Impact of National Institutes of Health and Food and Drug Administration Tobacco Research Funding: A Bibliometrics Analyses.

INTRODUCTION: Conduct bibliometric analyses documenting the output of National Institutes of Health (NIH) tobacco-related and Food and Drug Administration (FDA) tobacco regulatory science (FDA-TRS) research portfolios. AIMS AND METHODS: PubMed identifiers for publications between 2015 and 2020 citing tobacco funding by NIH and/or FDA were imported into NIH iCite generating measures of productivity and influence, including number of citations, journal, relative citation ratios (RCR), and comparison of research influence across Web of Science (WoS) disciplines. Coauthorship and measures of centrality among and between NIH and FDA-supported investigators gauged collaboration. RESULTS: Between FY 2015 and 2020, 8160 publications cited funding from NIH tobacco-related grants, 1776 cited FDA-TRS grants and 496 cited Common funding (ie, both NIH and FDA-TRS funding). The proportion of publications citing NIH grants declined while those citing FDA-TRS or Common funding rose significantly. Publications citing Common funding showed the highest influence (mean RCR = 2.52). Publications citing FDA-TRS funding displayed higher median RCRs than publications citing NIH funding in most WoS categories. Higher translational progress was estimated over time for FDA-TRS and Common publications compared to NIH publications. Authors citing Common funding scored highest across all collaboration measures. CONCLUSIONS: This study demonstrates the high bibliometric output of tobacco research overall. The rise in publications citing FDA-TRS and Common likely reflects increased funding for TRS research. Higher RCRs across WoS subject categories and trends towards human translation among FDA-TRS and Common publications indicate focus on research to inform regulation. This analysis suggests that FDA support for TRS has expanded the field of tobacco control resulting in sustained productivity, influence, and collaboration. IMPLICATIONS: This paper is the first effort to better describe the impact of tobacco research resulting from the addition of FDA funding for TRS in the past decade. The analysis provides impetus for further investigation into the publication topics and their focus which would offer insight into the specific evidence generated on tobacco control and regulation.

NIH Tobacco Research and the Emergence of Tobacco Regulatory Science.

INTRODUCTION: This study explores how the emergence of FDA-funded Tobacco Regulatory Science (TRS) research complements and perhaps influenced the direction of tobacco research supported by NIH. AIMS AND METHODS: New NIH- and FDA-funded tobacco projects awarded in fiscal years (FY) 2011-2020 were identified using internal NIH databases of awarded grants. Project abstracts and research aims were coded by the authors to characterize research domains and tobacco products studied. RESULTS: Between FY 2011 and 2020, NIH funded 1032 and FDA funded 322 new tobacco projects. For the years and grant activity codes studied, the number of new NIH tobacco projects declined while FDA's increased; combined the number of new projects held steady. Much of NIH research included smoking combustibles (43.7%). The most common products in FDA research were cigarettes (74.8%) and e-cigarettes/ENDS (48.1%). Most NIH (58.6%) and FDA (67.7%) projects included research on the determinants of tobacco use. Another area of apparent overlap was health effects (29.5% NIH and 30.1% FDA). Projects unique to NIH included treatment interventions (33.3%), disease pathology/progression (17.8%) and neurobiology (18.9%). A minority of both NIH and FDA projects included populations particularly vulnerable to tobacco product use. CONCLUSIONS: In total, support for new tobacco research supported by NIH and FDA combined remained steady for the time period covered, though there was a concomitant decline in NIH tobacco projects with the increase in FDA-funded TRS projects for the activity codes studied. Despite the apparent overlap in some areas, both NIH and FDA support research that is unique to their respective missions. IMPLICATIONS: NIH continues to support tobacco research that falls within and outside of FDA's regulatory authorities. This research still is needed not only to bolster the evidence base for regulatory decisions at the national and state levels, but also to advance a comprehensive scientific agenda that can inform multiple levels of influence on tobacco control, use and addiction. It will be important to continue monitoring FDA-funded TRS and NIH-funded tobacco research portfolios to ensure that the level of support for and focus of the research is sufficient to address the burden of tobacco-related morbidity and mortality.

