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Scott S Billecke, Pamela A Marcovitz Beaumont Health System, Ministrelli Women’s Heart Center, Royal Oak, MI, USA Abstract: The use of multiple drug regimens is increasingly recognized as a tacit requirement for the management of hypertension, a necessity fueled in part by rising rates of metabolic syndrome and diabetes. By targeting complementary pathways, combinations of antihypertensive drugs can be applied to provide effective blood pressure control while minimizing side effects and reducing exposure to high doses of individual medications. In addition, combination therapies, including angiotensin converting enzyme (ACE) inhibitors and calcium channel blockers (CCBs), have the added benefit of reducing cardiovascular mortality and morbidity over other dual therapies while providing equivalent blood pressure control. It is possible that angiotensin receptor blockers (ARBs), which unlike ACE inhibitors are minimally affected by upregulation of alternative pathways for angiotensin II accumulation following long-term treatment, would also provide such outcome benefits. At issue, however, is maintaining patient compliance, as adding medications is known to reduce adherence to treatment regimens. The purpose of this review is to summarize existing trial data for the long-term safety and efficacy of a recent addition to the armamentarium of dual-antihypertensive therapeutic options, the telmisartan/amlodipine single pill combination. The areas where long-term data are lacking, notably clinical information regarding minorities and women, will also be discussed. Keywords: Angiotensin receptor blocker, antihypertensive, calcium channel blocker, clinical trial, combination pill

Background: We sought to study the feasibility of establishing a comprehensive, mostly self-directed yoga program in a hospital and its dose-effect relationship on cardiovascular risk factors and quality of life (QoL) measures over six months., Methods: Yoga-based techniques (Advanced Yoga Practices; AYP; advancedyogapractices.com) were taught in 12 biweekly group sessions and self-directed practice at home was emphasized. Cardiovascular risk factors were elucidated by interview and review of medical history. Quality of life (QoL) outcomes included the SF-36, the Cohen Perceived Stress Scale (CPSS), and the Hospital Anxiety and Depression Scale (HADS). Risk factors and QoL measures were compared in participants at baseline and six months, as well as between those practicing ≥ 7 times versus < 7 times per week., Results: A total of 22 individuals (19 women, mean age 59 ± 8.7 years) completed the study. At six months, changes were noted in the Mental Component Scale (MCS) of the SF-36 (p=0.0004) and the CPSS (p = 0.022). A greater improvement in CPSS was noted in those practicing ≥ 7 times versus < 7 times a week (p=0.045). No changes were noted in cardiovascular risk factors., Conclusions: The prescription of a self-directed yoga program was feasible in a hospital setting and resulted in improvement in QoL measures at six months. Practicing more than seven times per week correlated with greater improvement in the perception of stress. Thus, at least a once-daily dose of AYP techniques for a significant improvement in perceived stress is an appropriate dose to employ and study in hospital settings.

Background: Exercise provides numerous cardiovascular (CV) benefits to women; however, identifying women who are likely to sustain musculoskeletal injury or discomfort is important when tailoring exercise activities., Purpose: To evaluate factors associated with the incidence of injury in women at risk for CV disease (CVD) during their participation in a structured exercise intervention for primary CVD prevention., Study Design: Retrospective comparative study., Methods: Baseline anthropometric measures, CV efficiency (via standardized submaximal treadmill testing), and questionnaire-based assessments of functional capacity (Duke Activity Status Index [DASI]), daytime sleepiness (Epworth Sleepiness Scale), and depression (9-item Patient Health Questionnaire) were compared between participants sustaining an injury or experiencing persistent pain, regardless of cause, with those reporting no such events., Results: Of 117 women enrolled in the study, 27 (23%) experienced an injury or activity-related musculoskeletal pain during the program (injured group). Baseline characteristics for the entire group were: age, 57.3 years; weight, 203 lb; body mass index, 34.8 kg/m 2 ; and waist circumference, 39.9 in. Characteristics did not differ significantly between the injured and non-injured groups for baseline demographics (P > 0.05), but obesity incidence (body mass index ≥ 30 kg/m 2 ) was more prevalent in the injured group (P = 0.02). On univariate analysis, 9-item Patient Health Questionnaire depression scores were higher for injured participants (5.6 vs 4.1, P = 0.16) and DASI activity scores were significantly lower for injured participants (31.3 vs 41.0, P = 0.004) despite there being no significant difference in baseline exercise capacity, expressed as metabolic equivalents (8.1 vs 7.5, P = 0.20) between groups. The DASI score was an independent predictor of injury by multivariate analysis (P = 0.0092)., Conclusion: Women study participants who were injured or experienced activity-related pain during a 6-month structured exercise intervention (injured group) had similar waist measurements, lipid/lipoprotein level profiles, and hemodynamic responses compared with women study participants who were event free (non-injured group); however, injured-group participants were more obese, more depressed, and reported lower functional capacity. Women who have the characteristics of the injured-group participants may be at greater risk for activity-induced injury when participating in exercise-based prevention programs for CV health.

