; 45 kcal% fat and 17 kcal% sucrose) diet for 16 weeks to induce obesity and mice were subsequently studied at the physiological, biochemical and molecular level. RESULTS: Obese MHC-ATGL mice were protected from increased intramyocardial TAG accumulation, despite similar increases in body weight and systemic insulin resistance as obese WT mice. Importantly, analysis of in vivo cardiac function using transthoracic echocardiography showed that ATGL overexpression protected from obesity-induced systolic and diastolic dysfunction and ventricular dilatation. Ex vivo working heart perfusions revealed impaired cardiac glucose oxidation following obesity in both WT and MHC-ATGL mice, which was consistent with similar impaired cardiac insulin signaling between genotypes. However, hearts from obese MHC-ATGL mice exhibited reduced reliance on palmitate oxidation when compared with the obese WT, which was accompanied by decreased expression of proteins involved in fatty acid uptake, storage and oxidation in MHC-ATGL hearts. CONCLUSION: These findings suggest that cardiomyocyte-specific ATGL overexpression was sufficient to prevent cardiac steatosis and decrease fatty acid utilization following HFHS diet feeding, leading to protection against obesity-induced cardiac dysfunction.

Are users of sulphonylureas at the time of an acute coronary syndrome at risk of poorer outcomes?

AIMS: Adenosine triphosphate sensitive potassium (K(ATP)) channel activity is cardioprotective during ischaemia. One of the purported mechanisms for sulphonylurea adverse effects is through inhibition of these channels. The purpose of this study is to examine whether patients using K(ATP) channel inhibitors at the time of an acute coronary syndrome are at greater risk of death or heart failure (HF) than those not exposed. METHODS: Using linked administrative databases we identified all adults who had an acute coronary syndrome between April 2002 and October 2006 (n = 21 023). RESULTS: Within 30 days of acute coronary syndrome, 5.3% of our cohort died and 15.6% were diagnosed with HF. Individuals with diabetes exhibited significantly higher risk of death (adjusted OR: 1.20, 95% CI: 1.03-1.40) and death or HF (aOR: 1.73, 95% CI: 1.59-1.89) than individuals without diabetes. However, there was no significantly increased risk of death (aOR: 1.00, 95% CI: 0.76-1.33) or death/HF (aOR: 1.06, 95% CI: 0.89-1.26) in patients exposed to K(ATP) channel inhibitors versus patients not exposed to K(ATP) channel inhibitors prior to their acute coronary syndrome. CONCLUSIONS: Diabetes is associated with an increased risk of death or HF within 30 days of an acute coronary syndrome. However, we did not find any excess risk of death or HF associated with use of K(ATP) channel inhibitors at the time of an acute coronary syndrome, raising doubts about the hypothesis that sulphonylureas inhibit the cardioprotective effects of myocardial K(ATP) channels.

Host-Microbe Interplay in the Cardiometabolic Benefits of Dietary Polyphenols.

Polyphenols are nonessential phytonutrients abundantly found in fruits and vegetables. A wealth of data from preclinical models and clinical trials consistently supports cardiometabolic benefits associated with dietary polyphenols in murine models and humans. Furthermore, a growing number of studies have shown that specific classes of polyphenols, such as proanthocyanidins (PACs) and ellagitannins, as well as the stilbenoid resveratrol, can alleviate several features of the metabolic syndrome. Moreover, mounting evidence points to the gut microbiota as a key mediator of the health benefits of polyphenols. In this review we summarize recent findings supporting the beneficial potential of polyphenols against cardiometabolic diseases, with a focus on the role of host-microbe interactions.

Characterisation of the Selective Reduced Uteroplacental Perfusion (sRUPP) Model of Preeclampsia.

Preeclampsia is a complication of pregnancy characterised by gestational hypertension, proteinuria and/or end organ disease. The reduced uteroplacental perfusion (RUPP) model, via partial occlusion of the lower abdominal aorta, mimics insufficient placental perfusion as a primary causal characteristic of preeclampsia. However, a major limitation of the RUPP model is that perfusion is reduced to the entire hindquarters of the rat resulting in hindlimb ischemia. We hypothesised that clipping the uterine and ovarian arteries in the selective (s)RUPP model would provoke signs of preeclampsia while avoiding systemic ischemia. Sham, RUPP or sRUPP procedures were performed in pregnant Sprague Dawley rats on gestational day (GD)14. On GD21 uterine blood flow was significantly reduced in both the RUPP and sRUPP models while aortic flow was reduced only in RUPP. Both models resulted in increased MAP, increased vascular oxidative stress (superoxide generation), increased pro-inflammatory (RANTES) and reduced pro-angiogenic (endoglin) mediators. Vascular compliance and constriction were unaltered in either RUPP or sRUPP groups. In summary, refinements to the RUPP model simultaneously maintain the characteristic phenotype of preeclampsia and avoid peripheral ischemia; providing a useful tool which may be used to increase our knowledge and bring us closer to a solution for women affected by preeclampsia.

