Decreased β2-adrenoceptor density and decreased isoproterenol induced c-AMP increase in juvenile type I diabetes mellitus: An additional cause of severe hypoglycaemia in childhood diabetes?

Little is known about the receptor and post receptor mechanisms of sympathoadrenal signal transmission in type I diabetes mellitus. Therefore, we examined the maximum binding of granulocyte ₂-adrenoceptors and the in vitro c-AMP accumulation in lymphocytes of 24 children and adolescents with diabetes mellitus and 14 similarly aged healthy subjects. The number of high affinity ₂-adrenoceptors on granulocytes correlated significantly with unstimulated (r=0.6,P<0.004) and with isoproterenol stimulated c-AMP values in lymphocytes (r=0.68,P<0.0007) showing the proportional changes of ₂-adrenoceptors and c-AMP in two different cells. The number of ₂-adrenoceptors on granulocytes was significantly reduced in diabetic as compared to healthy children (median 1397, range 599–3405 vs. 2205, 825–3200 ₂-adrenoceptors per granulocyte,P=0.014). Moreover, the percentage in vitro stimulation of c-AMP by isoproterenol in lymphocytes was significantly reduced in diabetic children as compared to healthy individuals (120%, 39%–278% vs. 225%, 66%–500%,P=0.012). These results indicate a decreased sympathoadrenergic signal transmission in peripheral blood cells as a model for the liver probably contributing to severe hypoglycaemia in diabetic children.     

Changes in stress relaxation property and softness of soft denture lining materials after cyclic loading

Objectives

Soft denture lining materials show varied and changeable stress relaxation property and softness under cyclic loading conditions. The purpose of this study was to determine the changes in the stress relaxation property and the softness of commercial soft lining materials after cyclic loading.

Methods

One plasticized acrylic-based material (Coe Soft: COS) and two silicone-based materials (Mucosoft: MUS; Sofreliner S: SFL) were investigated. For each material, 10 cylinder-shaped specimens were subjected to 29 days of cyclic loading, 1800 times per day; other 10 unloaded specimens were used as control groups. Stress relaxation ratio and softness were measured after 1, 4, 8, 15, 22 and 29 days of cyclic loading or storage.

Results

During 29 day period, the stress relaxation ratios of the cyclic loaded and unloaded COS, MUS and SFL specimens were 28.5-31.6% and 30.9-32.6%, 6.4-15.1% and 1.8-15.4%, 14.0-38.2% and 19.8-40.6%, respectively. The softness values of the cyclic loaded and unloaded COS, MUS and SFL specimens were 42.6-60.6% and 56.4-61.8%, 47.6-52.5% and 46.9-51.5%, 56.6-58.4% and 58.0-65.1%, respectively. Based on repeated-measures, 3-way ANOVA, the stress relaxation ratio was influenced by the loaded or stored period and the type of material (p < 0.05), but not by the application of cyclic loading; the softness value was influenced by the loaded or stored period and the application of cyclic loading (p < 0.05), but not by the type of material.

Significance

Soft lining materials showed the loaded or stored period and the material type dependent stress relaxation properties regardless of the application of cyclic loading. Softness value was influenced by the loaded or stored period and the application of cyclic loading.     

Nitric oxide metabolites are associated with survival in older patients

OBJECTIVES: To assess the efficacy of various vascular endocrinological substances, such as plasma nitric oxide metabolites (NOx), as surrogate markers of survival in older patients. DESIGN: Prospective cohort, observational. SETTING: Nagoya University Hospital and related hospitals, Japan. PARTICIPANTS: One hundred fifty patients aged 70 and older, recruited consecutively from the outpatient clinics of Nagoya University Hospital and related hospitals. MEASUREMENT: Serum biochemical analyses such as albumin and total cholesterol, various prognostic markers, such as tumor necrosis factor (TNF)-alpha, NOx, activities of daily living (ADLs), and instrumental ADLs (IADLs) were evaluated on enrollment. ADLs, IADLs, and comorbidities, especially depression and impaired cognition, were evaluated on enrollment. The main outcome was survival rate over 2.75 years. RESULTS: Forty-nine patients died during the follow-up period. Mann-Whitney U-test showed that hemoglobin, total protein, serum albumin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high sensitive c-reactive protein, NOx, B-type natriuretic peptide, interleukin-6, and TNF-alpha levels; ADLs; cognitive impairment; and depressive status were significantly different for subjects who survived and those who died. Of the dependent variables in the Cox proportional hazards regression analyses, only ADLs, NOx, and albumin were significantly different. In the Kaplan-Meier analyses of mortality, the prognosis of patients in the third and fourth quartiles of NOx was significantly worse than that of patients in the first or second quartile. The prognosis of patients with impaired ADLs was worse than that of other patients for the overall period. CONCLUSION: Lower levels of NOx may be associated with survival in older patients. It may be an effective marker, like ADLs, which is a well-known marker.     

