Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT₂ and 5-HT₇ receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca²⁺] in a dose-dependent manner (EC₅₀ = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]_i (EC₅₀ = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT_2α or Cv5-HT₇ in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT_2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT₇ receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT₇ in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca²⁺- and cAMP-signalling cascades.     

Short-Term Effects of Particulate Matter on Stroke Attack: Meta-Regression and Meta-Analyses

Background and Purpose

Currently there are more and more studies on the association between short-term effects of exposure to particulate matter (PM) and the morbidity of stroke attack, but few have focused on stroke subtypes. The objective of this study is to assess the relationship between PM and stroke subtypes attack, which is uncertain now.

Methods

Meta-analyses, meta-regression and subgroup analyses were conducted to investigate the association between short-term effects of exposure to PM and the morbidity of different stroke subtypes from a number of epidemiologic studies (from 1997 to 2012).

Results

Nineteen articles were identified. Odds ratio (OR) of stroke attack associated with particular matter (“thoracic particles” [PM₁₀]<10 µm in aerodynamic diameter, “fine particles” [PM_2.5]<2.5 µm in aerodynamic diameter) increment of 10 µg/m³ was as effect size. PM₁₀ exposure was related to an increase in risk of stroke attack (OR per 10 µg/m³ = 1.004, 95%CI: 1.001∼1.008) and PM_2.5 exposure was not significantly associated with stroke attack (OR per 10 µg/m³ = 0.999, 95%CI: 0.994∼1.003). But when focused on stroke subtypes, PM_2.5 (OR per 10 µg/m³ = 1.025; 95%CI, 1.001∼1.049) and PM₁₀ (OR per 10 µg/m³ = 1.013; 95%CI, 1.001∼1.025) exposure were statistically significantly associated with an increased risk of ischemic stroke attack, while PM_2.5 (all the studies showed no significant association) and PM₁₀ (OR per 10 µg/m³ = 1.007; 95%CI, 0.992∼1.022) exposure were not associated with an increased risk of hemorrhagic stroke attack. Meta-regression found study design and area were two effective covariates.

Conclusion

PM_2.5 and PM₁₀ had different effects on different stroke subtypes. In the future, it''s worthwhile to study the effects of PM to ischemic stroke and hemorrhagic stroke, respectively.     

TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

Background

RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.

Methods and Results

TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.

Conclusion

RP105 is a potent inhibitor of post-interventional neointima formation.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

The Mast Cell Degranulator Compound 48/80 Directly Activates Neurons

Background

Compound 48/80 is widely used in animal and tissue models as a “selective” mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents.

Methodology/Principal Findings

We used in vivo recordings from extrinsic intestinal afferents together with Ca⁺⁺ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca⁺⁺ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca⁺⁺ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H₁ and H₂ antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca⁺⁺ transients in mast cell-free enteric neuron cultures.

Conclusions/Significance

The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.     

99mTc-3P4-RGD2 Scintimammography in the Assessment of Breast Lesions: Comparative Study with 99mTc-MIBI

Purpose

To compare the potential application of ^99mTc-3P-Arg-Gly-Asp (^99mTc-3P₄-RGD₂) scintimammography (SMM) and ^99mTc-methoxyisobutylisonitrile (^99mTc-MIBI) SMM for the differentiation of malignant from benign breast lesions.

Method

Thirty-six patients with breast masses on physical examination and/or suspicious mammography results that required fine needle aspiration cytology biopsy (FNAB) were included in the study. ^99mTc-3P₄-RGD₂ and ^99mTc-MIBI SMM were performed with single photon emission computed tomography (SPECT) at 60 min and 20 min respectively after intravenous injection of 738±86 MBq radiotracers on a separate day. Images were evaluated by the tumor to non-tumor localization ratios (T/NT). Receiver operating characteristic (ROC) curve analysis was performed on each radiotracer to calculate the cut-off values of quantitative indices and to compare the diagnostic performance for the ability to differentiate malignant from benign diseases.