Regulation of NHE1 expression in L6 muscle cells.

We examined regulation of expression of the NHE1 promoter on rat L6 cells. Transient transfection of these cells showed that there are two regions critical for basal expression in this cell type. One is from bp -155 to -171 and second more proximal region is between bp -92 and -125. When cells were induced to differentiate by serum withdrawal, mRNA levels rose 2-3-fold. To investigate the mechanisms of this phenomenon a series of stable transfectants were made of the NHE1 promoter in L6 cells. Muscle differentiation caused a significant stimulation of transcriptional activity in the stable cells containing the more distal regions of the promoter. The results show that basal expression of the NHE1 promoter is mediated largely by two proximal regions of the gene. However, during the process of differentiation more distal regions of the gene are involved in elevation of the level of expression.

Induction of expression of the sodium-hydrogen exchanger in rat myocardium.

OBJECTIVE: The aim was to examine the regulation of the cardiac Na+/H+ exchanger NHE-1 isoform mRNA in response to ischaemia and acidosis in the mammalian myocardium. METHODS: Male Sprague Dawley rat hearts were perfused in a non-circulated retrograde fashion according to the Langendorff method. Hearts were perfused for 3 h at flow rates of either 10 ml.min-1 (control), or 3, 1, or 0 ml.min-1 (ischaemia) followed by 5 min of reperfusion. Hearts were immediately frozen in liquid N2, and stored at -80 degrees C until ready for RNA isolation. Northern blot analysis was used to examine expression of the NHE-1 isoform of the Na+/H+ exchanger message in these isolated perfused hearts. Activity of the Na+/H+ exchanger was assessed in primary cultures of neonatal rat myocytes under either control conditions or after treatment with chronic, low external pH. RESULTS: A decrease in developed tension and an increase in resting tension was observed which was dependent upon the severity of the ischaemic episode. Low flow ischaemia of 3 ml.min-1 caused increased Na+/H+ exchanger message levels, while perfusion at more reduced flow rates eliminated the increase. Treatment of primary cultures of isolated myocytes with low external pH resulted in increased ability to recover from an acute acid load. CONCLUSIONS: Low flow ischaemia can increase the Na+/H+ exchanger message in the intact mammalian myocardium. More severe ischaemia prevents the increase, suggesting that severely damaged tissue may not be capable of the ischaemic response. Primary cultures of isolated myocytes can respond to chronic low external pH by increasing Na+/H+ exchanger activity.

Identification of a small Na+/H+ exchanger-like message in the rabbit myocardium.

We examined the Na+/H+ exchanger message in isolated perfused rabbit hearts using Northern blot analysis with cDNA encoding for the rabbit cardiac Na+/H+ exchanger. A cDNA probe from the coding region of the rabbit myocardial Na+/H+ exchanger hybridized to mRNA of 5 kb under high stringency, and to a second 3.8 kb mRNA species under low stringency. When Northern blots were re-probed with a section of the 3'-untranslated region of the cDNA, the 5 kb message was apparent while the smaller 3.8 kb message was not. If isolated working rabbit hearts were subjected to ischemia we observed increases in the 3.8 kb message. Overall, the results show that a 3.8 kb mRNA product, which is homologous to the amiloride sensitive Na+/H+ exchanger, exists in the myocardium and increases during ischemia in the myocardium.

Autopsy analysis of disease frequency in Kinshasa, Republic of Zaire.

Between July 1973 and December 1974, 304 autopsies were done at Hopital Mama Yemo, Kinshasa, Republic of Zaire. The causes of death and sudden death were tabulated and analyzed. The frequency of various diseases and groups of diseases among all the individuals and a large subset of individuals who died traumatically are tabulated and discussed. The present report is the most quantitative, albeit only, current estimate of mortality and prevalence of diseases in Zaire.