Host-agent-vector-environment measures for electronic cigarette research used in NIH grants.

OBJECTIVE: The purpose of this study is to describe the focus and comprehensiveness of domains measured in e-cigarette research. METHODS: A portfolio analysis of National Institutes of Health grants focusing on e-cigarette research and funded between the fiscal years 2007 and 2015 was conducted. Grant proposals were retrieved using a government database and coded using the Host-Agent-Vector-Environment (HAVE) model as a framework to characterise the measures proposed. Eighty-one projects met the criteria for inclusion in the analysis. RESULTS: The primary HAVE focus most commonly found was Host (73%), followed by Agent (21%), Vector (6%) and Environment (0%). Intrapersonal measures and use trajectories were the most common measures in studies that include Host measures (n=59 and n=51, respectively). Product composition was the most common area of measurement in Agent studies (n=24), whereas Marketing (n=21) was the most common (n=21) area of Vector measurement. When Environment measures were examined as secondary measures in studies, they primarily focused on measuring Peer, Occupation and Social Networks (n=18). Although all studies mentioned research on e-cigarettes, most (n=52; 64%) did not specify the type of e-cigarette device or liquid solution under study. CONCLUSIONS: This analysis revealed a heavy focus on Host measures (73%) and a lack of focus on Environment measures. The predominant focus on Host measures may have the unintended effect of limiting the evidence base for tobacco control and regulatory science. Further, a lack of specificity about the e-cigarette product under study will make comparing results across studies and using the outcomes to inform tobacco policy difficult.

High Dietary ω-6:ω-3 PUFA Ratio Is Positively Associated with Excessive Adiposity and Waist Circumference.

OBJECTIVE: The aim of this study was to analyze dietary ω-6:ω-3 polyunsaturated fatty acid (PUFA) ratio and its association with adiposity and serum adiponectin levels in a Mexican population. METHODS: In this cross-sectional study, individuals with a BMI ≥ 18.5 kg/m2, were classified using four methods to measure adiposity. Parameters of body composition were measured by InBody 3.0. Diet intake was evaluated prospectively using a 3-day written food record. Serum high-molecular weight adiponectin isoform was measured using an ELISA assay. Biochemical and adiposity variables were analyzed by tertiles of dietary ω-6:ω-3 PUFA ratio. RESULTS: A total of 170 subjects were recruited with a mean age of 36.9 ± 11.8 years. The 73.5% of subjects were women. Subjects in the higher tertile of dietary ω-6:ω-3 PUFA ratio had more adiposity and higher levels of triglycerides, VLDL-c, glucose, insulin and HOMA-IR than those in the first tertile (p < 0.05). Adiponectin levels showed a trend according to dietary ω-6:ω-3 PUFA ratio (p = 0.06). A linear regression model showed that waist circumference, insulin, and HOMA-IR have positive associations with dietary ω-6:ω-3 PUFA ratio. CONCLUSION: This study suggests that high dietary ω-6:ω-3 PUFA ratio is positively associated with excessive adiposity and worse metabolic profile.

Associations of the lipid genetic variants Thr54 ( FABP2) and -493T ( MTTP) with total cholesterol and low-density lipoprotein cholesterol levels in Mexican subjects.

