Cardiac β-adrenoceptors and adenylyl cyclase activity in human left ventricular hypertrophy due to pressure overload

Summary— The effect of left ventricular hypertrophy (LVH) due to chronic pressure overload on right atrial (RA) and left ventricular (LV) myocardial β-adrenergic receptor (β-AR) density and subtypes, adenylyl cyclase (AC) activity and ADP-pertussis toxin ribosylated proteins was investigated in humans with LVH due to aortic stenosis and in patients without LVH undergoing heart surgery for mitral stenosis or coronary artery disease taken as controls. Both groups presented normal systolic function or plasma catecholamine levels. In LVH and controls, β-AR density was similar in RA (62 ± 6 vs 77 ± 12 fmol·mg^?1 protein) and LV (39 ± 7 vs 32 ± 2 fmol·mg^?1 protein). In LVH, β₁-AR percentage was < than in controls in LV (35 ± 11 vs 73 ± 5%, P < 0.05) but not in RA (79 ± 5 vs 73 ± 8%). Basal AC activity in RA (19 ± 4 vs 21 ± 6 pmol·;mg^?1 protein) and LV (22 ± 5 vs 27 ± 3 pmol·mg^?1 protein) was similar in LVH and in controls. Isoprenaline-induced stimulation of AC in RA was similar in LVH and in controls (51 ± 18 vs 36 ± 18%) but < in LV of LVH (7 ± 6 vs 45 ± 6%, P < 0.05). In the presence of ICI-118,551 (a β₂-adrenoceptor antagonist), isoprenaline failed to induce any increase in cAMP in LVH. The quantification of ADP-pertussis toxin ribosylated proteins indicated a lower concentration of substrates in LV myocardial membranes from LVH. These data indicate that in LVH due to pressure overload, there is a down-regulation of β₁-AR and an increase in β₂-AR density. This is associated with alterations of the transmembrane signalling marked by a decreased capacity of isoprenaline to stimulate AC and an impaired expression of Gi proteins.     

Defects of β2-adrenergic signal transduction in chronic lymphocytic leukaemia: relationship to disease progression

The second messenger 3′:5′-cyclic adenosine monophosphate (cAMP) inhibits the proliferation of human B lymphocytes. In lymphoid malignancies, cAMP levels or the number of β₂-adrenergic receptors seem to be decreased. In order to explore this phenomenon further, the function of the β₂-adrenergic receptor complex was examined in mononuclear leucocytes (MNLs) from patients with B-cell chronic lymphocytic leukaemia (CLL). Peripheral blood MNLs from 25 CLL patients (16 male, nine female; aged 62 ± 9 years) and 10 healthy volunteers (seven male, three female; aged 47 ± 19 years) were used. The binding characteristics of β₂-adrenergic receptors (β₂-AR) on MNLs were determined by radioligand binding assays with [¹²⁵I]-cyanopindolol ([¹²⁵I]-CYP). The number of high-affinity binding sites for [¹²⁵I]-CYP was significantly lower in CLL patients (313 ± 300 sites per cell; mean ± SD) than in control subjects (1479 ± 1268 sites per cell). Moreover, the density of β₂-AR decreased with disease progression, from Binet stage A (371 ± 236, n = 13) to B (236 ± 136, n = 7) and C (141 ± 59, n = 5) (P < 0.05; Kruskal–Wallis analysis). Functional analyses of the β₂-AR complex were performed by measuring the cellular cAMP content of MNLs in response to different stimulators. The cAMP production of MNLs upon isoprenaline stimulation (ISO; 10 min, 10^?4 mol L^?1) was slightly lower in CLL patients (12.5 ± 7.04 pmol 10^?6 cells) than in control subjects (15.91 ± 10.08 pmol 10^?6 cells), and decreased with CLL progression (stage A 14 ± 7; stage B 13.66 ± 3.91; stage C 3.07 ± 0.79 pmol 10^?6 cells). In contrast, cAMP accumulation in response to cholera toxin (CHO; 10^?4 g ml^?1, 120 min) was not different in control subjects (70.07 ± 31.30 pmol 10^?6 cells) and CLL patients (stage A 95.24 ± 123.07 , stage B 70.76 ± 57.37, stage C 33.21 ± 33.73 pmol 10^?6 cells). When stimulated with forskolin (100 μmol L^?1, 15 min), control MNLs produced about ten-fold more cAMP than CLL MNLs (188.56 ± 92.53 vs. 17.88 ± 10.32 pmol 10^?6 cells); this response was not stage dependent. Taken together, the results show that the β₂-AR transmembrane signalling is impaired in CLL patients. The correlation of some β₂-AR signalling defects with disease progression suggests that they may contribute to the disease progression of CLL patients.     