Construction of functional soft tissues from premodulated smooth muscle cells using a bioreactor system

Artificial smooth muscle tissues should be constructed with well-differentiated and aligned smooth muscle cells (SMCs) for proper functioning. In a previous study, we produced cell/scaffold hybrids composed of consistently aligned SMCs in a contractile state using cyclic mechanical strain. In this study, the preconditioned hybrids were organized as functional smooth muscle constructs, which had a high cellular density, using a bioreactor system. We determined that the alignment and contractile phenotype of the initially generated SMCs would be retained after a 7-day culture period in a bioreactor. Mechanical properties of the smooth muscle constructs were measured and compared with those of native smooth muscle tissues and acellular scaffolds. The constructs had a denser cell concentration than the preconditioned hybrids, although they were not fully filled with cells. The premodulated cell alignment and contractile phenotype were retained after culture in a bioreactor. The 7-day-cultured constructs had similar allowed stress levels to native tissues while their stiffness was much lower, suggesting that they had malleable and durable characteristics. These results suggest that functional smooth muscle tissues with mechanical stability can be produced using premodulated SMCs and a bioreactor system.     

Cellular pharmacokinetics and pharmacodynamics of dipyridamole in vascular smooth muscle cells

Hemodialysis arteriovenous grafts are often plagued by stenosis at the vein-graft anastomosis, which is due to the proliferation of venous smooth muscle cells (SMCs). Perivascular delivery of dipyridamole, a potent antiproliferative agent, has been proposed for the prevention of graft stenosis. In order to develop an optimal delivery system for dipyridamole, we examined its pharmacokinetics and pharmacodynamics in human and porcine venous and arterial SMCs in vitro. SMCs were incubated with dipyridamole for various durations, and visualized for the uptake and release by fluorescence microscopy, which were further quantified by fluorospectrometry. The antiproliferative effect of dipyridamole was examined by cell counting or the methylthiazoletetrazolium (MTT) dye-reduction assay. Cytotoxicity was examined by the lactate dehydrogenase (LDH)-release assay. The kinetics of dipyridamole transport through the cell membrane was compatible with a passive diffusion mechanism. Dipyridamole inhibited SMC proliferation in a dose-dependent manner and was more effective in venous than arterial cells in both species. The inhibition was completely reversible at 15microg/ml upon drug removal from the medium. At 25microg/ml, however, the effect was partially irreversible, which might be attributed to the cytotoxicity of dipyridamole. These data support the need for sustained delivery of dipyridamole to achieve the long-term inhibition of SMC proliferation in the prevention of stenosis since SMCs are continuously stimulated at the anastomosis of hemodialysis arteriovenous grafts.     

Increased nitric oxide production in eating disorders

Animal studies showed that nitric oxide (NO)/cyclic-GMP (cGMP) pathway is involved in the modulation of eating behavior. To address its role in eating disorders (ED), plasma nitrite and cGMP levels were studied in 50 ED patients (25 with Anorexia Nervosa, AN; 25 with Bulimia Nervosa, BN) and 20 sex- and age-matched controls (C). Nitrites (nmol/mg protein, mean+/-S.E.M.: any ED 1.01+/-0.29; AN 1.15+/-0.47; BN 0.88+/-0.36; C 0.25+/-0.07; p<0.01) and cGMP (nmol/ml plasma, mean+/-S.E.M.: any ED 2.58+/-0.60; AN 2.81+/-1.10; BN 2.41+/-0.70; C 0.11+/-0.05; p<0.01) were significantly higher in ED patients than in C. Nitrite and cGMP levels inversely correlated with BMI in AN patients (nitrites: r=-0.62 p<0.01; cGMP r=-0.45 p<0.05) but not in BN patients (nitrites: r=-0.15 p=0.49; cGMP: r=-0.05 p=0.13) or in control subjects (nitrites: r=0.11 p=0.98; cGMP r=0.37 p=0.32). Significant correlations were also present in bulimic patients between nitrite levels, frequency of binges and several psychopathological dimensions, as assessed through the EDE. This is the first evidence of an alteration of the NO pathway in ED patients. Further studies are needed to ascertain whether an increase in NO levels plays a possible role in the pathogenesis of ED.     