PIP: The causes of mortality and frequency of diseases were tabulated in 304 autopsies performed at Hopital Mama Yemo, Kinshasa, between July 1973 and December 1974. 78 of these autopsies were performed on subjects who died at Hopital Mama Yemo, 36 encompassed subjects from other hospitals, and 190 were of medicolegal cases in which the cause of death was not apparent from external examination. Men comprised 63.5% of autopsied cases. The mean age was 30.19 +or- 1.31 for men and 19.84 +or- 1.76 for women. 16.8% of deaths were due to homicide, 6.3% to suicide, and 8.9% to accidents, yielding an overall prevalence for trauma of 32%. Cancer accounted for only 3% of deaths, and cardiovascular diseases 8.2%. Bacterial infections (predominantly streptococcal disease, lobar pneumonia, and pulmonary tuberculosis) represented the largest single cause of death (17.4%). Parasitic infections comprised a further 6.3% of mortality and viral infections 7.2%, giving infectious diseases a combined frequency of 30.9%. Metabolic diseases were responsible for an additional 11.8% of deaths. Obstetric causes were identified in 3.9% of fatalities, and 95% of these cases represented hemorrhagic and septic complications of illegal abortion. Neonatal deaths (4.3%) were largely due to pneumonitis from aspirated amniotic fluid. A final 5.9% of deaths were unexplained. Also analyzed were cases of sudden death occurring outside the hospitals. 31.3% of these deaths were attributed to cardiovascular diseases and 46.3% to infection (including 2.5% due to septic abortion). Finally, the frequency of major diseases in this series was tabulated. Malaria was most frequently found (41.8%), followed by intravascular erythrocytic sickling (18.3%) and hypertension (16%). 12% of females in this series (20% of those dying traumatically) showed evidence of pelvic inflammatory disease. This series is considered to overestimate the frequency of trauma because of the large number of medicolegal cases that fall in this category. This selection for trauma further led to an oversampling of adult men. Nonetheless, it represents the 1st and best qualitative estimate of disease mortality and prevalence in Zaire. The trends in mortality and morbidity identified through this study provide a basis for planning health care and health education.

Fatty acid oxidation in the reperfused ischemic heart.

Myocardial ATP production is dependent chiefly on the oxidative decarboxylation of glucose and fatty acids. The co-utilization of these and other substrates is determined by both the amount of any given substrate supplied to the heart as well as by complex intracellular regulatory mechanisms. This regulated balance is altered during and after ischemia. During aerobic reperfusion of ischemic myocardium, a rapid recovery of energy production is desirable for the complete recovery of muscle contractile function. It is now clear that the type of energy substrate used by the heart during reperfusion will directly influence this contractile recovery. By increasing the relative proportion of glucose oxidized to that of fatty acids, the mechanical function of the reperfused heart can be improved. However, fatty acid oxidation recovers quickly during reperfusion and dominates as a source of oxygen consumption. These high rates of fatty acid oxidation occur at the expense of glucose oxidation, resulting in a decreased recovery of both cardiac function and efficiency during reperfusion. One contributory factor to these high rates of fatty acid oxidation is a decrease in myocardial malonyl-coenzyme A (CoA) levels. Malonyl-CoA, which is synthesized by acetyl-CoA carboxylase, is an essential metabolic intermediary in the regulation of fatty acid oxidation. A decrease in malonyl-CoA level results in an increase of carnitine palmitoyl transferase-1 mediated fatty acid uptake into the mitochondria. This mechanism seems important in the regulation of fatty acid oxidation in the postischemic heart and is discussed in detail in this review, with reference to specific clinical scenarios of ischemia and reperfusion and options for modulating cardiac energy metabolism.

Gene transfer and metabolic modulators as new therapies for pulmonary hypertension. Increasing expression and activity of potassium channels in rat and human models.