Results

The mean T/NT ratio of ^99mTc-3P₄-RGD₂ in malignant lesions was significantly higher than that in benign lesions (3.54±1.51 vs. 1.83±0.98, p<0.001). The sensitivity, specificity, and accuracy of ^99mTc-3P₄-RGD₂ SMM were 89.3%, 90.9% and 89.7%, respectively, with a T/NT cut-off value of 2.40. The mean T/NT ratio of ^99mTc-MIBI in malignant lesions was also significantly higher than that in benign lesions (2.86±0.99 vs. 1.51±0.61, p<0.001). The sensitivity, specificity and accuracy of ^99mTc-MIBI SMM were 87.5%, 72.7% and 82.1%, respectively, with a T/NT cut-off value of 1.45. According to the ROC analysis, the area under the curve for ^99mTc-3P₄-RGD₂ SMM (area = 0.851) was higher than that for ^99mTc-MIBI SMM (area = 0.781), but the statistical difference was not significant.

Conclusion

^99mTc-3P₄-RGD₂ SMM does not provide any significant advantage over the established ^99mTc-MIBI SMM for the detection of primary breast cancer. The T/NT ratio of ^99mTc-3P₄-RGD₂ SMM was significantly higher than that of ^99mTc-MIBI SMM. Both tracers could offer an alternative method for elucidating non-diagnostic mammograms.     

Calpain Inhibition Reduces Amplitude and Accelerates Decay of the Late Sodium Current in Ventricular Myocytes from Dogs with Chronic Heart Failure

Calpain is an intracellular Ca²⁺ -activated protease that is involved in numerous Ca²⁺ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca²⁺ -dependent increase of the late sodium current (I_NaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell I_NaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which I_NaL was activated by the presence of a higher (1μM) intracellular [Ca²⁺] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1–2 h before I_NaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of I_NaL decay evaluated by the two-exponential fit (τ₁ = 42±3.0 ms τ₂ = 435±27 ms, n = 6, in MDL vs. τ₁ = 52±2.1 ms τ₂ = 605±26 control no vehicle, n = 11, and vs. τ₁ = 52±2.8 ms τ₂ = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA). MDL significantly reduced I_NaL density recorded at –30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage relationships demonstrated that the I_NaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca²⁺ accumulation in the pulse train.

Conclusions

Calpain inhibition reverses I_NaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca²⁺. Specifically it decreases I_NaL density and accelerates I_NaL kinetics resulting in improvement of myocyte electrical response and Ca²⁺ handling as predicted by our in silico simulations.     

Impaired Chemosensitivity of Mouse Dorsal Raphe Serotonergic Neurons Overexpressing Serotonin 1A (Htr1a) Receptors

Background

Serotonergic system participates in a wide range of physiological processes and behaviors, but its role is generally considered as modulatory and noncrucial, especially concerning life-sustaining functions. We recently created a transgenic mouse line in which a functional deficit in serotonin homeostasis due to excessive serotonin autoinhibition was produced by inducing serotonin 1A receptor (Htr1a) overexpression selectively in serotonergic neurons (Htr1a raphe-overexpressing or Htr1a^RO mice). Htr1a^RO mice exhibit episodes of autonomic dysregulation, cardiovascular crises and death, resembling those of sudden infant death syndrome (SIDS) and revealing a life-supporting role of serotonergic system in autonomic control. Since midbrain serotonergic neurons are chemosensitive and are implicated in arousal we hypothesized that their chemosensitivity might be impaired in Htr1a^RO mice.

Principal findings

Loose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1a^RO mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO₂ produced an increase in firing rate of serotonergic neurons (0.260±0.041 Hz, n = 20, p = 0.0001) and application of 3% CO₂ decreased their firing rate (−0.142±0.025 Hz, n = 17, p = 0.0008). In contrast, in Htr1a^RO mice, firing rate of serotonergic neurons was not significantly changed by 9% CO₂ (0.021±0.034 Hz, n = 16, p = 0.49) and by 3% CO₂ (0.012±0.046 Hz, n = 12, p = 0.97).

