Exercise based assessment of cardiac autonomic function in type 1 versus type 2 diabetes mellitus.

BACKGROUND: Cardiac autonomic neuropathy (CAN) is a complication of diabetes mellitus (DM) that is associated with increased mortality. Exercise-based assessment of autonomic function has identified diminished parasympathetic reactivation after exercise in type 2 DM. It is postulated herein, that this would be more prominent among those with type 1 DM. METHODS: Sixteen subjects with type 1 DM (age 32.9 ± 10.1 years), 18 subjects with type 2 DM (55.4 ± 8.0 years) and 30 controls (44.0 ± 11.6 years) underwent exercise-based assessment of autonomic function. Two 16-min submaximal bicycle tests were performed followed by 45 min of recovery. On the second test, atropine (0.04 mg/kg) was administered near end-exercise so that all of the recovery occurred under parasympathetic blockade. Plasma epinephrine and norepinephrine levels were measured at rest, during exercise, and during recovery. RESULTS: There were no differences in resting or end-exercise heart rates in the three groups. Parasympathetic effect on RR-intervals during recovery (p < 0.03) and heart rate recovery (p = 0.02) were blunted in type 2 DM. Type 1 DM had higher baseline epinephrine and norepinephrine levels (p < 0.03), and exhibited persistent sympathoexcitation during recovery. CONCLUSIONS: Despite a longer duration of DM in the study patients with type 1 versus type 2 DM, diminished parasympathetic reactivation was not noted in type 1 DM. Instead, elevation in resting plasma catecholamines was noted compared to type 2 DM and controls. The variable pathophysiology for exercise-induced autonomic abnormalities in type 1 versus type 2 DM may impact prognosis.

Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure.

Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under ß3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the ß3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

An assessment of norepinephrine mediated hypertrophy to apoptosis transition in cardiac cells: a signal for cell death.

OBJECTIVES: Heart is an organ which is under a constant work load that generates numerous stress responses. Heart failure is associated with increased plasma norepinephrine (NE) and hypertrophic cell death. Within the current study we try to understand the concentration dependent molecular switch from hypertrophy to apoptosis under stress. METHODS: The effect of increasing concentration of NE on cell death was studied using MTT assay based on which further experimental conditions were decided. Trypan Blue staining and TUNEL assay were done at selected concentrations of NE. Cellular and nuclear morphology at these concentrations was studied using Haematoxylin-Eosin, DAPI and PI stains. The molecular switch between hypertrophy and cell death was studied by expression analysis of ß-MyHC and TNF-α. Rhodamine and DCFH-DA staining were done to evaluate the role of mitochondria and ROS under these conditions. Role of caspases under these transitions was also evaluated. RESULT: NE shows steep falls in cell viability at 50 µM and 100 µM concentrations. The cellular and nuclear morphology is altered at these concentrations along with alterations at molecular level showing a shift from hypertrophy towards cell death. Altered mitochondrial membrane potential and increase in ROS support this which leads to caspase dependent activation of cell death. CONCLUSION: We show that at 50 µM NE, there occurs a transition from cellular hypertrophy towards death. This could be beneficial to prevent hypertrophy induced cardiac cell death and evaluating cardio protective therapeutic targets in vitro.

Defining uremic arterial functional abnormalities in patients recently started on haemodialysis: combined in vivo and ex vivo assessment.