The challenge of managing hypertension is exemplified by the limited success of monotherapy and necessity for multiple drug regimens targeting complimentary pathways. Recent evidence suggests that combination therapy including angiotensin-converting enzyme (ACE) inhibitors and calcium channel blockers (CCBs) provides blood pressure control while reducing cardiovascular mortality and morbidity over ACE inhibitor/diuretic therapy. However, CCBs, ACE inhibitors and angiotensin receptor blockers all increase plasma renin activity (PRA), promoting angiotensin I accumulation and angiotensin II production through alternative pathways. While the clinical ramifications of this and other compensatory pathways activating the renin-angiotensin-aldosterone system are unclear, the recently approved aliskiren/amlodipine antihypertensive combination pill has been shown to decrease PRA via aliskiren's direct inhibition of renin. The purpose of this monograph is to review the mechanisms of action, pharmacodynamics, and safety and efficacy profile of the aliskiren/amlodipine combination pill., (Copyright 2011 Prous Science, S.A.U. or its licensors. All rights reserved.)

The clinical presentation of Takotsubo syndrome, or apical ballooning syndrome, resembles an extensive anterolateral myocardial infarction with chest pain symptoms and electrocardiographic ST-elevation or T-wave inversion noted in most patients. However, coronary arteries are invariably found to be normal or to display minimal atherosclerotic disease despite modest elevation of cardiac enzymes. Since most cases of Takotsubo syndrome occur after intense physical and/or emotional stress, catecholamine surge appears to be a common underlying mechanism. We present a case of Takotsubo syndrome, which presented with unusual symptoms and was found to be caused by pheochromocytoma. A sudden rise in blood pressure moments after completion of echocardiographic stress testing aided in uncovering the diagnosis., (©2010, Wiley Periodicals, Inc.)

Breast arterial calcium (BAC) has been suggested as a marker and predictor of cardiovascular risk and coronary artery disease (CAD). However, an association between BAC and these cardiovascular end points has not been fully elucidated in patients undergoing cardiac catheterization. Consecutive patients undergoing mammography and cardiac catheterization within a 36-month period were retrospectively evaluated through chart review. Cardiac catheterization films and mammograms from 94 patients were independently reviewed for the presence of CAD and BAC, respectively. Cardiovascular risk factors, history of revascularization, and history of myocardial infarction were compared between women with and without BAC. BAC was more prevalent in older women (mean age 69 + or - 10 vs 63 + or - 11 years, p = 0.02). Aside from an inverse correlation with smoking, there was no difference in the presence of CAD or cardiovascular risk factors between patients with and without BAC. Patients with BAC had a lesser history of acute myocardial infarction (21% vs 41%, p <0.05) and were less likely to undergo revascularization (23% vs 43%, p <0.05). In conclusion, BAC was not positively associated with cardiovascular risk factors, documented CAD, or acute cardiovascular events, suggesting that the presence of BAC as determined by mammography is not a useful predictor of CAD in intermediate- to high-risk patients., (Copyright 2010. Published by Elsevier Inc.)

Background: Plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is significantly elevated in patients with kidney disease and is a potential risk factor for cardiovascular disease. Here, we tested whether human whole blood (WB), as in rodent blood, can accumulate free ADMA and whether this accumulation is a function of disease burden., Methods: In 16 healthy control subjects (CO), 18 patients with ESRD and 18 matched hypertensive patients with normal renal function (HTN), we compared using high-pressure liquid chromatography baseline plasma and WB supernatant (WBSUP) ADMA and symmetrical dimethylarginine (SDMA) concentrations and accumulation during a 5-h incubation. We measured protein turnover in incubated WBSUP to determine if proteolytic processes drive ADMA accumulation., Results: Elevated plasma ADMA was confirmed in ESRD and HTN populations while basal WBSUP ADMA was significantly higher in ESRD subjects than controls (P = 0.05 versus CO; P = 0.02 versus HTN). Plasma SDMA followed a similar pattern. Incubation of WBSUP resulted in ADMA release from protein-incorporated stores while SDMA was unaffected. ADMA accumulation in ESRD samples was significantly greater than that in HTN (P = 0.03). CO and HTN men showed significantly greater ADMA accumulation than women (P = 0.01 and P = 0.003, respectively) but no gender difference was observed in the ESRD group (P = 0.26). ADMA accumulation correlated with ex vivo protein turnover (R = 0.76, P < 0.0001)., Conclusions: Human blood is capable of releasing physiologically significant quantities of ADMA via proteolytic pathways. Dysregulated ADMA release from WB reservoirs may contribute to the distinctly high plasma ADMA levels in ESRD populations.