The nitric oxide-guanosine 3',5'-cyclic monophosphate pathway regulates dopamine efflux in the medial preoptic area and copulation in male rats

Dopamine in the medial preoptic area (MPOA) plays a significant role in regulation of male copulation. One mediator of the MPOA dopamine level is nitric oxide. In the current study, we investigated the role of the nitric oxide-guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the regulation of MPOA dopamine and copulation in male rats. The reverse-dialysis of a membrane-permeable analog, 8-Br-cGMP, increased, while a soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), significantly reduced basal dopamine and its metabolite levels. ODQ successfully blocked a nitric oxide donor-induced increase in dopamine levels, while a neuronal nitric oxide synthase (nNOS) inhibitor was ineffective in blocking an 8-Br-cGMP-induced increase in dopamine, indicating that cGMP is "downstream" of nitric oxide. Furthermore, 8-Br-cGMP facilitated, while ODQ inhibited copulation. Given the steroid-sensitive nature of nNOS functions and the multiple roles nitric oxide plays in the MPOA, we propose that nitric oxide provides important integration of various neurochemical and neuroendocrine signals. The involvement of the central nitric oxide-cGMP pathway in the regulation of copulation also raises an interesting therapeutic possibility, as the manipulation of the same pathway in peripheral tissue is already utilized in treatment of male sexual dysfunction.     

Functional status of beta-2-adrenoceptor in isolated membranes of mature erythrocytes from patients with cirrhosis and oesophageal varices

Propranolol is a widely used drug for prophylaxis of variceal bleeding in patients with cirrhosis, but not all patients show an adequate clinical response. This variability may be in relation to beta adrenoceptor activity, but no information is available in this setting. Thirty-nine patients with advanced cirrhosis and presence of oesophageal varices were sequentially included. We studied the function of beta-2-adrenoceptor in isolated membranes of mature erythrocytes obtained from patients by measuring cyclic AMP (cAMP) production before and after isoproterenol. Blood samples obtained from 11 healthy volunteers were used as control. Patients showed a six-fold increase in the mean basal cAMP production as compared to healthy volunteers. Isoproterenol produced a small, non-significantly and highly variable increase in the AC activity in patients compared with controls. cAMP values remain stable after three months of continuous treatment with oral beta-blockers in both groups. Patients without antecedent of variceal bleeding or with an active alcohol intake showed a significantly higher isoproterenol effect. In conclusion, beta-receptor function in human erythrocytes membranes is altered in patients with cirrhosis and oesophageal varices.     

Tyrosine kinase effects on adrenoceptor-stimulated cyclic AMP accumulation in preoptic area and hypothalamus of female rats: modulation by estradiol

These studies examined the functional interactions between adrenergic G-protein coupled receptors and protein tyrosine kinases in the preoptic area and hypothalamus, brain regions that regulate reproductive function in female rats, and evaluated whether in vivo treatment with estradiol for 2 days modulates the cross-talk between these two signaling pathways. In hypothalamic slices genistein, a general tyrosine kinase inhibitor, enhances norepinephrine-stimulated cAMP synthesis independent of estradiol treatment. Genistein appears to act by increasing beta-adrenoceptor signaling. At high norepinephrine concentrations, estradiol potentiates genistein enhancement of the cAMP response in hypothalamic slices. This interaction between estradiol and genistein appears to involve modification of alpha(2)-adrenoceptor signaling mechanisms. In preoptic area slices, genistein enhancement of norepinephrine-stimulated cAMP synthesis is only observed in estradiol-treated rats. In this brain region, genistein enhances cAMP accumulation by modifying alpha(1)- and/or alpha(2)-adrenoceptor rather than beta-adrenoceptor signaling. Genistein amplification of norepinephrine-stimulated cAMP synthesis is not mediated by interactions with estrogen receptors, or by regulation of adenylyl cyclase or phosphodiesterase activities. At the concentration used, genistein inhibits tyrosine phosphorylation in slices from both brain regions. Daidzein, an inactive analogue of genistein, fails to enhance the norepinephrine-stimulated cAMP response in either brain region independent of hormone treatment. These results suggest that protein tyrosine kinases regulate adrenergic responses in the hypothalamus and preoptic area. Moreover, the functional interaction between adrenergic G-protein coupled receptor signaling and protein tyrosine kinases is modified in a brain region and receptor subtype specific manner by estradiol.