Endothelial dysfunction is a key initiating event in vascular disease in chronic kidney disease (CKD) patients and haemodialysis (HD) patients exhibit significant vascular abnormalities. To understand this further, we examined how ex vivo intrinsic function in isolated arteries correlates with in vivo assessments of cardiovascular status in HD patients. Abdominal fat biopsies were obtained from 11 HD patients and 26 non-uremic controls. Subcutaneous arteries were dissected and mounted on a wire myograph, and cumulative concentration-response curves to noradrenalin, endothelin-1, a thromboxane A2 agonist (U46619), angiotensin II, vasopressin, bradykinin (BK), acetylcholine (ACh) and sodium nitroprusside (SNP) were constructed. Pulse wave velocity and blood pressure were measured in HD patients. Enhanced (P<0.05-0.0001) maximal contractile responses (Rmax) to all spasmogens (particularly vasopressin) were observed in arteries from HD patients compared to controls, and this effect was more pronounced in arteries with an internal diameter>600 µm. The potency (pEC50) of U46619 (P<0.01) and vasopressin (P<0.001) was also increased in arteries>600 µm of HD patients. The maximal relaxant response to the endothelium-dependent dilators ACh and BK were lower in HD patients (P<0.01-P<0.0001) (worse for ACh than BK); however the endothelium-independent dilator SNP was similar in both groups. PWV was significantly correlated with the vasoconstrictor response to vasopressin (P = 0.042) in HD patients. HD patients are primed for hypertension and end organ demand ischaemia by a highly sensitised pressor response. The failure of arterial relaxation is mediated by endothelial dysfunction. Intrinsic vascular abnormalities may be important in sensitising HD patients to recurrent cumulative ischaemic end organ injury.

Noninvasive identification and assessment of functional brown adipose tissue in rodents using hyperpolarized ¹³C imaging.

OBJECTIVE: The recent identification of functional depots of brown adipose tissue (BAT) in adult humans has potential implications for the treatment of obesity. In order to evaluate new therapies aimed at inducing the production of more BAT or activating BAT in humans, it will be important to develop noninvasive methods to assess the functional state of the tissue in vivo. In this study, we investigate the feasibility of using hyperpolarized (13)C imaging to noninvasively identify functional, activated BAT in an in vivo rodent model, in less than 1 min, following an infusion of pre-polarized [1-(13)C] pyruvate. DESIGN: Hyperpolarized (13)C imaging was used to monitor BAT metabolic conversion of pre-polarized [1-(13)C] pyruvate in rats during baseline and norepinephrine (NE)-stimulated conditions. RESULTS: Activated BAT, stimulated by NE injection, can be detected in rats by increased conversion of pre-polarized [1-(13)C] pyruvate into its downstream products (13)C bicarbonate and [1-(13)C] lactate. The colocalization of the (13)C signal to interscapular BAT was validated using hematoxylin-eosin histological staining. CONCLUSION: The radiation-free nature and recent translation into the clinic of the hyperpolarized (13)C-imaging test may potentially facilitate trials of therapeutics targeting BAT activation in humans.

Combining 123I-metaiodobenzylguanidine SPECT/CT and 18F-FDG PET/CT for the assessment of brown adipose tissue activity in humans during cold exposure.

UNLABELLED: Brown adipose tissue (BAT) has become a focus of research in the hope of finding a new target to fight obesity. Metabolic BAT activity can be visualized with (18)F-FDG PET/CT. Furthermore, the sympathetic innervation of BAT can be visualized with the radiolabeled norepinephrine analog (123)I-metaiodobenzylguanidine ((123)I-MIBG). We aimed to determine whether (123)I-MIBG SPECT/CT and (18)F-FDG PET/CT identify the same anatomic regions as active BAT in adult humans. Furthermore, we investigated whether the magnitude of BAT activity measured by these techniques correlated. Finally, we tried to establish the optimal time interval between (123)I-MIBG administration and subsequent SPECT/CT acquisition to visualize sympathetic stimulation of BAT. METHODS: Ten lean (body mass index, 19-25 kg/m(2)), healthy Caucasian men (age, 18-32 y) underwent one (18)F-FDG PET/CT and two (123)I-MIBG-SPECT/CT scans within a 2-wk interval. On 2 separate occasions, the subjects were exposed to mild cold (17°C) for 2 h after an overnight fast. After 1 h of cold exposure, (18)F-FDG (one occasion) or (123)I-MIBG (other occasion) was administered. (18)F-FDG PET/CT was performed at 1 h after (18)F-FDG administration, and (123)I-MIBG-SPECT/CT was performed at 4 and 24 h after (123)I-MIBG injection. RESULTS: (18)F-FDG uptake in BAT was observed in 8 of 10 subjects, whereas (123)I-MIBG uptake was observed in 7 of 10 subjects in both the SPECT/CT scans acquired at 4 h after (123)I-MIBG administration and the SPECT/CT scans acquired at 24 h after (123)I-MIBG administration. All subjects who showed (123)I-MIBG uptake in BAT also showed (18)F-FDG uptake in BAT. There was no statistically significant correlation between maximal standardized uptake value of (18)F-FDG and semiquantitative uptake of (123)I-MIBG at 4 h after administration. However, a positive correlation was found between the maximal standardized uptake value of (18)F-FDG and semiquantitative uptake of (123)I-MIBG at 24 h after administration (r = 0.64, P = 0.04). CONCLUSION: (123)I-MIBG SPECT/CT, as a marker of sympathetic activity, and (18)F-FDG PET/CT, as a marker of metabolic activity, identified the same anatomic regions as active BAT. Moreover, when (123)I-MIBG SPECT/CT was performed at 24 h after (123)I-MIBG administration, the magnitude of BAT activity measured with these techniques correlated strongly. This finding not only supports that BAT activity in humans is sympathetically influenced but also identifies (123)I-MIBG SPECT/CT, when performed 24 h after (123)I-MIBG injection, as a method to visualize and quantify sympathetic stimulation of BAT.