Mammalian paraoxonases (PONs 1, 2 and 3) are a highly conserved family of esterases, with uncertain physiological functions and natural substrates. Here we characterize the ability of purified recombinant human PONs to hydrolyze estrogen esters, a class of compounds previously not known to be PON substrates. PONs hydrolyzed estrogen mono- and diesters at position 3 of the steroid A-ring. Diesters were better substrates for the PONs and were very efficiently hydrolyzed, particularly by PON3. Esters at position 17 were not cleaved by the PONs unless an adjacent double bound was present. Purified human serum butyryl cholinesterase also hydrolyzed estrogen esters, however it preferably hydrolyzed the mono-esters. The ability of the PONs' to effectively hydrolyze a variety of estrogen esters provides further insight into the structure of their active sites and suggests that natural compounds with aromatic ester groups might be relevant substrates for the PONs.

The endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is elevated in many patients and may contribute to the initiation and progression of their disease. While some mechanistic pathways have been identified, tissue-specific contributions to ADMA control remain unclear. We sought to determine if whole blood (WB) could participate in ADMA control ex vivo. Anesthetized male Sprague-Dawley rats underwent exsanguinations, and WB preparations were incubated at 37 degrees C for 5 h. ADMA and symmetrical dimethylarginine were analyzed by high-pressure liquid chromatography. Incubation of lysed red blood cell (RBC) supernatant yielded a significant decrease in ADMA that was blocked by 4124W, a synthetic inhibitor of dimethylarginine dimethylaminohydrolase, the only reported enzyme to hydrolyze ADMA. Hydrolysis of ADMA was diminished by addition of physiologically relevant concentrations of zinc (i.e., 20 microM). Conversely, when rat WB or WB supernatant was incubated at 37 degrees C, it liberated quantities of free ADMA (1-2 microM) that in vivo would likely have pathological consequences. Addition of arginine methyltransferase inhibitors to these incubations did not reduce ADMA release, indicating no dominant role for active protein methylation during these incubations. This ADMA liberation was significantly reduced by addition of protease inhibitors, indicating a dependence on peptide bond hydrolysis. Total ADMA (protein incorporated plus free) was determined by acid hydrolysis and found to be 43.18 +/- 4.79 microM in WB with approximately 95% of this in RBCs. These ex vivo data demonstrate the potential of blood to control the NO-NOS system by modulating free ADMA.

Elevated plasma concentrations of symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA) are repeatedly associated with kidney failure. Both ADMA and SDMA can be excreted in urine. We tested whether renal excretion is necessary for acute, short-term maintenance of plasma ADMA and SDMA. Sprague-Dawley rats underwent sham operation, bilateral nephrectomy (NPX), ureteral ligation, or ureteral section under isoflurane anesthesia. Tail-snip blood samples (250 microl) were taken before and at 6- or 12-h intervals for 72 h after operation. Plasma clearance was assessed in intact and NPX rats. High-performance liquid chromatography determined SDMA and ADMA concentrations. Sodium, potassium, creatinine, blood urea nitrogen (BUN), and body weight were also measured. Forty-eight hours after NPX, SDMA increased 25 times (0.23 +/- 0.03 to 5.68 +/- 0.30 microM), whereas ADMA decreased (1.17 +/- 0.08 to 0.73 +/- 0.08 microM) by 38%. Creatinine and BUN increased, paralleling SDMA. Sham-operated animals showed no significant changes. Increased SDMA confirms continuous systemic production of SDMA and its obligatory renal excretion, much like creatinine. In contrast, decreased plasma ADMA suggests that acute total NPX either reduced systemic ADMA formation and/or systemic hydrolysis of ADMA increased 48-h post-NPX. However, plasma clearance of ADMA appeared unchanged 48 h after NPX. We conclude that renal excretory function is needed for SDMA elimination but not needed for acute, short-term ADMA elimination in that systemic hydrolysis is fully capable of clearing plasma ADMA.