UNLABELLED: Chronic Hypoxic Pulmonary Hypertension (CH-PHT) is characterized by pulmonary artery (PA) vasoconstriction and cell proliferation/hypertrophy. PA smooth muscle cell (PASMC) contractility and proliferation are controlled by cytosolic Ca++ levels, which are largely determined by membrane potential (E(M)). E(M) is depolarized in CH-PHT due to decreased expression and functional inhibition of several redox-regulated, 4-aminopyridine (4-AP) sensitive, voltage-gated K+ channels (Kv1.5 and Kv2.1). Humans with Pulmonary Arterial Hypertension (PAH) also have decreased PASMC expression of Kv1.5 and Kv2.1. We speculate this "K+-channelopathy" contributes to PASMC depolarization and Ca++ overload thus promoting vasoconstriction and PASMC proliferation. We hypothesized that restoration of Kv channel expression in PHT and might eventually be beneficial. METHODS: Two strategies were used to increase Kv channel expression in PASMCs: oral administration of a metabolic modulator drug (Dichloroacetate, DCA) and direct Kv gene transfer using an adenovirus (Ad5-Kv2.1). DCA a pyruvate dehydrogenase kinase inhibitor, promotes a more oxidized redox state mimicking normoxia and previously has been noted to increase K+ current in myocytes. Rats were given DCA in the drinking water after the development of CH-PHT and hemodynamics were measured approximately 5 days later. We also tested the ability of Ad5-Kv2.1 to increase Kv2.1 channel expression and function in human PAs ex vivo. RESULTS: The DCA-treated rats had decreased PVR, RVH and PA remodeling compared to the control CH-PHT rats (n=5/group, p<0.05). DCA restored Kv2.1 expression and PASMC Kv current density to near normoxic levels. Adenoviral gene transfer increased expression of Kv2.1 channels and enhanced 4-AP constriction in human PAs. CONCLUSION: Increasing Kv channel function in PAs is feasible and might be beneficial.

Maternal resveratrol treatment during pregnancy improves adverse fetal outcomes in a rat model of severe hypoxia.

Prenatal hypoxia is a common complication in pregnancy. We sought to determine whether resveratrol, a phytoalexin shown to improve health in several species, improves fetal outcomes associated with prenatal hypoxia in rats. Supplementation of maternal diets with resveratrol (4 g/kg diet) from gestational day (GD) 7 to GD21 almost completely reversed fetal demise in hypoxic (8.5% oxygen) pregnancies. We also show that resveratrol crosses the placenta, and may affect the fetus directly.

Adiponectin gene therapy ameliorates high-fat, high-sucrose diet-induced metabolic perturbations in mice.

BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance. METHODS AND RESULTS: The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed significantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins. CONCLUSION: Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity.

Specific activation of the Na+/H+ exchanger gene during neuronal differentiation of embryonal carcinoma cells.

We examined the regulation of the Na+/H+ exchanger gene during differentiation of the P19 mouse embryonal carcinoma cells. Treatment of P19 cells with retinoic acid induces the development of neurons, astroglia, and microglia cells. Upon retinoic acid-induced differentiation of P19 cells, there was an early and rapid 10-fold increase in NHE1 transcription. A proximal cis-acting AP-2 site of the NHE1 promoter was sufficient for stimulation of transcription of the gene by differentiation. Bandshift experiments demonstrated that in retinoic acid-treated cells there was an elevated level of AP-2 transcription factor binding to the AP-2 consensus site of the Na+/H+ exchanger gene. In the differentiation defective mutant RAC65, the effect of differentiation on Na+/H+ exchanger gene expression was reduced by 60%. Examination of Na+/H+ exchanger activity showed that retinoic acid-treated P19 cells recovered from an acid load at a rate approximately three times greater than untreated cells. The increases in gene expression and protein activity preceded major changes in cell morphology, suggesting that the initiation of differentiation is linked to NHE1 gene expression. Our findings show for the first time that the NHE1 gene is activated early in cell differentiation and that this activation may play an important role in the process of neuronal cell differentiation.

Glucose metabolism, H+ production and Na+/H+-exchanger mRNA levels in ischemic hearts from diabetic rats.

Glycolysis uncoupled from glucose oxidation is a major reason for the intracellular acidosis that occurs during severe myocardial ischemia. The imbalance between glycolysis and glucose oxidation, and the resultant H+ produced from glycolytically derived ATP hydrolysis in the diabetic rat heart is the focus of this study. Isolated working hearts from 6 week streptozotocin diabetic rat hearts were perfused with 11 mM glucose and 1.2 mM palmitate and subjected to a 25 min period of global ischemia. A second series of experiments were also performed in which hearts from control, diabetic, and islet-transplanted diabetic rats were subjected to a 30 min aerobic perfusion, followed by a 60 min period of low-flow ischemia (coronary flow = 0.5 ml/min) and 30 min of aerobic reperfusion. H+ production from glucose metabolism was measured throughout the two protocols by simultaneous measurement of glycolysis and glucose oxidation using perfusate labelled with [5-3H/U-14C]-glucose. Rates of H+ production were calculated by measuring the difference between glycolysis and glucose oxidation. The H+ production throughout the perfusion was generally lower in diabetic rat hearts compared to control hearts, while islet-transplantation of diabetic rats increased H+ production to rates similar to those seen in control hearts. This occurred primarily due to a dramatic increase in the rates of glycolysis. Despite the difference in H+ production between control, diabetic and islet-transplanted diabetic rat hearts, no difference in mRNA levels of the cardiac Na+/H+-exchanger (NHE-1) was seen. This suggests that alterations in the source of protons (i.e. glucose metabolism) are as important as alterations in the fate of protons, when considering diabetes-induced changes in cellular pH. Furthermore, our data suggests that alterations in Na+/H+-exchange activity in the diabetic rat heart occur at a post-translational level, possibly due to direct alterations in the sarcolemmal membranes.