Assessment of antidiabetogenic potential of fermented soybean extracts in streptozotocin-induced diabetic rat.

Most of the available drugs for the treatment of diabetes mellitus (DM) produce detrimental side effects, which has prompted an ongoing search for plant with the antidiabetic potential. The present study investigated the effect of soybean extracts fermented with Bacillus subtilis MORI, fermented soybean extracts (BTD-1) was investigated in streptozotocin (STZ)-induced diabetic rats. The possible effects of BTD-1 against hyperglycemia and free radical-mediated oxidative stress was investigated by assaying the plasma glucose level and the activity of enzymatic antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA). A significant increase in the levels of both plasma glucose and reactive oxygen species (ROS) was observed in the diabetic rats when compared to normal control group. After administration of BTD-1 (500 and 1000 mg/kg/day), the elevated plasma glucose level was significantly reduced while the plasma insulin level and the activities of SOD, GSH-Px, CAT and MDA were significantly increased. The results suggest that administration of BTD-1 can inhibit hyperglycemia and free radical-mediated oxidative stress. The administration of BTD-1 also inhibited the contractile response by norepinephrine (10(-10)-10(-5) M) in the presence of endothelium, and caused significant relaxation by carbachol (10(-8)-10(-5) M) in rat aorta. These findings indicate that BTD-1 improves vascular functions on STZ-induced diabetic rats. Therefore, subchronic administration of BTD-1 could prevent the functional changes in vascular reactivity in STZ-induced diabetic rats. The collective findings support that administration of BTD-1 may prevent some diabetes-related changes in vascular reactivity directly and/or indirectly due to its hypoglycaemic effect and inhibition of production of ROS.

Assessment of an uncalibrated pressure waveform device's ability to track cardiac output changes due to norepinephrine dose adjustments in patients with septic shock: a comparison with Doppler echocardiography.

OBJECTIVES: The FloTrac Vigileo (FTV) estimates cardiac output (CO) on the basis of an uncalibrated arterial pressure waveform. To assess the ability of the third-generation of FTV (v.3.02) to track changes in CO following norepinephrine dose adjustment in patients with septic shock, we performed a comparative study using Doppler echocardiography (DE). STUDY DESIGN: Prospective observational study. PATIENTS: We prospectively included 20 mechanically ventilated patients receiving norepinephrine and monitored with the FTV. Five minutes after each change in norepinephrine dose (decided by the attending physician), CO was measured simultaneously with the FTV (CO(FTV)) and DE (CO(DE)). The changes in CO were compared. ROC curves were built to assess the ability of FTV to detect significant changes in CO(DE) of at least 15%. RESULTS: Ninety pairs of CO variations measurements were made. The intertechnique correlation coefficient for changes in CO of at least 15% was r=0.59; P=0.0009. The AUC of a ROC curve built to test the FTV's ability to detect a CO(DE) increase of 15% or more was 0.783 (±0.083) (P=0.005). A CO(FTV) threshold value of 15% had a sensitivity of 54% (25-81) and a specificity of 87% (77-94). For a CO(DE) decrease of 15% or more, the ROC curve had an AUC of 0.616 (±0.075) (P=0.12) and a CO(FTV) threshold value of 13% yielded a sensitivity of 53% (27-79) and a specificity of 72% (60-82). CONCLUSIONS: The FTV was unable to accurately track changes in CO following norepinephrine dose adjustments in critically ill patients with septic shock.