It is established that neuronal NO synthase (nNOS) is ubiquitinated and proteasomally degraded. The metabolism-based inactivation of nNOS and the inhibition of heat shock protein 90 (hsp90)-based chaperones, which are known to regulate nNOS, both lead to enhanced proteasomal degradation of nNOS. The mechanism of this selective proteolytic degradation, or in essence how the nNOS becomes labilized and recognized for ubiquitination and subsequent degradation, has not been determined. In the current study, we used a crude preparation of reticulocyte proteins, which contains ubiquitin-conjugating enzymes and the proteasome, to determine how nNOS is labilized. We found that the inactive monomeric heme-deficient nNOS (apo-nNOS) is rapidly degraded in vitro, consistent with the finding that both metabolism-based inactivation and inhibition of hsp90-based chaperones cause the formation of apo-nNOS and enhance its degradation in vivo. In the current study, we discovered that destabilization of the dimeric nNOS, as determined by measuring the SDS-resistant dimer, is sufficient to trigger ubiquitin-proteasomal degradation. Treatment of nNOS with NG-nitro-L-arginine or 7-nitroindazole led to stabilization of the dimeric nNOS and decreased proteasomal degradation of the enzyme, consistent with that observed in cells. Thus, it seems that the dimeric structure is a major determinant of nNOS stability and proteolysis.

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S., Bender, A. T., Kanelakis, K. C., Murphy, P. J. M., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 278, 15465-15468). In this work, we examine heme binding by apo-nNOS to form the active enzyme in a cell-free system. We show that cytosol from Sf9 cells facilitates heme-dependent apo-nNOS activation by promoting functional heme insertion into the enzyme. Sf9 cytosol also converts the glucocorticoid receptor (GR) to a state where the hydrophobic ligand binding cleft is open to access by steroid. Both cell-free heme activation of purified nNOS and activation of steroid binding activity of the immunopurified GR are inhibited by radicicol treatment of Sf9 cells prior to cytosol preparation, and addition of purified hsp90 to cytosol partially overcomes this inhibition. Although there is an hsp90-dependent machinery in Sf9 cytosol that facilitates heme binding by apo-nNOS, it is clearly different from the machinery that facilitates steroid binding by the GR. hsp90 regulation of apo-nNOS heme activation is very dynamic and requires higher concentrations of radicicol for its inhibition, whereas GR steroid binding is determined by assembly of stable GR.hsp90 heterocomplexes that are formed by a purified five-chaperone machinery that does not activate apo-nNOS.

It has been shown that administration of cigarette smoke to rats leads to loss of neuronal nitric-oxide synthase (nNOS) activity and nNOS protein in penile tissue. The exact mechanism for this loss of activity and protein is not known. In the current study, we investigated whether extracts prepared from cigarette smoke or from the cigarette itself could directly inhibit nNOS activity. We discovered that the cigarette smoke extract and the cigarette extract cause a time-, concentration-, and calmodulin-dependent inactivation of nNOS in an in vitro system containing the purified enzyme. L-Arginine, but not D-arginine, protects nNOS from this time-dependent inactivation, suggesting an active site directed event. The kinetics of inactivation are consistent with the metabolism-based or suicide inactivation of nNOS. Based on studies with other metabolism-based inactivators, this cigarette-mediated inactivation may render nNOS more susceptible to proteasomal degradation and thereby may explain the loss of nNOS protein in vivo. The component(s) responsible for nNOS inactivation is not volatile, is not retained by a 3,000 molecular weight cut-off membrane, binds to activated charcoal, and is highly water-soluble under both acidic and basic conditions. The discovery of a direct inactivation of nNOS by an organic, cationic compound(s) present in tobacco and tobacco smoke provides a basis for further study of not only the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of nNOS.

Nitric oxide synthase (NOS) is a highly regulated enzyme that produces nitric oxide, a critical messenger in many physiological processes. In this perspective, we explore the role of proteolytic degradation of NOS, in particular the inducible and neuronal isoforms of NOS, as a mechanism of regulation of the enzyme. The ubiquitin-proteasome and calpain pathways are the major proteolytic systems identified to date that are responsible for this regulated degradation. The degradation of NOS is affected by diverse agents, including glucocorticoids, caveolin, neurotoxic compounds, and certain NOS inhibitors. Some irreversible inactivators of NOS enhance the proteolytic degradation of the enzyme, and this property may be of great importance in understanding the biological effects of these inhibitors, some of which are being developed for clinical use. Analogies with the regulated degradation of liver microsomal cytochromes P450, which are related to NOS, provide a framework for understanding these processes. Finally, a new perspective on the regulation of NOS by hsp90-based chaperones is presented that involves facilitated heme insertion into the enzyme.