Conclusions

Our findings support the hypothesis that chemosensitivity of midbrain serotonergic neurons provides a physiological mechanism for arousal responses to life-threatening episodes of hypercapnia and that functional impairment, such as excessive autoinhibition, of midbrain serotonergic neuron responses to hypercapnia may contribute to sudden death.     

The Effects of Ca2+ and MgADP on Force Development during and after Muscle Length Changes

The goal of this study was to compare the effects of Ca²⁺ and MgADP activation on force development in skeletal muscles during and after imposed length changes. Single fibres dissected from the rabbit psoas were (i) activated in pCa²⁺4.5 and pCa²⁺6.0, or (ii) activated in pCa²⁺4.5 before and after administration of 10 mM MgADP. Fibres were activated in sarcomere lengths (SL) of 2.65 µm and 2.95 µm, and subsequently stretched or shortened (5%SL at 1.0 SL.s⁻¹) to reach a final SL of 2.80 µm. The kinetics of force during stretch were not altered by pCa²⁺ or MgADP, but the fast change in the slope of force development (P₁) observed during shortening and the corresponding SL extension required to reach the change (L₁) were higher in pCa²⁺6.0 (P₁ = 0.22±0.02 P_o; L₁ = 5.26±0.24 nm.HS^.1) than in pCa²⁺4.5 (P₁ = 0.15±0.01 P_o; L₁ = 4.48±0.25 nm.HS^.1). L₁ was also increased by MgADP activation during shortening. Force enhancement after stretch was lower in pCa²⁺4.5 (14.9±5.4%) than in pCa²⁺6.0 (38.8±7.5%), while force depression after shortening was similar in both Ca²⁺ concentrations. The stiffness accompanied the force behavior after length changes in all situations. MgADP did not affect the force behavior after length changes, and stiffness did not accompany the changes in force development after stretch. Altogether, these results suggest that the mechanisms of force generation during and after stretch are different from those obtained during and after shortening.     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Macroscopic Fluorescence Imaging: A Novel Technique to Monitor Retention and Distribution of Injected Microspheres in an Experimental Model of Ischemic Heart Failure

Background

The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections.

Methods and Results

A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5×10⁵) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78×10⁵±0.31×10⁵ in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74×10⁵±0.18×10⁵; p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90×10⁵±0.20×10⁵) and the right (1.07×10⁵±0.17×10⁵) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups.

Conclusions

We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs.     

Improved Glycaemia Correlates with Liver Fat Reduction in Obese,Type 2 Diabetes,Patients Given Glucagon-Like Peptide-1 (GLP-1) Receptor Agonists

Glucagon-like peptide-1 receptor agonists (GLP-1 RA) are effective for obese patients with type 2 diabetes mellitus (T2DM) because they concomitantly target obesity and dysglycaemia. Considering the high prevalence of non-alcoholic fatty liver disease (NAFLD) in patients with T2DM, we determined the impact of 6 months’ GLP-1 RA therapy on intrahepatic lipid (IHL) in obese, T2DM patients with hepatic steatosis, and evaluated the inter-relationship between changes in IHL with those in glycosylated haemoglobin (HbA₁c), body weight, and volume of abdominal visceral and subcutaneous adipose tissue (VAT and SAT). We prospectively studied 25 (12 male) patients, age 50±10 years, BMI 38.4±5.6 kg/m² (mean ± SD) with baseline IHL of 28.2% (16.5 to 43.1%) and HbA₁c of 9.6% (7.9 to 10.7%) (median and interquartile range). Patients treated with metformin and sulphonylureas/DPP-IV inhibitors were given 6 months GLP-1 RA (exenatide, n = 19; liraglutide, n = 6). IHL was quantified by liver proton magnetic resonance spectroscopy (¹H MRS) and VAT and SAT by whole body magnetic resonance imaging (MRI). Treatment was associated with mean weight loss of 5.0 kg (95% CI 3.5,6.5 kg), mean HbA_1c reduction of 1·6% (17 mmol/mol) (0·8,2·4%) and a 42% relative reduction in IHL (−59.3, −16.5%). The relative reduction in IHL correlated with that in HbA₁c (ρ = 0.49; p = 0.01) but was not significantly correlated with that in total body weight, VAT or SAT. The greatest IHL reduction occurred in individuals with highest pre-treatment levels. Mechanistic studies are needed to determine potential direct effects of GLP-1 RA on human liver lipid metabolism.     