Regulação autonômica das propriedades mecânicas em bioprótese valvar porcina / Autonomic regulation of mechanical properties in porcine mitral valve cusps / Regulación autonómica de las propiedades mecánicas en bioprótesis valvular porcina

FUNDAMENTO: A presença de nervos nas válvulas cardíacas foi demonstrada pela primeira vez há décadas e identificadas em subpopulações: simpáticas e parassimpáticas, e, portanto, é esperado que as válvulas sejam grandemente afetadas pelos nervos autônomos. Entretanto, poucos estudos têm se concentrado na regulação de válvulas cardíacas pelo sistema nervoso autônomo. OBJETIVO: Buscamos identificar o papel do sistema nervoso autônomo na regulação das propriedades mecânicas dos tecidos de válvulas mitrais porcinas. MÉTODOS: As propriedades mecânicas dos folhetos de válvulas mitrais porcinas foram avaliados em resposta à norepinefrina (NE) e acetilcolina (ACH), os principais neurotransmissores. Ao mesmo tempo, fentolamina (FENT), metoprolol (Metop), atropina (Atrop) e desnudamento endotelial foram adicionados ao sistema reativo. RESULTADOS: Sob condições fisiológicas, a rigidez não foi afetada pelo desnudamento endotelial (p > 0,05). A NE significantemente aumentou a rigidez valvar por aumento de 10 vezes na concentração (10-6 vs 10-7, p < 0,05; 10-5 vs 10-6, p < 0,05). Essa resposta foi amenizada por FENT, Metop ou desnudamento endotelial (p < 0,05); entretanto, manteve-se aumentada de maneira significante quando comparada aos Controles (p < 0,05). A ACH causou uma diminuição na rigidez acompanhada por um aumento em sua concentração (alteração significante na rigidez por aumento de 10 vezes na concentração de ACH, 10-6 vs Controle, p < 0,05; 10-5 vs 10-6, p < 0,05), que foi revertida pelo desnudamento endotelial e Atrop (p > 0,05 vs Controle). CONCLUSÃO: Esses achados ressaltam o papel do sistema nervoso autônomo na regulação das propriedades mecânicas das cúspides de válvula mitral porcina, o que reforça a importância do estado nervoso autônomo no funcionamento ideal da válvula.

BACKGROUND: The presence of nerves in heart valves was first depicted decades ago and identified into subpopulations: sympathetic, parasympathetic. So valves are expected to be greatly affected by the autonomic nerves. However, few studies have focused on the regulation of heart valves by the autonomic nervous system. OBJECTIVE: We sought to identify the role of the autonomic nervous system in the regulation of the mechanical properties of porcine mitral valve tissues. METHODS: Mechanical properties of porcine mitral valve leaflets were evaluated in response to norepinephrine (NE) and acetylcholine (ACH), the main neurotransmitters. At the same time, phentolamine (Phent), metoprolol (Metop), atropine (Atrop) and endothelial denudation were added to the reactive system. RESULTS: Under physiological conditions, the stiffness was not affected by endothelial denudation (p > 0.05). NE elevated the valve stiffness significantly per 10-fold increase in concentration (10-6 vs 10-7, p < 0.05; 10-5 vs 10-6, p < 0.05). This response was mitigated by Phent, Metop or endothelial denudation (p < 0.05), however, it was still increased significantly when compared to Controls (p < 0.05). ACH caused a decrease in stiffness accompanied by an increase in its concentration (significant change in stiffness per 10-fold increase in ACH concentration, 10-6 vs Control, p < 0.05; 10-5 vs 10-6, p < 0.05), which were reversed by endothelial denudation and Atrop (p > 0.05 vs Control). CONCLUSION: These findings highlight the role of the autonomic nervous system in the regulation of the mechanical properties of porcine mitral valve cusps, which underline the importance of autonomic nervous status for optimal valve function.