It is established that neuronal NO synthase (nNOS) is associated with the chaperone hsp90, although the functional role for this interaction has not been defined. We have discovered that inhibition of hsp90 by radicicol or geldanamycin nearly prevents the heme-mediated activation and assembly of heme-deficient apo-nNOS in insect cells. This effect is concentration-dependent with over 75% inhibition achieved at 20 microm radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme insertion is prevented. We propose that the hsp90-based chaperone machinery facilitates functional heme entry into apo-nNOS by the opening of the hydrophobic heme-binding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor geldanamycin uncouples endothelial NOS activity and increases endothelial NOS-dependent O(2)() production. Geldanamycin is an ansamycin benzoquinone, and we show here that it causes oxidant production from nNOS in insect cells as well as with the purified protein. At a concentration of 20 microm, geldanamycin causes a 3-fold increase in NADPH oxidation and hydrogen peroxide formation from purified nNOS, whereas the non-quinone hsp90 inhibitor radicicol had no effect. Thus, consistent with the known propensity of other quinones, geldanamycin directly redox cycles with nNOS by a process independent of any action on hsp90, cautioning against the use of geldanamycin as a specific inhibitor of hsp90 in redox-active systems.

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect low-density lipoprotein (LDL) against oxidation. These properties are influenced by a well-characterized polymorphism (Q192R) in human PON1. We now report the identification and characterization of a phenotypically similar, but genetically distinct polymorphism in rabbit PON1. This polymorphism in rabbits was detected by phenotyping sera obtained from 16 inbred rabbit strains and 20 outbred New Zealand White rabbits by paraoxonase/arylesterase activity. The genetic basis of the rabbit polymorphism was determined by DNA sequencing and found to reside in a region distinct from the human Q192R and M55L polymorphisms. Three variant nucleotides within exon 4 (corresponding to P82S, K93E and S1O1G) were found to segregate with the observed rabbit PON1 phenotypes (rPON1A and rPON1B). The rPON1A and rPON1B proteins were purified and compared to the two human isoforms (192Q and 192R). The human and rabbit PON1s displayed similar characteristics with respect to physical properties and substrate specificity. However, rPON1A and rPON1B hydrolysed a variety of substrates at different rates. The rPON1A was also at least three-fold more efficient at protecting LDL from oxidation than rPON1B. Our characterization of a rabbit PON1 polymorphism provides useful insights into important functional residues in PON1. In addition, due to the observed similarities between the rabbit and human polymorphisms, the rabbit may serve as a good model to examine the effect of human PON1 polymorphisms in disease development.

The paraoxonase gene family contains at least three members: PON1, PON2, and PON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g. lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (approximately 39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

Recent evidence has been acquired that implicates an important role for several histidine residues in the hydrolytic mechanisms of human paraoxonase/arylesterase (PON1). Following titration with diethylpyrocarbonate (DEPC), both human serum and recombinant human type Q PON1 were inhibited in respect to their hydrolytic activity in a dose-responsive manner. Human PON1 treated with varying concentrations lost hydrolytic activity, and with each histidine modified, there was an exponential drop in hydrolytic activity. The reaction was followed spectrophotometrically at 244 nm. Recombinant wild-type and C283A PON1 enzymes inhibited with DEPC and subsequently treated with hydroxylamine had partial restoration of activity. The C283A mutant lacks a free sulfhydryl group, indicating that its inactivation is due to histidine specific modification. The dose response and time course of inactivation as well as the extent of reactivation by hydroxylamine were similar for both the wild-type and mutant recombinant enzymes. Mutants of PON1 containing an asparagine substituted for each of several conserved histidine residues lost hydrolytic activity for each single substitution. The mutants of PON1 constructed and assayed for arylesterase activity were H114N, H133N, and H284N. Each single aminoacid substitution rendered the enzyme catalytically inactive. These two pieces of evidence implicate an important role for several histidine residues in the hydrolytic mechanism of PON1. Although it is unusual for a calcium dependent enzyme to require histidines for its catalytic activity, acquired data suggest such a circumstance.