Myocardial β-adrenergic reactivity in pressure overload-induced cardiac hypertrophy in the rat

Summary— The purpose of this study was to evaluate the changes in myocardial β-adrenergic reactivity in animals undergoing a 4 week cardiac pressure-overload. Abdominal aortic constriction (AAC) or sham operation (sham) were performed in male Wistar rats, and 4 weeks later, isoprenaline dose-effects (chronotropic, inotropic and lusitropic properties) were studied after pithing. Noradrenaline (NA) and adrenaline (A) concentrations and NA turn-over index (DHPG /NE ratio) were evaluated in heart ventricles, while β-adrenoceptor characteristics in ventricle homogenates and slices with [¹²⁵I]iodocyanopindolol and the β(1)/β(2)-adrenoceptor ratio were estimated. Four weeks of cardiac pressure overload resulted in a 70% increase in ventricle weight/body weight ratio (from 2.5 ± 0.1 to 4.2 ± 0.3 mg/g in sham and AAC rats, respectively) and a 24% increase in protein contents (from 11.3 ± 0.7 to 14.0 ± 1.1 mg/100 mg ventricle in sham and AAC rats respectively). The ventricle NA content was similar in AAC and sham, while the ventricle A content and NA ***turn-over index were significantly increased in AAC rats (35 and 80% vs sham, respectively). Dose response of isoprenaline was significantly shifted to the right for all studied effects in AAC rats. However, maximal response (in relative values) was similar in AAC and sham rats only for heart rate but not for parameters depending on left ventricle contractile response. The β-adrenoceptor density was significantly decreased in AAC by 30% without apparent affinity change and due to decreases in β(1)-sites in septum and to β(1)- and β(2)-adrenoceptors in left ventricle endocardium. Decreases in isoprenaline-induced cardiac responses in AAC rats are associated with β(1)-adrenoceptor density reduction and modification of β(1)- and β(2)-adrenoceptor ratio. These modifications are not the only reason for such dose response changes, at least for contractile response.     

Altered skeletal muscle glucose transport and blood lipid levels in habitual cigarette smokers

We determined whether habitual cigarette smoking alters insulin-stimulated glucose transport and GLUT4 protein expression in skeletal muscle. Vastus lateralis muscle was obtained from 10 habitual cigarette smokers and 10 control subjects using an open muscle biopsy procedure. Basal 3-O-methylglucose transport was twofold higher (P > 0·01) in muscle from habitual smokers (0·05 ± 0·08 vs. 1·04 ± 0·19 μmol ml^?1 h^?1; controls vs. smokers respectively). Insulin (600 pmol l^?1) increased glucose transport 2·6-fold (P > 0·05) in muscle from control subjects, whereas no significant increase was noted in habitual smokers. Skeletal muscle GLUT4 protein expression was similar between the groups. FFA levels were elevated in the smokers (264 ± 49 vs. 748 ± 138 μmol l^?1 for control subjects vs. smokers; P < 0·05), and serum triglyceride levels were increased in the smokers (0·9 ± 0·2 vs. 2·3 ± 0·6 mmol l^?1 for control subjects vs. smokers; P < 0·05). Skeletal muscle carnitine palmitil (acyl) transferase activity was similar between the groups, indicating that FFA transport into the mitochondria was unaltered by cigarette smoking. In conclusion, cigarette smoking appears to have a profound effect on glucose transport in skeletal muscle. Basal glucose transport is markedly elevated, whereas insulin-stimulated glucose transport is impaired. These changes cannot be explained by altered protein expression of GLUT4, but may be related to increased serum FFA and triglyceride levels. These findings highlight the importance of identifying habitual cigarette smokers in studies aimed at assessing factors that lead to alterations in lipid and glucose homeostasis in people with non-insulin-dependent diabetes mellitus (NIDDM).     

β-Adrenoceptor density in patients with left-sided valvular regurgitation

Summary— Aortic regurgitation differs from mitral regurgitation in that it is a result of combined volume and pressure overload, while the latter represents an almost pure volume overload. In this study, we tested the possibility that these two forms of left ventricular volume overload exert different effects on β-adrenoceptor density. Lymphocyte (n = 33) and myocardial (n = 22) β-adrenoceptor densities were evaluated by [¹²⁵I]-iodocyanopindolol binding in volume-overloaded patients with left heart valvular disease, compared with 31 healthy donor blood and 15 donor heart controls, made available as a result of failing to get matching recipient. The total lymphocyte (LC) β-adrenoceptor density decreased from 43.4 ± 5.5 fmol mg^?1 protein in controls to 9.2 ± 2.7 fmol (P < 0.001) in heart valvular patients. In the myocardial controls, the left ventricular (LV)-receptor density was 126.7 ± 19.5 fmol; right ventricular (RV), 123.1 ± 14.6 fmol; left atrial (LA), 81.6 ± 10.5 fmol; and right atrial (RA), 108.1 ± 14.5 fmol mg^?1 protein. Compared to this group, the total LV-receptor density of the patients decreased by 63%, RV by 54%, LA by 31% and RA by 34%. The decrease in receptor density exhibited a positive correlation with increasing ejection fractions in both the left (r = 0.38) and right (r = 0.44) ventricles, indicating that the former was dependent on the extent of the disease. These changes were accompanied by a 44% increase in plasma epinephrine, 13% in norepinephrine and a 27% decrease in dopamine levels. Based on the predominant left ventricular volume overload classified as aortic regurgitation (AVR), mitral regurgitation (MVR) and mixed aortic and mitral regurgitation (MOL), the attenuation in myocardial-receptor densities showed the following trend: MOL > MVR > AVR. The results show a global reduction in myocardial and LC β-adrenoceptor density, which depends on the origin and the gravity of the LV volume overload.     