FUNDAMENTO: La presencia de nervios en las válvulas cardíacas quedó demostrada por primera vez hace algunas décadas e identificadas en sub-poblaciones: simpáticas y parasimpáticas y por lo tanto, lo que se espera es que las válvulas reciban una gran afectación de los nervios autónomos. Sin embargo, pocos estudios se han concentrado en la regulación de válvulas cardíacas a través del sistema nervioso autónomo. OBJETIVO: Buscamos identificar el papel del sistema nervioso autónomo en la regulación de las propiedades mecánicas de los tejidos de las válvulas mitrales porcinas. MÉTODOS: Las propiedades mecánicas de las capas de válvulas mitrales porcinas fueron evaluadas en respuesta a la norepinefrina (NE) y a la acetilcolina (ACH), los principales neurotransmisores. Igualmente, la fentolamina (FENT), el metoprolol (Metop), la atropina (Atrop) y la denudación endotelial también se añadieron al sistema reactivo. RESULTADOS: Bajo condiciones fisiológicas, la rigidez no se afectó por el denudación endotelial (p > 0,05). La NE aumentó significativamente la rigidez valvular con un aumento de 10 veces en la concentración (10-6 vs 10-7, p < 0,05; 10-5 vs 10-6, p < 0,05). Esa respuesta fue amenizada por FENT, Metop o denudación endotelial (p < 0,05); pero se mantuvo aumentada de manera significativa cuando se le comparó con los Controles (p < 0,05). La ACH causó una disminución en la rigidez acompañada por un aumento en su concentración (alteración significativa en la rigidez por el aumento en 10 veces de la concentración de ACH, 10-6 vs Control, p < 0,05; 10-5 vs 10-6, p < 0,05), que fue revertida por la denudación endotelial y Atrop (p > 0,05 vs Control). CONCLUSIÓN: Esos hallazgos destacan el rol del sistema nervioso autónomo en la regulación de las propiedades mecánicas de las cúspides de la válvula mitral porcina, lo que refuerza la importancia del estado nervioso autónomo en el funcionamiento ideal de la válvula.

[Efficacy, safety and cost of sedation with sevoflurane in intensive care unit]. / Efficacité, tolérance et coût d'une sédation par sévoflurane en réanimation.

OBJECTIVES: To study efficacy, systemic and cerebral haemodynamic, and cost of sedation with sevoflurane after midazolam failure. STUDY DESIGN: Prospective observational study in a mixed intensive care unit. PATIENTS AND METHODS: Mechanically ventiled patients in whom deep sedation failed (Ramsay score<5 despite midazolam 10mg/h and fentanyl 400µg/h) were enrolled. Sedation with sevoflurane and fentanyl (200µg/h) was performed during 48 hours. Sevoflurane was administered with a dedicated filter (AnaConDa™) and sevoflurane infusion rate was adjusted in order to achieve a Ramsay score ≥5. Ramsay score, mean arterial blood pressure, norepinephrine dose/24h, intracranial pressure and cerebral perfusion pressure in patients with brain injury were measured. Directs costs for sedation were calculated. An analysis of variance for repeated measures compared values between D0 (intravenous sedation), D1 and D2 (inhaled sedation). RESULTS: Twenty-five patients (age=51 [38-63], SAPS II=43 [33-49]) were enrolled. Ramsay score was 4 [4,5] at D0 and 6 [6] at D1 and D2 (P<0.05 vs D0). Mean arterial pressure was significantly lower at D1 (80 [73-86] mmHg) as compared to D0 (84 [77-92] mmHg) and D2 (84 [78-91] mmHg) (P<0,05). Norepinephrine consumption was lower at D2 as compared to D1 (P<0,05). Intracranial pressure was lower at D1 (9 [5-13] mmHg) and D2 (11 [7-15] mmHg) as compared to D0 (12 [7-17] mmHg) (P<0.05). PPC was stable at D1 and increased at D2. Directs costs were significantly increased with sevoflurane. CONCLUSION: Sevoflurane is an effective and safe alternative to midazolam in ICU patients associated with a moderate increase in costs.

Assessment of blood flow changes in human skin by microdialysis urea clearance.