Characteristics of Glucose Metabolism in Nordic and South Asian Subjects with Type 2 Diabetes

Background

Insulin resistance and type 2 diabetes are more prevalent in people of South Asian ethnicity than in people of Western European origin. To investigate the source of these differences, we compared insulin sensitivity, insulin secretion, glucose and lipid metabolism in South Asian and Nordic subjects with type 2 diabetes.

Methods

Forty-three Nordic and 19 South Asian subjects with type 2 diabetes were examined with intra-venous glucose tolerance test, euglycemic clamp including measurement of endogenous glucose production, indirect calorimetry measuring glucose and lipid oxidation, and dual x-ray absorptiometry measuring body composition.

Results

Despite younger mean ± SD age (49.7±9.4 vs 58.3±8.3 years, p = 0.001), subjects of South Asian ethnicity had the same diabetes duration (9.3±5.5 vs 9.6±7.0 years, p = 0.86), significantly higher median [inter-quartile range] HbA_1c (8.5 [1.6] vs 7.3 [1.6] %, p = 0.024) and lower BMI (28.7±4.0 vs 33.2±4.7 kg/m², p<0.001). The South Asian group exhibited significantly higher basal endogenous glucose production (19.1 [9.1] vs 14.4 [6.8] µmol/kgFFM⋅min, p = 0.003). There were no significant differences between the groups in total glucose disposal (39.1±20.4 vs 39.2±17.6 µmol/kgFFM⋅min, p = 0.99) or first phase insulin secretion (AUC_{0–8 min}: 220 [302] vs 124 [275] pM, p = 0.35). In South Asian subjects there was a tendency towards positive correlations between endogenous glucose production and resting and clamp energy expenditure.

Conclusions

Subjects of South Asian ethnicity with type 2 diabetes, despite being younger and leaner, had higher basal endogenous glucose production, indicating higher hepatic insulin resistance, and a trend towards higher use of carbohydrates as fasting energy substrate compared to Nordic subjects. These findings may contribute to the understanding of the observed differences in prevalence of type 2 diabetes between the ethnic groups.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Whole-Body Insulin Sensitivity Rather than Body-Mass-Index Determines Fasting and Post-Glucose-Load Growth Hormone Concentrations

Background

Obese, non-acromegalic persons show lower growth hormone (GH) concentrations at fasting and reduced GH nadir during an oral glucose tolerance test (OGTT). However, this finding has never been studied with regard to whole-body insulin-sensitivity as a possible regulator.

Methods

In this retrospective analysis, non-acromegalic (NonACRO, n = 161) and acromegalic (ACRO, n = 35), non-diabetic subjects were subdivided into insulin-sensitive (IS) and –resistant (IR) groups according to the Clamp-like Index (CLIX)-threshold of 5 mg·kg⁻¹·min⁻¹ from the OGTT.