Influence of portacaval shunt on bile formation in the rat

Abstract. Bile flow and bile acid secretion were measured in rats 21 to 28 days after a portacaval shunt and in sham-operated and normal animals. The following results were obtained. (1) Bile flow was significantly lower (6.65 ± SEM 0.36 μl-min^?1 · 100 g^?1) in the shunted rats than in the sham-operated animals (8.21 ± SEM 0.21 μl · min^?1 · 100 g^?1; P < 0.01). (2) Bile acid excretion was not significantly different in the shunted rats (0.27 ± SEM 0.03 μmol · min^?1 · 100 g^?1) and the sham-operated rats (0.26 ± SEM 0.02 μmol · min^?1 · 100 g^?1; NS). (3) During bile acid infusions, there was a linear relationship between bile flow and bile acid excretion in both groups of animals. The slope of the relationship was similar, suggesting that the osmotic activity of the bile acids was not modified in the shunted animals, and the bile acid-independent flow, estimated by the extrapolation of this relationship to a zero bile acid excretion, was significantly lower in the rats with a portacaval shunt (5.20 ± SEM 0.40 μl · min^?1 · 100 g^?1) than in the sham-operated animals (6.50 ± SEM 0.30 μl · min^?1 · 100 g^?1; P < 0.02). (4) The liver weight was significantly lower in the rats with a portacaval shunt than in the sham-operated animals and there was a parallel decrease of liver weight (-17%) and of the bile acid-independent flow (-22%). No difference was found between the sham-operated rats and the normal rats. It is concluded that portacaval shunt in the rat results in a decreased bile flow, due to a decrease in the bile acid-independent flow. Since bile acid secretion rate remained unchanged, it is suggested that the secretion of bile acids on the one hand and the bile acid-independent flow on the other are regulated by separate mechanisms.     

Triiodothyronine and amiodarone effects on β3-adrenoceptor density and lipolytic response to the β3-adrenergic agonist BRL 37344 in rat white adipocytes

Summary— The β-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T₃) effect on β-adrenergic receptors (β-ARs) in adipose tissue. AM decreases the expression of some T₃ sensitive genes in various tissues and antagonizes the effect of T₃ on its nuclear receptors. In this study, the T₃, AM and AM + T₃ effects on the β1- and β₃-AR density were assessed on rat white adipocytes by radioligand binding using [³H]CGP 12177 after characterization of these subtypes by displacement of [³H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study β₃-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (E_max × 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T₃ effect on lipolysis. The β₁-binding characteristics (receptor density [B_max]: 45 ± 4 fmol/mg of proteins; dissociation factor [K_d]: 0.96 ± 0.10 nM) were not modified by either compound. Finally, T₃ significantly increased β₃-AR density (587 ± 69 versus 363 ± 25 fmol/mg of proteins) and K_d (38 ± 2 versus 23 ± 3 nM), while AM alone had no effect and did not antagonize the T₃ effect on β₃-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific β₃-agonist and increased the β₃-AR density without changing in β₁-AR number and affinity. Furthermore, the lack of antagonism between AM and T₃ on β₃-AR expression suggests that T₃ does not work directly on the β₃-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.     

MRI tumor characterization using Gd‐GlyMe‐DOTA‐perfluorooctyl‐mannose‐conjugate (Gadofluorine M™), a protein‐avid contrast agent

The rationale and objectives were to define the MRI tumor‐characterizing potential of a new protein‐avid contrast agent, Gd‐GlyMe‐DOTA‐perfluorooctyl‐mannose‐conjugate (Gadofluorine M?; Schering AG, Berlin, Germany) in a chemically induced tumor model of varying malignancy. Because of the tendency for this agent to form large micelles in water and to bind strongly to hydrophobic sites on proteins, it was hypothesized that patterns of dynamic tumor enhancement could be used to differentiate benign from malignant lesions, to grade the severity of malignancies and to define areas of tumor necrosis. Gadofluorine M, 0.05 mmol Gd kg^?1, was administered intravenously to 28 anesthetized rats that had developed over 10 months mammary tumors of varying degrees of malignancy as a consequence of intraperitoneal administration of N‐ethyl‐N‐nitrosourea (ENU), 45–250 mg kg^?1. These tumors ranged histologically from benign fibroadenomas to highly undifferentiated adenocarcinomas. Dynamic enhancement data were analyzed kinetically using a two‐compartment tumor model to generate estimates of fractional plasma volume (fPV), apparent fractional extracellular volume (fEV*) and an endothelial transfer coefficient (K^PS) for this contrast agent. Tumors were examined microscopically for tumor type, degree of malignancy (Scarff–Bloom–Richardson score) and location of necrosis. Eighteen tumor‐bearing rats were successfully imaged. MRI data showed an immediate strong and gradually increasing tumor enhancement. K^PS and fEV*, but not fPV obtained from tumors correlated significantly (p < 0.05) with the SBR tumor grade, r = 0.65 and 0.56, respectively. Estimates for K^PS and fEV* but not fPV were significantly lower in a group consisting of benign and low‐grade malignant tumors compared with the group of less‐differentiated high‐grade tumors (1.61 ± 0.64 vs 3.37 ± 1.49, p < 0.01; 0.45 ± 0.17 vs 0.78 ± 0.24, p < 0.01; and 0.076 ± 0.048 vs 0.121 ± 0.088, p = 0.24, respectively). It is concluded that the protein‐avid MRI contrast agent Gadofluorine M enhances tumors of varying malignancy depending on the tumor grade, higher contrast agent accumulation for more malignant lesions. The results show potential utility for differentiating benign and low‐grade malignant lesions from high‐grade cancers. Copyright © 2006 John Wiley & Sons, Ltd.     