OBJECTIVE: The aim of this study was to evaluate the urea clearance technique for the measurement of drug-induced blood flow changes in human skin and compare it to two non-invasive techniques: polarization light spectroscopy and laser Doppler perfusion imaging. METHODS: Fifteen microdialysis catheters were placed intracutaneously on the volar aspect of the forearms of healthy human subjects and were perfused with nitroglycerine, noradrenaline, and again nitroglycerine to induce local tissue hyperemia, hypoperfusion, and hyperemia, respectively. RESULTS: Urea clearance, but not the other techniques, detected the changes in blood flow during changes in flow. The last hyperemic response was detected by all three methods. CONCLUSION: Urea clearance can be used as a relatively simple method to estimate blood flow changes during microdialysis of vasoactive substances, in particular when the tissue is preconditioned in order to enhance the contrast between baseline and the responses to the provocations. Our results support that, in the model described, urea clearance was superior to the optical methods as it detected both the increases and decrease in blood flow, and the returns to baseline between these periods.

Tissue viability imaging for assessment of pharmacologically induced vasodilation and vasoconstriction in human skin.

Tissue viability imaging (TIVI) is a novel polarization spectroscopy method for assessing dermal vascular viability. The purpose of the present study was to compare TIVI with laser Doppler flowmetry (LDF) for assessment of pharmacologically induced vasodilation and vasoconstriction in human skin. Eight individual skin sites on the backs of seven healthy volunteers were randomized to receive an intradermal injection of prostaglandin E2 (PGE2, 10(-6) to 10(-9)M), norepinephrine (NE, 10(-5) to 10(-7)M), or vehicle. Vascular responses were measured by TIVI and LDF at the injection sites at 1-min intervals starting 2min before and ending 15min after the skin challenge. TIVI and LDF demonstrated significant dose-dependent and time-related vasodilator responses to PGE2 and vasoconstrictor responses to NE, respectively (p<0.001). The time course and dose-response functions for LDF and TIVI showed notable differences. Dose-response data showed a significant reduction in TIVI signal with NE 10-7M (10(-6) NE with LDF) whereas PGE2 10(-6)M was required to elicit a significant increase in TIVI signal (10(-8)M PGE2 with LDF). TIVI demonstrated relative vascular response changes of 0.79 to 1.63 of baseline values at 15min with NE 10(-5)M or PGE2 10(-6)M compared to values of 0.59 to 8.38 with LDF. There was a modest though significant correlation between relative changes in vascular responses measured by the two methods (p<0.0001, r(2)=0.521). A Bland-Altman difference plot demonstrated significant underestimation of relative increase versus baseline measured by TIVI (r(2)=0.99, p<0.0001). We conclude that TIVI polarization spectroscopy is a sensitive method for measurement of NE-induced vascular responses but that it is less sensitive than LDF for measurement of the PGE2-induced reactions.

Computational mapping of the conformational transitions in agonist selective pathways of a G-protein coupled receptor.

The active state conformation of a G-protein coupled receptor (GPCR) is influenced by the chemical structure and the efficacy of the bound ligand. Insight into the active state conformation as well as the activation pathway for ligands with different efficacies is critical in designing functionally specific drugs for GPCRs. Starting from the crystal structure of the beta2-adrenergic receptor, we have used coarse grain computational methods to understand the modulation of the potential energy landscape of the receptor by two full agonists, two partial agonists, and an inverse agonist. Our coarse grain method involves a systematic conformational spanning of the receptor transmembrane helices followed by an energy minimization and ligand redocking in each sampled conformation. We have derived the activation pathways for several agonists and partial agonists, using a Monte Carlo algorithm, and these are in agreement with fluorescence spectroscopy measurements. The calculated pathways for the full agonists start with an energy downhill step leading to a stable intermediate followed by a barrier crossing leading to the active state. We find that the barrier crossing involves breaking of an interhelical hydrogen bond between helix5 and helix6, and polarization of the binding site residues by water facilitates the barrier crossing. The uphill step in the partial agonist salbutamol induced activation is distinct from full agonist norepinephrine, and originates from steric hindrance with the aromatic residues on helix6. Virtual ligand screening with the salbutamol-stabilized conformation shows enrichment of noncatechol agonists over the norepinephrine-stabilized conformation. Our computational method provides an unprecedented opportunity to derive hypotheses for experiments and also understand activation mechanisms in GPCRs.