Activation of the Na+/H+ exchanger gene by the transcription factor AP-2.

We have isolated and characterized regions important for expression of the mouse Na+/H+ exchanger gene. A 1.1-kilobase fragment upstream of the 5'-untranslated region contains specific DNA motifs characteristic of promoter and enhancer elements including a TATA box, two CAAT boxes, an SP-1 site, a cyclic AMP response element-binding site, and an AP-2-like site. This 1.1-kilobase fragment directs transcription of a luciferase reporter gene in mouse fibroblasts (NIH 3T3) and human Hep G2 cells. Deletion or mutation of an AP-2-like site 100 base pairs from the start site of transcription resulted in loss of most of the reporter plasmid activity. In addition, cotransfection of an AP-2 expression plasmid and the mouse promoter/luciferase plasmid increased the amount of Na+/H+ exchanger-directed transcription in AP-2-deficient Hep G2 cells. Moreover, mobility shift analysis indicated that a putative AP-2-binding site is capable of binding purified AP-2 protein and a specific protein from nuclear extracts of NIH 3T3 cells. The results show that the transcription factor AP-2 may play an important role in regulation of transcription of the mouse Na+/H+ exchanger gene.

Characterization of the placental brush border membrane Na+/H+ exchanger: identification of thiol-dependent transitions in apparent molecular size.

We examined the protein and mRNA encoding the amiloride-sensitive Na+/H+ exchanger from human placenta. Reverse transcriptase PCR of human placental RNA and a human choriocarcinoma cell line showed that the message for the amiloride-sensitive Na+/H+ exchanger from human placenta. Reverse transcriptase PCR of human placental RNA and a human choriocarcinoma cell line showed that the message for the amiloride-sensitive Na+/H+ exchanger is present in the placenta and its derived cell line. Northern blot analysis showed only one species of Na+/H+ exchanger mRNA, of about 5 kb in size. To examine the Na+/H+ exchanger protein two different affinity-purified antibodies were produced against the C-terminal cytoplasmic region of the Na+/H+ exchanger. The antibodies both identified a 105 kDa protein in human placental brush border membrane vesicles. Under non-reducing conditions the amount of 105 kDa protein was greatly decreased, while a 205 kDa protein became apparent. This is probably a dimer of the 105 kDa protein. The monomer-to-dimer transition was dependent on the concentration of beta-mercaptoethanol. The results show that the amiloride-sensitive Na+/H+ exchanger is relatively abundant in human placenta and that it can exist as a larger 205 kDa protein linked by disulphide bonds.

Regulation of NHE-1 promoter in mammalian myocardium.

The Na+/H exchanger (NHE-1) is an integral membrane protein responsible for intracellular pH regulation in the myocardium and other tissues. The NHE-1 isoform is universally distributed in mammalian cells. We examined regulation of a 1.1-kb fragment of the NHE-1 promoter in neonatal rat cardiomyocytes. Deletion of most of the promoter up to an AP-2 site reduced activity 75%. Further deletion of the promoter or mutation of the AP-2 site reduced or eliminated activity almost completely. Gel mobility shift assay showed that purified AP-2 protein or AP-2-like protein from nuclear extracts of isolated myocytes can bind to DNA of the NHE-1 protein. External acidosis did not cause increased transcription from the promoter. Removal of serum from the medium reduced activity of the NHE-1 promoter. The elements responsible for activation of the promoter by serum were contained within both the 1.1-kb and AP-2-containing region. The results show that the cis-acting putative AP-2 site and the presence of serum are important in NHE-1 expression, whereas external acidosis had no direct effect on the promoter.