Formation of prostacyclin during administration of β1-selective and non-selective β-adrenergic antagonists in healthy humans

Summary. Vascular formation of prostacyclin is increased by propranolol in patients with essential hypertension. However the possible effect of β-adrenoceptor blocking drugs in healthy subjects is, however, not known. We studied this issue by analysis of the urinary excretion of the prostacyclin metabolite, 2,3-dinor-6-keto-prostaglandin F_la during intake of a (β₁-selective (metoprolol) or a non-selective (propranolol) (3-adrenoceptor antagonist. After 14 days of metoprolol treatment (100 mg d^-1) the urinary excretion of PGI-M did not differ from control (253 ± 77 vs. 220 ± 33 pg mg^-1 creatinine, respectively). Five days of randomized cross-over treatment with propranolol (80 mg day^-1) or placebo did not affect urinary PGI-M significantly either (177 ± 11 vs. 202 ± 11 pg mg^-1 creatinine, respectively). Furthermore, a daily increasing dose of propranolol (80–480 mg) progressively lowered resting blood pressure and heart rate, but failed to influence urinary excretion of PGI-M. The data demonstrate that metoprolol and propranolol do not affect basal cardiovascular formation of prostacyclin in healthy subjects. Thus, the biosynthesis of prostacyclin does not appear to be regulated by p-adrenoceptor activity under normal conditions.     

Moderate hyperthyroidism reduces liver amino nitrogen conversion,muscle nitrogen contents and overall nitrogen balance in rats

There are conflicting data on the effect of thyroid hormones on nitrogen metabolism. We determined the basal blood amino nitrogen (amino-N) concentrations, the urea nitrogen (urea-N) synthesis rate and the maximum hepatic capacity of urea nitrogen synthesis during saturating infusion of alanine, in moderately acutely (24 h) and chronically (7 days) hyperthyroid rats and compared this with changes in organ nitrogen contents in muscles and kidney, nitrogen excretion and nitrogen balance. Forty-three rats were made acutely hyperthyroid through administration of 5 μg 100 g^?1 triiodothyronine twice daily (T₃: 2.2 ± 0.7 vs. 0.87 ± 0.04 nmol L^?1, P < 0.01). Fifty-one rats were made chronically hyperthyroid through administration of 12.5 μg 100 g^?1 thyroxine twice daily (T₃: 2.63 ± 0.18 vs. 0.87 ± 0.04 nmol L^?1, P < 0.01). Weight gain was halved in this group. Both acute and chronic hyperthyroidism increased basal blood amino-N concentration in both groups by 16% (4.5 ± 0.15 vs. 3.9 ± 0.13 mmol L^?1 and 4.7 ± 0.12 vs. 3.9 ± 0.13 mmol L^?1, respectively, P < 0.01), and decreased basal urea-N synthesis rate in both groups by 30% [2.7 ± 03 vs. 4.1 ± 0.3 μmol (min ×100 g)^?1 and 3.1 ± 0.3 vs. 4.1 ± 0.3μmol (min ×100 g g)^?1, respectively, P < 0.01]. The capacity of urea-N synthesis during saturation fell in both groups by 35% compared with controls [6.5 ± 0.4 vs. 9.3 ± 0.5 μmol (min ×100 g)^?1 and 5.7 ± 0 .5 vs. 9.3 ± 0.6 μmol (min ×100 g)^?1, respectively, P < 0.01]. Nitrogen contents in the muscles, soleus and extensor digitorum longus, of chronically hyperthyroid rats decreased by 22% and 11%, respectively, whereas kidney N-content increased by 12% (P < 0.05). N-balance and urinary urea-N excretion fell by 30%, whereas faeces-N excretion increased by 80% in hyperthyroid rats. Overall liver function assessed by galactose elimination capacity did not differ among groups. Both acute and chronic moderate hyperthyroidism increase blood amino-N and decrease basal and maximum rate of urea formation. Furthermore, chronic hyperthyroidism reduces N-contents of muscles, urinary urea-N excretion and N-balance. Thyroid hormones thus mobilize muscle-N, whereas amino-N in the liver is spared from irretrievable conversion into urea.     