Vasorelaxant effect in rat aortic rings through calcium channel blockage: a preliminary in vitro assessment of a 1,3,4-oxadiazole derivative.

The study was undertaken on the basis of several reports in the literature that relaxation of vascular smooth muscles is a good treatment strategy in hypertension, angina and other cardiovascular disorders. Oxadiazoles have been reported to have effect on vascular smooth muscles and calcium influx. The goals of our current in vitro study were to investigate the effect of a 1,3,4-oxadiazole derivative on vascular smooth muscles in rat aorta, and to elucidate the associated signaling pathway. NOX-1 induced a relaxation of vascular smooth muscles in both endothelium intact and denuded rat aortic rings precontracted with norepinephrine or phenylephrine or KCl. NOX-1 also significantly antagonized cumulative dose-response effect of norepinephrine, phenylephrine, KCl or calcium with reduction in submaximal contractions. Verapamil, an L-type of calcium channel blocker, effectively attenuated phenylephrine and calcium induced contractions in aortic rings. Incubation with NOX-1 and verapamil did not significantly alter the dose-response curve of phenylephrine or calcium compared to verapamil treatment alone indicating L-type Ca2+ channel blockage leads to loss of NOX-1 activity. Hence it can be concluded NOX-1 exhibited vasorelaxant action by inhibiting calcium influx from extracellular space to intracellular space through L-type of calcium channels.

Cyclonatsudamine A, a new vasodilator cyclic peptide from Citrus natsudaidai.

A new cyclic heptapeptide, cyclonatsudamine A (1), cyclo (-Gly-Tyr-Leu-Leu-Pro-Pro-Ser-), has been isolated from the peels of Citrus natsudaidai and the structure was elucidated by 2D NMR analysis and chemical degradation. Cyclonatsudamine A (1) relaxed norepinephrine-induced contractions of rat aorta, which may be mediated through the increased release of NO from endothelial cells.

Assessment of vasoconstrictive potential of D-lysergic acid using an isolated bovine lateral saphenous vein bioassay.

Vasoconstriction has been associated with several symptoms of fescue toxicosis thought to be alkaloid induced. Lysergic acid, an ergot alkaloid, has been proposed as a toxic component of endophyte-infected tall fescue. The objective of this study was to examine the vasoconstrictive potential of D-lysergic acid using a bovine lateral (cranial branch) saphenous vein bioassay. Before testing lysergic acid, validation of the bovine lateral saphenous vein bioassay for use with a multimyograph apparatus was conducted using a dose-response to norepinephrine to evaluate the effects of limb of origin (right vs. left) and overnight storage on vessel contractile response. Segments (2 to 3 cm) of the cranial branch of the lateral saphenous vein were collected from healthy mixed breed cattle (n = 12 and n = 7 for the lysergic acid and norepinephrine experiments, respectively) at local abattoirs. Tissue was placed in modified Krebs-Henseleit, oxygenated buffer and kept on ice or stored at 2 to 8 degrees C until used. Veins were trimmed of excess fat and connective tissue, sliced into 2- to 3-mm sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O2, 5% CO2; pH = 7.4; 37 degrees C). Tissue was allowed to equilibrate at 1 g of tension for 90 min before initiation of treatment additions. Increasing doses of norepinephrine (1 x 10(-8) to 5 x 10(-4) M) or lysergic acid (1 x 10(-11) to 1 x 10(-4) M) were administered every 15 min after buffer replacement. Data were normalized as a percentage of the contractile response induced by a reference dose of norepinephrine. Veins from both left and right limbs demonstrated contractions in a dose-dependent manner (P < 0.01) but did not differ between limbs. There were no differences in dose-response to norepinephrine between tissue tested the day of dissection and tissue tested 24 h later. Exposure of vein segments to increasing concentrations of lysergic acid did not result in an appreciable contractile response until the addition of 1 x 10(-4) M lysergic acid (15.6 +/- 2.3% of the 1 x 10(-4) M norepinephrine response). These data indicate that only highly elevated concentrations of lysergic acid result in vasoconstriction. Thus, in relation to the symptoms associated with vasoconstriction, lysergic acid may only play a minor role in the manifestation of fescue toxicosis.