Results

Non-acromegalic IS (CLIX: 8.8±0.4 mg·kg⁻¹·min⁻¹) persons with similar age and sex distribution, but lower (p<0.001) body-mass-index (BMI = 25±0 kg/m², 84% females, 56±1 years) had 59% and 70%, respectively, higher (p<0.03) fasting GH and OGTT GH area under the curve concentrations than IR (CLIX: 3.5±0.1 mg·kg⁻¹·min⁻¹, p<0.001) subjects (BMI = 29±1 kg/m², 73% females, 58±1 years). When comparing on average overweight non-acromegalic IS and IR with similar anthropometry (IS: BMI: 27±0 kg/m², 82% females, 58±2 years; IR: BMI: 27±0 kg/m², 71% females, 60±1 years), but different CLIX (IS: 8.7±0.9 vs. IR: 3.8±0.1 mg·kg⁻¹·min⁻¹, p<0.001), the results remained almost the same. In addition, when adjusted for OGTT-mediated glucose rise, GH fall was less pronounced in IR. In contrast, in acromegalic subjects, no difference was found between IS and IR patients with regard to fasting and post-glucose-load GH concentrations.

Conclusions

Circulating GH concentrations at fasting and during the OGTT are lower in non-acromegalic insulin-resistant subjects. This study seems the first to demonstrate that insulin sensitivity rather than body-mass modulates fasting and post-glucose-load GH concentrations in non-diabetic non–acromegalic subjects.     

Linkage between Increased Nociception and Olfaction via a SCN9A Haplotype

Background and Aims

Mutations reducing the function of Na_v1.7 sodium channels entail diminished pain perception and olfactory acuity, suggesting a link between nociception and olfaction at ion channel level. We hypothesized that if such link exists, it should work in both directions and gain-of-function Na_v1.7 mutations known to be associated with increased pain perception should also increase olfactory acuity.

Methods

SCN9A variants were assessed known to enhance pain perception and found more frequently in the average population. Specifically, carriers of SCN9A variants rs41268673C>A (P610T; n = 14) or rs6746030C>T (R1150W; n = 21) were compared with non-carriers (n = 40). Olfactory function was quantified by assessing odor threshold, odor discrimination and odor identification using an established olfactory test. Nociception was assessed by measuring pain thresholds to experimental nociceptive stimuli (punctate and blunt mechanical pressure, heat and electrical stimuli).

Results

The number of carried alleles of the non-mutated SCN9A haplotype rs41268673C/rs6746030C was significantly associated with the comparatively highest olfactory threshold (0 alleles: threshold at phenylethylethanol dilution step 12 of 16 (n = 1), 1 allele: 10.6±2.6 (n = 34), 2 alleles: 9.5±2.1 (n = 40)). The same SCN9A haplotype determined the pain threshold to blunt pressure stimuli (0 alleles: 21.1 N/m², 1 allele: 29.8±10.4 N/m², 2 alleles: 33.5±10.2 N/m²).

Conclusions

The findings established a working link between nociception and olfaction via Na_v1.7 in the gain-of-function direction. Hence, together with the known reduced olfaction and pain in loss-of-function mutations, a bidirectional genetic functional association between nociception and olfaction exists at Na_v1.7 level.     

Measurement of Pulmonary Flow Reserve and Pulmonary Index of Microcirculatory Resistance for Detection of Pulmonary Microvascular Obstruction

Background

The pulmonary microcirculation is the chief regulatory site for resistance in the pulmonary circuit. Despite pulmonary microvascular dysfunction being implicated in the pathogenesis of several pulmonary vascular conditions, there are currently no techniques for the specific assessment of pulmonary microvascular integrity in humans. Peak hyperemic flow assessment using thermodilution-derived mean transit-time (T_mn) facilitate accurate coronary microcirculatory evaluation, but remain unvalidated in the lung circulation. Using a high primate model, we aimed to explore the use of T_mn as a surrogate of pulmonary blood flow for the purpose of measuring the novel indices Pulmonary Flow Reserve [PFR = (maximum hyperemic)/(basal flow)] and Pulmonary Index of Microcirculatory Resistance [PIMR = (maximum hyperemic distal pulmonary artery pressure)×(maximum hyperemic T_mn)]. Ultimately, we aimed to investigate the effect of progressive pulmonary microvascular obstruction on PFR and PIMR.