Influence of patient related factors on number of mesenchymal stromal cells reached after in vitro culture expansion for clinical treatment

Background: Number of stromal cells injected in patients with ischaemic heart disease (IHD) may be of importance for the treatment efficacy, which in turn may be influenced by various patient-related factors. In this study, we investigate whether patient-related factors influence the number of autologous stromal cells reached after in vitro culture expansion for clinical therapy.

Methods: Culture expansion data from 111 patients with IHD treated with autologous stromal cells in three clinical trials were used. We correlated the final cell count after two passages of cultivation with different patient factors.

Results: There was a significant relation between body mass index (BMI) and the number of adipose derived stromal cells (ASCs) reached after culture expansion and for all patients included into the three studies (r?=?0.375, p?=?.019 and r?=?0.200, p?=?.036, respectively). Moreover, there was a significantly higher number of ASCs reached in patients with hypertension compared to those without hypertension and for all patients overall (68.8?±?39.6?×?10⁶ vs. 39.1?±?23.6?×?10⁶, p?=?.020 and 62.0?±?55.0?×?10⁶ vs. 29.0?±?19.3?×?10⁶, p?<?.001, respectively). The same tendency was seen with bone marrow derived mesenchymal stromal cells (MSCs) in patients with hypertension compared to those without hypertension (58.4?±?61.8?×?10⁶ vs. 22.6?±?13.3?×?10⁶, p?<?.001) and in males compared to females (56.4?±?61.5?×?10⁶ vs. 30.9?±?27.9?×?10⁶, p?=?.041). Moreover, a significant negative correlation between left ventricular ejection fraction and number of MSCs was found (r?=??0.287, p?=?.017).

Conclusions: Patient related factors such as BMI, hypertension and gender may influence the number of MSCs reached after in vitro culture expansion.     

Clinical correlates of endothelial function in chronic heart failure

Objective

There is a close link between heart failure and endothelial dysfunction. Brachial flow-mediated dilation (FMD) is a validated non-invasive measure of endothelial function. The aim of this study was to investigate the clinical correlates of FMD in patients with chronic heart failure (CHF).

Design, setting, patients

We evaluated 60 CHF outpatients (age 62?±?14?years; 49 males, NYHA class 2.2?±?0.7, left ventricular ejection fraction, LVEF, 33?±?8%) taking conventional medical therapy (ACE-inhibitors and/or ARBs 93%, beta-blockers 95%) and in stable clinical conditions.

Main outcome measures

The maximum recovery value of FMD was calculated as the ratio of the change in diameter (maximum-baseline) over the baseline value.

Results

As compared with patients with a higher FMD, those with FMD below the median value (4.3%) were more frequently affected by ischemic cardiopathy (50 vs. 23%; p?=?0.032) and diabetes mellitus (20 vs. 3%; p?=?0.044), had a higher NYHA class (2.5?±?0.5 vs. 1.9?±?0.7; p?<?0.001) and NT-proBNP (2,690?±?3,690 vs. 822?±?1,060; p?=?0.001), lower glomerular filtration rate estimated by Cockcroft-Gault (GFRCG: 63?±?28 vs. 78?±?25; p?=?0.001) and LVEF (29?±?8 vs. 37?±?9; p?=?0.001), as well as more frequently showing a restrictive pattern (40 vs. 7%; p?=?0.002). In a multivariate regression model (R ²?=?0.48; p?<?0.001), FMD remained associated only with the NYHA class (p?=?0.039) and diabetes mellitus (p?=?0.024).

Conclusions

This study demonstrates that a better functional status and absence of diabetes mellitus are associated to higher FMD regardless of the etiology of the cardiac disease.     

Dynamic fluorescent imaging with indocyanine green for monitoring the therapeutic effects of photoimmunotherapy

A new type of monoclonal antibody (mAb)‐based, highly specific phototherapy (photoimmunotherapy; PIT) that uses a near‐infrared (NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with an mAb, has recently been described. NIR light exposure leads to immediate, target‐selective necrotic cell death. However, tumor shrinkage takes several days to occur, making it difficult to detect earlier changes in the tumor. In this study, Panitumumab targeting the epidermal growth factor receptor (EGFR1) conjugated to IR700 was used to treat EGFR‐expressing A431 tumor cells and in vivo xenografts. PIT was performed at varying doses of NIR light (10, 30, 50 and 100 J cm^?2) in xenograft tumors in mice. Indocyanine green (ICG) dynamic imaging was evaluated for monitoring cytotoxic effects for the first hour after PIT. Our results demonstrated a statistical difference (p < 0.05) in ICG intensity between control and PIT treated tumors in the higher light exposure groups (50 J cm^?2: 2.94 ± 0.35 vs 5.22 ± 0.92, p = 0.02; and 100 J cm^?2: 3.56 ± 0.96 vs 5.71 ± 1.43, p = 0.008) as early as 20 min post ICG injection. However, no significant difference (p > 0.05) in ICG intensity between control and PIT treated tumors was evident in the lower light exposure group at any time points up to 60 min (10 J cm^?2: 1.92 ± 0.49 vs 1.71 ± 0.3, p = 0.44; and 30 J cm^?2: 1.57 ± 0.35 vs 2.75 ± 0.59, p = 0.07). Similarly, the retention index (background to corrected uptake ratio of ICG) varied with light exposure. In conclusion, ICG may serve as a potential indicator of acute cytotoxic effects of mAb‐IR700‐induced PIT even before morphological changes can be seen in targeted tumors. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Influence of methoctramine on the acetylcholine-isoprenaline functional antagonism in the guinea pig isolated trachea