An assessment of the role of reactive oxygen species and redox signaling in norepinephrine-induced apoptosis and hypertrophy of H9c2 cardiac myoblasts.

Cardiac myocytes, upon exposure to increasing doses of norepinephrine (NE), transit from hypertrophic to apoptotic phenotype. Since reactive oxygen species (ROS) generation is attributed to both phenomena, the authors tested whether an elevation in intracellular ROS level causes such transition. H9c2 cardiac myoblasts upon treatment with hypertrophic and apoptotic doses of NE (2 and 100 microM, respectively) transiently induced intracellular ROS at a comparable level, while 200 microM H(2)O(2), another proapoptotic agonist, showed robust and sustained ROS generation. Upon analysis of a number of redox-responsive transcription factors as the downstream targets of ROS signaling, the authors observed that NE (2 and 100 microM) and H(2)O(2) (200 microM) were ineffective in inducing NF-kappaB while both the agonists upregulated AP-1 and Nrf-2. However, the extents of induction of AP-1 and Nrf-2 were not in direct correlation with the respective ROS levels. Also, AP-1 activities induced by two doses of NE were intrinsically different, since at 2 microM, it primarily induced FosB, and at 100 microM it activated Fra-1. Differential induction of FosB and Fra-1 was also reiterated in adult rat myocardium injected with increasing doses of NE. Therefore, NE induces hypertrophy and apoptosis in cardiac myocytes by distinct redox-signaling rather than a general surge of ROS.

Thiazide-like diuretics attenuate agonist-induced vasoconstriction by calcium desensitization linked to Rho kinase.

Lowering blood pressure using thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, has been proven to be effective in clinical studies. However, the mechanisms by which thiazide-like diuretics lower blood pressure are still poorly understood. To evaluate whether thiazide-like diuretics cause calcium desensitization in smooth muscle cells, we measured their effects on agonist-induced increase of blood pressure in Wistar rats in vivo and on agonist-induced vasoconstriction of aortic rings, DNA synthesis, and protein synthesis, RhoA, Rho kinase, and intracellular calcium in vascular smooth muscle cells in vitro. Thiazide-like diuretics significantly attenuated angiotensin II-induced or norepinephrine-induced increase of systolic blood pressure in rats. Thiazide-like diuretics inhibited agonist-induced vasoconstriction of aortic rings in a concentration-dependent manner in the presence and absence of endothelium. The inhibitory effects of thiazide-like diuretics were similar to that of the specific Rho kinase inhibitor Y27632. RT-PCR and immunoblotting showed that RhoA and Rho kinase were significantly reduced in vascular smooth muscle cells after administration of thiazide-like diuretics. In contrast, thiazide-like diuretics did not affect protein tyrosine phosphatase-2 (SHP-2) expression. Agonist-induced changes of intracellular calcium were not affected by thiazide-like diuretics. The study indicates that thiazide-like diuretics inhibit agonist-induced vasoconstriction by calcium desensitization in smooth muscle cells linked to the Rho-Rho kinase pathway.

Functional assessment of cryopreserved human femoral arteries for pharmaceutical research.

An established method for cryopreservation that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. HFAs were harvested from multiorgan donors and stored at 4 degrees C in saline solution before cryostorage. Thirty HFA rings were isolated and randomly assigned to one control group of unfrozen HFAs (eight rings) and one group of cryopreserved HFAs (22 rings). Cryopreservation was performed in RPMI solution containing dimethylsulfoxide (DMSO) and the rate of cooling was -1 degrees C/min until -40 degrees C and faster rates until -150 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs. After thawing, the maximal contractile responses to the contracting agonist tested (noradrenaline) were in the range of 43% of the responses in unfrozen HFAs. The endothelium-independent responses to sodium nitroprusside were not altered whereas the endothelium-dependent relaxant responses to acetylcholine were weakly altered. The cryopreservation method used provided a limited preservation of contractility of HFAs, a good preservation of the endothelium-independent relaxant responses, and a good preservation of endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of DMSO, might allow better post-thaw functional recovery.

Functional assessment of cryopreserved pig aortas for pharmaceutical research.

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached. After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.