Objective Mexico has one of the world's highest rates of obesity, which is influenced by lipid-genetic and lifestyle factors. This study aimed to determine whether FABP2 (Ala54Thr) and MTTP (-493 G/T) genetic polymorphisms are associated with metabolic disorders in Mexican subjects. Methods A total of 523 subjects participated in a cross-sectional study. Genotyping for FABP2 and MTTP was performed using real-time RT-PCR. Biochemical and anthropometric data were evaluated. Results The genetically at-risk group (Thr54/-493T) was associated with significantly higher total and low-density lipoprotein cholesterol levels (difference between genetically at-risk group and wild-type group: 10.6 mg/dL and 8.94 mg/dL, respectively). Carriers within the genetically at-risk group had a significantly higher prevalence rate of hypercholesterolaemia (42.5% vs. 32.0%) and higher LDL-C levels (37.6% vs. 26.4%) than did non-carriers. Conclusions Subjects who are genetically at risk (Thr54/-493T) have higher total cholesterol levels, low-density lipoprotein cholesterol levels, and prevalence rate of hypercholesterolaemia. These findings highlight the importance of basing nutritional intervention strategies for preventing and treating chronic diseases on individual genetic characteristics.

Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Dietary supplement research portfolio at the NIH, 2009-2011.

The U.S. dietary supplement market increased by 7.5% in 2012 compared with 2011, reaching $32.5 billion in sales. Therefore, federally supported research on dietary supplements is important to determine their health effects, safety, and efficacy. A portfolio analysis was performed across the NIH and the Office of Dietary Supplements (ODS) for fiscal years (FYs) 2009-2011 by using the databases Human Nutrition Research Information Management (HNRIM) and Computer Access to Research on Dietary Supplements (CARDS). The results indicated that total NIH dietary supplement-related funding for FYs 2009-2011 was $855 million ($295 million in 2009, $311 million in 2010, and $249 million in 2011). The institutes and centers with the highest investment in dietary supplement research were as follows: the National Heart, Lung, and Blood Institute ($135 million); the National Cancer Institute ($188 million); the National Center for Complementary and Alternative Medicine ($99 million); the National Institute of Diabetes and Digestive and Kidney Diseases ($68 million); the National Institute of Environmental Health Sciences ($58 million); and the ODS ($32 million). The dietary supplement ingredients receiving the most funding were botanicals (22%), vitamins (20%), lipids (14%), and minerals and trace elements (10%). The top 3 outcome research areas were cancer (61% of total dietary supplement investment), cardiovascular disease (47%), and women's reproductive health (38%). In FYs 2009, 2010, and 2011, the ODS provided 3.5%, 3.6%, and 4.1%, respectively, of the NIH investment in dietary supplement research. ODS funding focused on cellular, enzymatic, or molecular mechanisms (64% of total ODS funding). This portfolio analysis demonstrates that the NIH has committed substantial funding to dietary supplement research in an effort to expand the scientific knowledge base on the efficacy and safety of dietary supplements.

Perinatal exercise improves glucose homeostasis in adult offspring.

Emerging research has shown that subtle factors during pregnancy and gestation can influence long-term health in offspring. In an attempt to be proactive, we set out to explore whether a nonpharmacological intervention, perinatal exercise, might improve offspring health. Female mice were separated into sedentary or exercise cohorts, with the exercise cohort having voluntary access to a running wheel prior to mating and during pregnancy and nursing. Offspring were weaned, and analyses were performed on the mature offspring that did not have access to running wheels during any portion of their lives. Perinatal exercise caused improved glucose disposal following an oral glucose challenge in both female and male adult offspring (P < 0.05 for both). Blood glucose concentrations were reduced to lower values in response to an intraperitoneal insulin tolerance test for both female and male adult offspring of parents with access to running wheels (P < 0.05 and P < 0.01, respectively). Male offspring from exercised dams showed increased percent lean mass and decreased fat mass percent compared with male offspring from sedentary dams (P < 0.01 for both), but these parameters were unchanged in female offspring. These data suggest that short-term maternal voluntary exercise prior to and during healthy pregnancy and nursing can enhance long-term glucose homeostasis in offspring.

Augmented phosphorylation of cardiac troponin I in hypertensive heart failure.

An altered cardiac myofilament response to activating Ca(2+) is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca(2+) sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser(22/23) and protein kinase C (PKC) at Ser(22/23), Ser(42/44), and Thr(143). Whereas the functional significance of Ser(22/23) phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser(22/23) and Ser(42/44) in SHR. Enhanced Ser(22/23) phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCε, but no changes in PKA or PKC-ß levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser(42/44) (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.