Summary— It has been suggested that activation of muscarinic M₂ receptors is one of the components of the functional antagonism between muscarinic and β-adrenoceptor agonists in canine and guinea pig tracheal smooth muscle. The aim of the present study was to determine in the guinea pig trachea the importance of this component according to the magnitude of the acetylcholine-induced contraction. Cumulative concentration-response curves for isoprenaline were obtained in the absence or presence of the muscarinic M₂ receptor antagonist methoctramine (3 × 10^?7 M) in tracheal rings under basal tension or precontracted by acetylcholine 2 × 10^?7, 3 × 10^?6 and 10^?4 M, giving contractions of 25, 50 or 75%, respectively, of the maximal tension induced by acetylcholine 3 × 10^?3 M. In the absence of methoctramine, acetylcholine induced a concentration-dependent shift of the concentration-response curves of isoprenaline (-log EC₅₀ of isoprenaline are 8.09 ± 0.07, 7.85 ± 0.08, 7.38 ± 0.12 and 6.49 ± 0.12, n = 6 for basal tension and for acetylcholine concentrations of 2 × 10^?7, 3 × 10^?6 and 10^?4 M, respectively). In the presence of methoctramine, the basal -log EC₅₀ of isoprenaline was unmodified, whereas the acetylcholine-induced shifts of concentration-response curves of isoprenaline were abolished for low levels of contraction (25%) and significantly reduced to 50 and 75% levels of contraction. Under similar conditions, acetylcholine-induced shifts of concentration-response curves of isoprenaline were unmodified by the muscarinic M₁ receptor antagonist pirenzepine (10^?7 M). These results suggest that the inhibitory effect of M₂ receptors on β-adrenoceptor agonists effects is important for low contraction levels induced by acetylcholine, and that this effect becomes less important for higher concentrations of acetylcholine.     

Improved assessment of arterial stiffness using corrected cardio-ankle vascular index (CAVI0) in overweight adolescents with white-coat and essential hypertension

Arterial stiffness is a marker of vascular damage. Although adiposity increases cardiovascular risk, the relationship between paediatric overweight and arterial stiffness is unclear. The study aimed to evaluate the simultaneous effect of hypertension and overweight on arterial stiffness using cardio-ankle vascular index (CAVI) and related novel, theoretically blood pressure (BP)-independent, index CAVI₀. CAVI and CAVI₀ were measured in 140 adolescent boys (16.0?±?1.9?years) divided into age-matched groups: normal-weight normotensives, overweight normotensives, overweight white-coat hypertensives, and overweight essential hypertensives. Overweight normotensives had significantly lower CAVI and CAVI₀ compared to normal-weight normotensives (4.81?±?0.64 vs. 5.33?±?0.66, p?<?.01; 7.10?±?0.99 vs. 7.81?±?1.00, p?<?.01, respectively). CAVI and CAVI₀ in overweight essential hypertensives showed no significant difference compared to normal-weight normotensives and were significantly higher compared to overweight normotensives (5.32?±?0.77 vs. 4.81?±?0.64, p?<?.01; 7.77?±?1.19 vs. 7.10?±?0.99, p?<?.01, respectively). CAVI, but not CAVI₀, was associated positively with diastolic pressure (0.022?mmHg^?1, p?=?.002) and negatively with pulse pressure (?0.022?mmHg^?1, p?=?.001), and it was significantly higher in overweight white-coat hypertensives compared to overweight normotensives (5.20?±?0.63 vs. 4.81?±?0.64, p?<?.05). The lowering effect of overweight on arterial stiffness indexed by CAVI and CAVI₀ in hypertensive adolescents seems to counterbalance the early arteriosclerotic effect of essential hypertension. The increase in CAVI, but not CAVI₀, in overweight white-coat hypertensives could be attributable to residual BP dependence of CAVI, which is not present in CAVI₀. Under certain conditions, CAVI₀ may offer a clinically relevant improved assessment of arterial stiffness superior to CAVI.     

