Involvement of alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.     

Stress-shielding induced bone remodeling in cementless shoulder resurfacing arthroplasty: a finite element analysis and in vivo results

Cementless surface replacement arthroplasty (CSRA) of the shoulder was designed to preserve the individual anatomy and humeral bone stock. A matter of concern in resurfacing implants remains the stress shielding and bone remodeling processes. The bone remodeling processes of two different CSRA fixation designs, conical-crown (Epoca RH) and central-stem (Copeland), were studied by three-dimensional (3-D) finite element analysis (FEA) as well as evaluation of contact radiographs from human CSRA retrievals. FEA included one native humerus model with a normal and one with a reduced bone stock quality. Compressive strains were evaluated before and after virtual CSRA implantation and the results were then compared to the bone remodeling and stress-shielding pattern of eight human CSRA retrievals (Epoca RH n=4 and Copeland n=4). FEA revealed for both bone stock models increased compressive strains at the stem and outer implant rim for both CSRA designs indicating an increased bone formation at those locations. Unloading of the bone was seen for both designs under the central implant shell (conical-crown 50–85%, central-stem 31–93%) indicating high bone resorption. Those effects appeared more pronounced for the reduced than for the normal bone stock model. The assumptions of the FEA were confirmed in the CSRA retrieval analysis which showed bone apposition at the outer implant rim and stems with highly reduced bone stock below the central implant shell. Overall, clear signs of stress shielding were observed for both CSRAs designs in the in vitro FEA and human retrieval analysis. Especially in the central part of both implant designs the bone stock was highly resorbed. The impact of these bone remodeling processes on the clinical outcome as well as long-term stability requires further evaluation.     

Two-leg alternate loading model – A different approach to biomechanical investigations of fixation methods of the injured pelvic ring with focus on the pubic symphysis

The dorsal component of the pelvic ring is considered to be the most essential element for the stability of the pelvic ring. None of the current biomechanical set-ups include the effect of shear stresses by alternating loads that the pelvic ring has to withstand during walking. We hypothesize that a biomechanical test set-up with two-leg alternate loading will lead to stress imitation at the pubic symphysis that are more similar to existing strains than other test set-ups, and would, therefore, be more adequate for biomechanical testing of fixation methods.     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Multimodality palliative treatment of (111)In-pentetreotide negative/(123)I-MIBG positive metastatic carcinoid - a case report

Patients with carcinoid tumours frequently present with metastatic disease. There are only a few therapeutic options for these patients, and the main goal of palliative treatment is to reduce symptoms and thus to improve quality of life. Current therapy includes surgical resection, hepatic artery embolisation, chemotherapy and somatostatin analogue treatment; however, all these options have limitations. It seems probable that therapeutic modalities based on radiopharmaceuticals may provide better therapy, not only in relation to symptom reduction but may also improve patient survival. In this case report we present a 46-year-old woman with a symptomatic carcinoid, who at the time of diagnosis had liver and abdominal lymph node metastases, the primary tumour being located in the terminal ileum. (111)In-pentetreotide scanning was negative, whereas (123)I-MIBG scanning showed high avidity in the tumour tissue. After right hemicolectomy, two courses of (131)I-MIBG treatment were given (12.95 GBq and 12 GBq, respectively). After the second dose of (131)I-MIBG temporary pancytopenia was present. Octreotide therapy was given empirically only for a short time and was stopped because of drug intolerance. The patient underwent tricuspid and pulmonary valve replacement because of her carcinoid heart disease, followed by two courses of embolisation of liver metastases. While (131)I-MIBG therapy reduced the patient's symptoms of flushing and diarrhoea, there has not yet been any effect on tumour response or 5-HIAA production. This case illustrates the multimodality and multidisciplinary approach to such patients.     

Association Studies on Ghrelin and Ghrelin Receptor Gene Polymorphisms With Obesity

Ghrelin exerts a stimulatory effect on appetite and regulates energy homeostasis. Ghrelin gene variants have been shown to be associated with metabolic traits, although there is evidence suggesting linkage and association with obesity and the ghrelin receptor (GHSR). We hypothesized that these genes are good candidates for susceptibility to obesity. Direct sequencing identified 12 ghrelin single‐nucleotide polymorphisms (SNPs) and 8 GHSR SNPs. The 10 common SNPs were genotyped in 1,275 obese subjects and in 1,059 subjects from a general population cohort of European origin. In the obesity case‐control study, the GHSR SNP rs572169 was found to be associated with obesity (P = 0.007 in additive model, P = 0.001 in dominant model, odds ratio (OR) 1.73, 95% confidence interval (1.23–2.44)). The ghrelin variant, g.A265T (rs4684677), showed an association with obesity (P = 0.009, BMI adjusted for age and sex) in obese families. The ghrelin variant, g.A‐604G (rs27647), showed an association with insulin levels at 2‐h post‐oral glucose tolerance test (OGTT) (P = 0.009) in obese families. We found an association between the eating behavior “overeating” and the GHSR SNP rs2232169 (P = 0.02) in obese subjects. However, none of these associations remained significant when corrected for multiple comparisons. Replication of the nominal associations with obesity could not be confirmed in a German genome‐wide association (GWA) study for rs4684677 and rs572169 polymorphisms. Our data suggest that common polymorphisms in ghrelin and its receptor genes are not major contributors to the development of polygenic obesity, although common variants may alter body weight and eating behavior and contribute to insulin resistance, in particular in the context of early‐onset obesity.     

The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of alpha5beta1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the beta1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in alpha5beta1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the beta1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the alpha5beta1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.