Mitochondrial isolation from skeletal muscle.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles. Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37°C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 µg) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5 mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 µM) is added to start state 3. Oligomycin (1 µM), an ATPase synthase blocker, is used to estimate state. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 µM) is added to measurestate, or uncoupled respiration. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR ≥ 4 is considered as evidence of a viable mitochondria preparation. In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

Rat diaphragm mitochondria have lower intrinsic respiratory rates than mitochondria in limb muscles.

The mitochondrial content of skeletal muscles is proportional to activity level, with the assumption that intrinsic mitochondrial function is the same in all muscles. This may not hold true for all muscles. For example, the diaphragm is a constantly active muscle; it is possible that its mitochondria are intrinsically different compared with other muscles. This study tested the hypothesis that mitochondrial respiration rates are greater in the diaphragm compared with triceps surae (TS, a limb muscle). We isolated mitochondria from diaphragm and TS of adult male Sprague Dawley rats. Mitochondrial respiration was measured by polarography. The contents of respiratory complexes, uncoupling proteins 1, 2, and 3 (UCP1, UCP2, and UCP3), and voltage-dependent anion channel 1 (VDAC1) were determined by immunoblotting. Complex IV activity was measured by spectrophotometry. Mitochondrial respiration states 3 (substrate and ADP driven) and 5 (uncoupled) were 27 ± 8% and 24 ± 10%, respectively, lower in diaphragm than in TS (P < 0.05 for both comparisons). However, the contents of respiratory complexes III, IV, and V, UCP1, and VDAC1 were higher in diaphragm mitochondria (23 ± 6, 30 ± 8, 25 ± 8, 36 ± 15, and 18 ± 8% respectively, P ≤ 0.04 for all comparisons). Complex IV activity was 64 ± 16% higher in diaphragm mitochondria (P ≤ 0.01). Mitochondrial UCP2 and UCP3 content and complex I activity were not different between TS and diaphragm. These data indicate that diaphragm mitochondria respire at lower rates, despite a higher content of respiratory complexes. The results invalidate our initial hypothesis and indicate that mitochondrial content is not the only determinant of aerobic capacity in the diaphragm. We propose that UCP1 and VDAC1 play a role in regulating diaphragm aerobic capacity.

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments.

Efficient and specific phosphorylation of PKA substrates, elicited in response to ß-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Chronic hypoxia increases insulin-stimulated glucose uptake in mouse soleus muscle.

People living at high altitude appear to have lower blood glucose levels and decreased incidence of diabetes. Faster glucose uptake and increased insulin sensitivity are likely explanations for these findings: skeletal muscle is the largest glucose sink in the body, and its adaptation to the hypoxia of altitude may influence glucose uptake and insulin sensitivity. This study tested the hypothesis that chronic normobaric hypoxia increases insulin-stimulated glucose uptake in soleus muscles and decreases plasma glucose levels. Adult male C57BL/6J mice were kept in normoxia [fraction of inspired O2 = 21% (Control)] or normobaric hypoxia [fraction of inspired O2 = 10% (Hypoxia)] for 4 wk. Then blood glucose and insulin levels, in vitro muscle glucose uptake, and indexes of insulin signaling were measured. Chronic hypoxia lowered blood glucose and plasma insulin [glucose: 14.3 ± 0.65 mM in Control vs. 9.9 ± 0.83 mM in Hypoxia (P < 0.001); insulin: 1.2 ± 0.2 ng/ml in Control vs. 0.7 ± 0.1 ng/ml in Hypoxia (P < 0.05)] and increased insulin sensitivity determined by homeostatic model assessment 2 [21.5 ± 3.8 in Control vs. 39.3 ± 5.7 in Hypoxia (P < 0.03)]. There was no significant difference in basal glucose uptake in vitro in soleus muscle (1.59 ± 0.24 and 1.71 ± 0.15 µmol·g⁻¹·h⁻¹ in Control and Hypoxia, respectively). However, insulin-stimulated glucose uptake was 30% higher in the soleus after 4 wk of hypoxia than Control (6.24 ± 0.23 vs. 4.87 ± 0.37 µmol·g⁻¹·h⁻¹, P < 0.02). Muscle glycogen content was not significantly different between the two groups. Levels of glucose transporters 4 and 1, phosphoinositide 3-kinase, glycogen synthase kinase 3, protein kinase B/Akt, and AMP-activated protein kinase were not affected by chronic hypoxia. Akt phosphorylation following insulin stimulation in soleus muscle was significantly (25%) higher in Hypoxia than Control (P < 0.05). Neither glycogen synthase kinase 3 nor AMP-activated protein kinase phosphorylation changed after 4 wk of hypoxia. These results demonstrate that the adaptation of skeletal muscles to chronic hypoxia includes increased insulin-stimulated glucose uptake.