Longitudinal Changes in Glucose Metabolism of Denervated Muscle after Complete Peripheral Nerve Injury

Purpose

Electrodiagnostic studies can obtain information 2 or 3 weeks after an acute nerve injury. Previous studies have shown increased glucose metabolism in denervated muscles 1 week after injury using 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) positron emission tomography (PET). Therefore, this study aimed to evaluate the changes in glucose metabolism in denervated muscles using serial monitoring by [¹⁸F]FDG PET scans.

Procedures

Denervation was induced in eight male Sprague-Dawley rats (aged 7 weeks old) weighing 200–250 g. The right legs of the rats were denervated by resecting the sciatic nerve in the thigh after the initial skin incision. Two rats were sacrificed 1 and 10 weeks after denervation. Skeletal muscles (gastrocnemius and tibialis anterior) were excised from both the right and left legs of the rats. Staining with hematoxylin and eosin, glucose transporter (GLUT)-1, GLUT-4, and hexokinase II was undertaken. PET/computed tomography (CT) scans were performed on the six remaining rats a total of five times at 1, 2, 5, 8, and 10 weeks after denervation. Regions of interest were drawn on integrated PET/CT images to measure the degree of [¹⁸F]FDG uptake in the right and left lower leg muscles. Target-to-background ratios (TBRs) were calculated by dividing the FDG uptake of the lower leg muscles by that of the upper leg muscles.

Results

The TBRs of the denervated muscles were higher than those of the control muscles at both 1 (6.84?±?1.98 vs. 1.18?±?0.11, p?=?0.009) and 2 (4.10?±?2.05 vs. 1.86?±?0.73, p?=?0.0374) weeks after denervation. After 5 (2.18?±?0.78 vs. 1.35?±?0.47, p?=?0.1489), 8 (1.76?±?0.18 vs. 1.69?±?0.18, p?=?0.5127), and 10 (1.76?±?0.52 vs. 1.56?±?0.37, p?=?0.5637) weeks, the difference in the TBRs between the denervated and controls became non-significant.

Conclusions

[¹⁸F]FDG PET can visualize increased glucose metabolism in a denervated muscle early as 1 week after injury. Therefore, PET could be adopted as a noninvasive imaging modality for acute nerve injuries. In addition, [¹⁸F]FDG PET may help to understand the role of the nervous system in the control of peripheral tissues.

     

Daily variation in circulating cytokines and acute-phase proteins correlates with clinical and laboratory indices in community-acquired pneumonia

Our objective was to investigate the initial levels of circulating proinflammatory cytokines, such as interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF-α), of certain acute-phase proteins, such as C-reactive protein (CRP), fibrinogen (FBN) and albumin, and of the glycoprotein fibronectin at presentation and their daily variation during the clinical course of community-acquired pneumonia (CAP) in relation to clinical and laboratory indices of infection. Thirty otherwise healthy hospitalized patients aged 48 ± 3 years (mean ± SEM) and with bacteriologically confirmed CAP were studied prospectively. IL-1β and IL-6 were found to be 15-fold higher on admission (122 ± 9 pg mL^?1 and 60 ± 4 pg mL^?1 respectively), whereas TNF-α was three-fold higher (102 ± 5 pg mL^?1) than those of controls, all of them showing a decline towards normal. Initial CRP levels were increased 90-fold (416 ± 1 mg L^?1), whereas fibronectin levels were reduced (242 ± 9 mg dL^?1). The presence of parapneumonic effusion was associated with a higher TNF-α serum level (127 ± 7 vs. 86 ± 4 pg mL^?1, P = 0.0002), a more rapid daily decline in TNF-α (–7.2 ± 0.7 vs. ?3.8 ± 0.5 pg mL^?1 day^?1, P = 0.0005), a slower rate of decline in CRP (?42.8 ± 3.0 vs. ?54.6 ± 3.0 mg L^?1 day^?1, P = 0.02) and a slower rate of increase in FBN (5.9 ± 1.0 vs. 11.7 ± 1.0 mg dL^?1 day^?1), P = 0.001]. Furthermore, daily progression of serum levels of cytokines and acute-phase proteins correlated strongly with pyrexia, erythrocyte sedimentation rate (ESR), neutrophil count, alveolar–arterial oxygen difference and radiographic resolution, clinically manifested by improvement in the patients' condition.     

Menopause-associated changes in plasma lipids, insulin-like growth factor I and blood pressure: a longitudinal study