Methods and Results

Temperature- and pressure-sensor guidewires (TPSG) were placed in segmental pulmonary arteries (SPA) of 13 baboons and intravascular temperature measured. T_mn and hemodynamics were recorded at rest and following intra-SPA administration of the vasodilator agents adenosine (10–400 µg/kg/min) and papaverine (3–24 mg). Temperature did not vary with intra-SPA sensor position (0.010±0.009 v 0.010±0.009°C; distal v proximal; p = 0.1), supporting T_mn use in lung for the purpose of hemodynamic indices derivation. Adenosine (to 200 µg/kg/min) & papaverine (to 24 mg) induced dose-dependent flow augmentations (40±7% & 35±13% T_mn reductions v baseline, respectively; p<0.0001). PFR and PIMR were then calculated before and after progressive administration of ceramic microspheres into the SPA. Cumulative microsphere doses progressively reduced PFR (1.41±0.06, 1.26±0.19, 1.17±0.07 & 1.01±0.03; for 0, 10⁴, 10⁵ & 10⁶ microspheres; p = 0.009) and increased PIMR (5.7±0.6, 6.3±1.0, 6.8±0.6 & 7.6±0.6 mmHg.sec; p = 0.0048).

Conclusions

Thermodilution-derived mean transit time can be accurately and reproducibly measured in the pulmonary circulation using TPSG. Mean transit time-derived PFR and PIMR can be assessed using a TPSG and adenosine or papaverine as hyperemic agents. These novel indices detect progressive pulmonary microvascular obstruction and thus have with a potential role for pulmonary microcirculatory assessment in humans.     

Brain MRI CO2 Stress Testing: A Pilot Study in Patients with Concussion

Background

There is a real need for quantifiable neuro-imaging biomarkers in concussion. Here we outline a brain BOLD-MRI CO₂ stress test to assess the condition.

Methods

This study was approved by the REB at the University of Manitoba. A group of volunteers without prior concussion were compared to post-concussion syndrome (PCS) patients – both symptomatic and recovered asymptomatic. Five 3-minute periods of BOLD imaging at 3.0 T were studied – baseline 1 (BL1– at basal CO₂ tension), hypocapnia (CO₂ decreased ∼5 mmHg), BL2, hypercapnia (CO₂ increased ∼10 mmHg) and BL3. Data were processed using statistical parametric mapping (SPM) for 1^st level analysis to compare each subject’s response to the CO₂ stress at the p = 0.001 level. A 2^nd level analysis compared each PCS patient’s response to the mean response of the control subjects at the p = 0.05 level.

Results

We report on 5 control subjects, 8 symptomatic and 4 asymptomatic PCS patients. Both increased and decreased response to CO₂ was seen in all PCS patients in the 2^nd level analysis. The responses were quantified as reactive voxel counts: whole brain voxel counts (2.0±1.6%, p = 0.012 for symptomatic patients for CO₂ response < controls and 3.0±5.1%, p = 0.139 for CO₂ response > controls: 0.49±0.31%, p = 0.053 for asymptomatic patients for CO₂ response < controls and 4.4±6.8%, p = 0.281 for CO₂ response > controls).

Conclusions

Quantifiable alterations in regional cerebrovascular responsiveness are present in concussion patients during provocative CO₂ challenge and BOLD MRI and not in healthy controls. Future longitudinal studies must aim to clarify the relationship between CO₂ responsiveness and individual patient symptoms and outcomes.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Specific Localization of β-Arrestin2 in Myenteric Plexus of Mouse Gastrointestinal Tract

β-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of β-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of β-arrestin2 within the gastrointestinal wall is not known. In this study we found that β-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of β-arestin2 was significantly higher in the ileum than the colon. We identified four variants of β-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing β-arrestin2 in the murine myenteric plexus and the co-localization of MOR₁ and β-arrestin2. β-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, β-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of β-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC₅₀: wild type = 5.8 vs. β-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of β-arrestin2 in the myenteric plexus within MOR₁-expressing neurons and provide a relation for direct intracellular crosstalk between MOR₁ receptor activation and β-arrestin2 signaling in the myenteric neurons. β-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.