Glucose uptake in rat extraocular muscles: effect of insulin and contractile activity.

PURPOSE: Extraocular muscles show specific adaptations to fulfill the metabolic demands imposed by their constant activity. One aspect that has not been explored is the availability of substrate for energy pathways in extraocular muscles. In limb muscles, glucose enters by way of GLUT1 and GLUT4 transporters in a process regulated by insulin and contractile activity to match metabolic supply to demand. This mechanism may not apply to extraocular muscles because their constant activity may require high basal (insulin- and activity-independent) glucose uptake. The authors tested the hypothesis that glucose uptake by extraocular muscles is not regulated by insulin or contractile activity. METHODS: Extraocular muscles from adult male Sprague-Dawley rats were incubated with 100 nM insulin or were electrically stimulated to contract (activity); glucose uptake was measured with 2-deoxy-d[1,2-(3)H]glucose. The contents of GLUT1, GLUT4, total and phosphorylated protein kinase B (Akt), phosphorylated AMP-activated protein kinase (AMPK), and glycogen synthase kinase 3 (GSK3) underwent Western blot analysis. RESULTS: Insulin and activity increased glucose uptake over the basal rate to 108% and 78%, respectively. GLUT1 and GLUT4 were detectable in extraocular muscles. Phosphorylated AKT/total AKT increased by twofold after insulin stimulation, but there was no change with activity. AMPK phosphorylation increased 35% with activity. Phosphorylated-GSK3/total GSK3 did not change with insulin or activity. CONCLUSIONS: Glucose uptake in extraocular muscles is regulated by insulin and contractile activity. There is evidence of differences in the insulin signaling pathway that may explain the low glycogen content in these muscles.

Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

Ca(2+) desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (DeltaK210) in cardiac troponin T (cTnT). DeltaK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the deletion. In this study we investigate the impact of K210 deletion on phosphorylation propensity of sarcomeric proteins. Analysis of cardiac myofibrils isolated from DeltaK210 hearts identified a decrease in phosphorylation of cTnI (46%), cTnT (30%) and MyBP-C (32%) compared with wild-type controls. Interestingly, immunoblot analyses with phospho-specific antibodies show augmented phosphorylation of cTnT-Thr(203) (28%) and decreased phosphorylation of cTnI-Ser(23/24) (41%) in mutant myocardium. In vitro kinase assays indicate that DeltaK210 increases phosphorylation propensity of cTnT-Thr(203) three-fold, without changing cTnI-Ser(23/24) phosphorylation. Molecular modeling of cTnT-DeltaK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr(203), which may explain the enhanced PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-DeltaK210 binds stronger to cTnI compared with cTnT-wt. Collectively, our observations suggest that cardiomyopathy-causing DeltaK210 has far-reaching effects influencing cTnI-cTnT binding and posttranslational modifications of key sarcomeric proteins.

Impact of cardiac troponin T N-terminal deletion and phosphorylation on myofilament function.