We examined the effects of the menopause transition on plasma lipids, insulin-like growth factor I (IGF-I) and blood pressure. An initial cohort of 38, non-smoking, healthy premenopausal women (44–48 years) were examined at baseline and after a 6-year follow-up period. At follow-up, 18 women had spontaneously stopped menstruating, whereas 17 women remained premenopausal. Women who experienced natural menopause showed a greater decline in high-density lipoproteins (?5 ± 4 mg dL^?1 vs. ?1 ± 3 mg dL^?1; P < 0.01) and a greater increase in low-density lipoproteins (13 ± 12 vs. 5 ± 10 mg dL^?1; P < 0.05) and fasting triglycerides (14 ± 15 vs. 5 ± 11 mg dL^?1; P < 0.05) than women who remained premenopausal. No menopause effect was noted for total cholesterol. We noted a greater decline in IGF-I levels in women who experienced a natural menopause (?21 ± 11 ng mL^?1) than women who remained postmenopausal (?4 ± 10 ng mL^?1). Systolic blood pressure increased in postmenopausal (13 ± 10 mmHg) compared with premenopausal women (5 ± 4 mmHg; P < 0.01), whereas no menopause effect was noted for diastolic blood pressure. The increase in the waist-to-hip ratio was related to a decrease in high-density lipoprotein (r = ?0.49; P ? 0.05) and increase in low-density lipoproteins (r = 0.48; P < 0.05). The decline in IGF-I was related to the decline in reported leisure time physical activity (0.44; P < 0.05). We conclude that the natural menopause transition is associated with a worsening of the lipid profile and decline in IGF-I, which might be mitigated by deleterious changes in body fat distribution and physical activity.     

Skeletal muscle ischemia–reperfusion injury and cyclosporine A in the aging rat

Old patients exhibit muscle impairments and increased perioperative risk during vascular surgery procedures. Although aging generally impairs protective mechanisms, data are lacking concerning skeletal muscle in elderly. We tested whether cyclosporine A (CsA), which protects skeletal muscle from ischemia–reperfusion (IR) in young rats, might reduce skeletal muscle mitochondrial dysfunction and oxidative stress in aging rats submitted to hindlimb IR. Wistar rats aged 71–73 weeks were randomized to IR (3 h unilateral tourniquet application and 2 h reperfusion) or IR + CsA (10 mg/kg cyclosporine IV before reperfusion). Maximal oxidative capacity (V_Max), acceptor control ratio (ACR), and relative contribution of the mitochondrial respiratory chain complexes II, III, IV (V_Succ), and IV (V_TMPD/Asc), together with calcium retention capacity (CRC) a marker of apoptosis, and tissue reactive oxygen species (ROS) production were determined in gastrocnemius muscles from both hindlimbs. Compared to the nonischemic hindlimb, IR significantly reduced mitochondrial coupling, V_Max (from 7.34 ± 1.50 to 2.87 ± 1.22 μMO₂/min/g; P < 0.05; ?70%), and V_Succ (from 6.14 ± 1.07 to 3.82 ± 0.83 μMO₂/min/g; P < 0.05; ?42%) but not V_TMPD/_Asc. IR also decreased the CRC from 15.58 ± 3.85 to 6.19 ± 0.86 μMCa²⁺/min/g; P < 0.05; ?42%). These alterations were not corrected by CsA (?77%, ?49%, and ?32% after IR for V_Max, V_Succ, and CRC, respectively). Further, CsA significantly increased ROS production in both hindlimbs (P < 0.05; +73%). In old rats, hindlimb IR impairs skeletal muscle mitochondrial function and increases oxidative stress. Cyclosporine A did not show protective effects.     

Ammonia uptake by skeletal muscle in the hyperammonaemic rat

Abstract. A two-stage surgical occlusion of the portal vein was employed to produce hyperammonaemia in the rat. The procedure resulted in a significant rise of arterial blood ammonia level from 70·5 ± 6·5 μmol/l (mean ± SEM, n= 10) to 214·0 ± 37·7 μmol/l and in a rise of venous blood ammonia from 65·0 ± 9·4 μmol/l to 122·2 ± 7·4 μmol/l during the first day following the complete vein occlusion. A marked increase of the arteriovenous difference of ammonia concentration from virtually zero in sham-operated controls to 72 ± 9 (n= 8) μmol/l in rats 1 day after the surgical manipulation suggested uptake of ammonia by skeletal muscle. Rat muscle glutamine synthetase activity increased from 0·46 ± 0·06 u/mg (n= 7) in controls to 2·7 ± 0·3 u/mg (n= 7) on the fourth day following portal vein ligation, and muscle branched chain amino acids aminotransferase increased from 0·2 ± 0·05 u/mg in controls to 0·96 ± 0·1 u/mg (n= 7) during the first day of ligation. Glutamine dehydrogenase and aspartate aminotransferase activities were not affected by the surgical procedure. These observations suggest that ammonia trapping in skeletal muscle is coupled to glutamine formation via amination of glutamic acid. This conclusion was further supported by the finding that ammonia uptake correlated (r= 0·92) with enahnced release of glutamine from muscle and that treatment with methionine sulfoximine, a potent inhibitor of glutamine synthetase, changed the arteriovenous difference of glutamine from –0·92 ± 0·01 mmol/l in ligated animals (net release) to ± 0·12 ± 0·01 mmol/l (net uptake) in ligated and inhibitor-treated animals. Similarly, the inhibitor also abolished the arterio-venous difference of ammonia. Thus, the animal model of hyperammonaemia and the muscle enzyme assays reveal that skeletal muscle is involved in the regulation of blood ammonia level by conversion of ammonia, via glutamic acid, to glutamine.