Cardiac troponin T (cTnT) is a phosphoprotein that modulates cardiac muscle contraction through its extensive and diverse interactions with neighboring thin filament proteins. Its N-terminal half is the "glue" that anchors the troponin complex to tropomyosin-actin. Until now, studies aimed at investigating the role of the N-terminal tail region have not considered the effects of phosphorylation. To understand better the regulatory role of the N-terminal tail region of phosphorylated cTnT, we investigated the functional effects of N-terminal deletion (amino acids 1-91) and phosphorylation on Ca(2+) dependence of myofilament isometric force production, isometric ATPase rate, and thin filament sliding speed. Chemomechanical profiles were assessed in detergent permeabilized fiber preparations where the native troponin (cTn) was exchanged with recombinant cTn engineered to contain modified cTnT (truncated, phosphorylated) in the presence of wild-type cTnI and cTnC. Removal of the cTnT N-terminal amino acids 1-91 (cTnT-del) enhances myofilament responsiveness to nonsaturating Ca(2+) levels (the physiological range in cardiac myocytes). However, at saturating Ca(2+) levels, there is a reduction in isometric tension and ATPase rate. On one hand, phosphorylation of cTnT-del attenuates the sensitizing effect induced by truncation of the N-terminal tail, "resetting" myofilament Ca(2+) responsiveness back to control levels. On the other hand, it impairs isometric tension development and ATPase rate. Interestingly, phosphorylation of cTnT (cTnT-P) differentially regulates tension cost (an index of cross-bridge cycling rate): increased by cTn-del-P and decreased by intact cTn-wt-P. Like the isometric fiber data, sliding speed of thin filaments regulated by cTn-del is more sensitive to Ca(2+) compared with cTn-wt. Phosphorylation of cTnT (whether cTnT-del or -wt) depresses sliding speed and is associated with Ca(2+) desensitization of thin filament sliding speed.

The alpha1D-adrenergic receptor induces vascular smooth muscle apoptosis via a p53-dependent mechanism.

Activation of the endogenous alpha1-adrenergic receptor (AR) associated with human aortic smooth muscle cells resulted in a dose- and time-dependent increase in the levels of mitochondrial reactive oxygen species (ROS). ROS increases were apparent within 10 min and maximal after 45 min. Prolonged activation (>4 h) of the alpha1-AR resulted in smooth muscle cell apoptosis. Both the increase in ROS and apoptotic cell death were blocked by the nonselective alpha1-AR antagonist prazosin as well as the selective alpha1D-AR antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7, 9-dione (BMY 7378). Increases in ROS and apoptosis produced by alpha1-AR activation were also blocked by the p38 mitogen-activated protein kinase inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190) and the NAPDH oxidase inhibitor apocynin. The extracellular signal-regulated kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD 98059) or the c-Jun NH2-terminal kinase inhibitor 1, 9-pyrazoloanthrone anthra(1, 9-cd)pyrazol-6(2H)-one (SP 600125) was without effect on increases in ROS levels or apoptosis. Pifithrin-alpha, an inhibitor of the tumor suppressor protein p53, had no effect on ROS generation but did block alpha1D-AR-induced apoptosis. Activation of the alpha1D-AR resulted in translocation of p53 to the mitochondria. The mitochondrial translocation of p53 was blocked by prazosin, BMY 7378, apocynin, SB 202190, and pifithrin-alpha. Apoptosis was also blocked by small interfering RNA directed against p53. These data show that the alpha1D-AR is coupled to the generation of mitochondrial ROS by a pathway involving p38 and NADPH oxidase. Sustained activation of the alpha1D-AR results in smooth muscle cell apoptosis in a pathway that involves the tumor suppressor protein p53 and the mitochondrial translocation of p53. The data also provide evidence of a linkage between the alpha1D-AR and p53.

The alpha1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells.

BACKGROUND: The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. RESULTS: The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR. CONCLUSION: Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.