Serotonin receptor 1A promoter polymorphism, rs6295, modulates human anxiety levels via altering parasympathetic nervous activity.

OBJECTIVE: The G-allele of the -1019C/G (rs6295) promoter polymorphism of the serotonin receptor 1A (HTR1A) gene has been implicated in anxiety; however, the underlying neurophysiological processes are still not fully understood. Recent evidence indicates that low parasympathetic (vagal) tone is predictive of anxiety. We thus conducted a structural equation model (SEM) to examine whether the HTR1A rs6295 variant can affect anxiety by altering parasympathetic nervous activity. METHOD: A sample of 1141 drug-free healthy Han Chinese was recruited for HTR1A genotyping. Autonomic nervous function was assessed by short-term spectral analysis of heart rate variability (HRV). Anxiety and stress levels were evaluated by the Beck Anxiety Inventory (BAI) and the Perceived Stress Scale (PSS) respectively. RESULTS: The number of the HTR1A G allele was inversely correlated with high-frequency power (HF), a parasympathetic index of HRV. The HF index was negatively associated with BAI scores. Furthermore, the good-fitting SEM, adjusting for confounding variables (e.g., age and PSS levels), revealed a significant pathway linking rs6295 variant to BAI scores via HF index modulation. CONCLUSION: These results are the first to show that HTR1A -1019C/G polymorphism influences anxiety levels by modulating parasympathetic tone, providing a neurophysiological insight into the role of HTR1A in human anxiety.

Calcitonin alleviates hyperalgesia in osteoporotic rats by modulating serotonin transporter activity.

Calcitonin may relieve pain by modulating central serotonin activity. Calcitonin partly reversed the hypersensitivity to pain induced by ovariectomy. This suggests that the anti-nociceptive effects of calcitonin in the treatment of osteoporosis may be mediated by alterations in neural serotonin transporter (SERT) activity. INTRODUCTION: This study used a rat model of osteoporosis to evaluate the role of the cerebral serotonin system in the anti-nociceptive effect of calcitonin, a drug used to treat post-menopausal osteoporosis. METHODS: Osteoporosis was induced in rats by ovariectomy (OVX). Rats were then randomized to the following four groups: sham operation, OVX, OVX plus calcitonin, or OVX plus alendronate. RESULTS: OVX led to alterations in bone micro-architecture; alendronate strongly reversed this effect, and calcitonin moderately reversed this effect. OVX increased hyperalgesia (determined as the time for hind paw withdrawal from a heat source); calcitonin reduced this effect, but alendronate had no effect. OVX increased the expression of c-Fos (a neuronal marker of pain) in the thalamus; calcitonin strongly reversed this effect, and alendronate moderately reversed this effect. OVX also reduced SERT but increased 5-HT1A receptor expression and activity; calcitonin aggravated this effect, but alendronate had no effect on recovery of SERT/5-HT1A activity and expression. CONCLUSIONS: Our study of a rat model of osteoporosis suggests that OVX-induced enhancement of the serotonergic system may protect against hyperalgesia. However, the anti-nociceptive effects of calcitonin in osteoporosis may be mediated by decreased neural SERT activity and increased activation of 5-HT1 receptors in the thalamus.

Mild portal endotoxaemia induces subacute hepatic inflammation and pancreatic beta-cell dysfunction in rats.

BACKGROUND: Portal endotoxaemia has been speculated to be crucially involved in the pathogenesis of chronic hepatic inflammation, which is highly associated with the development of type 2 diabetes mellitus. This study tests whether portal endotoxaemia is a pathogenic link between chronic subacute hepatic inflammation and pancreatic beta-cell dysfunction. MATERIALS AND METHODS: Rats were randomly assigned into two groups: rats with intraportal saline or low-dose lipopolysaccharide (LPS) infusion for 4 weeks. Pathological changes in the liver were evaluated via histological and biochemical examination. Pancreatic insulin secretion was evaluated by in vivo hyperglycaemic clamp study. RESULTS: White blood cell count was significantly increased after intraportal LPS infusion for 4 weeks. Plasma amylase and chemoluminescence counts indicating superoxide levels were significantly increased after LPS treatments for 2 and 4 weeks. Intraportal low-dose LPS infusion significantly increased tumour necrosis factor-alpha and interleukin-6 contents in liver and pancreas. Circulating C-reactive protein, thiobarbituric acid reactive substances (TBARS) and endotoxin levels were not different among groups. The first- and second-phase insulin secretions in hyperglycaemic clamp were significantly decreased in LPS-treated rats. The histopathological scores, de novo production of reactive oxygen substrate and TBARS contents in the liver and pancreas were significantly increased in LPS-infused rats. Leucocyte infiltration was clearly visible in pancreatic islets of LPS-treated rats. CONCLUSIONS: The present study demonstrated that mild portal endotoxaemia caused subacute hepatic inflammation and impaired pancreatic insulin secretion, implicating that portal endotoxaemia is a potential risk factor to link chronic subacute hepatic inflammation and pancreatic beta-cell dysfunction.

Selective COX2 inhibition improves whole body and muscular insulin resistance in fructose-fed rats.

BACKGROUND: The effects of cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) inhibition on insulin resistance in subjects with the metabolic syndrome remain elusive. Aims of this study were to examine the effects of COX1 and COX2 inhibitors on whole body and muscular insulin resistance in fructose-fed rats, an animal model of the metabolic syndrome. MATERIALS AND METHODS: The rats on regular or 60% fructose-enriched diets for 6 weeks were further divided into rats combined with or without piroxicam (a selective COX1 inhibitor) or celecoxib (a selective COX2 inhibitor) treatment for an additional 2 weeks. Euglycaemic hyperinsulinaemic clamp (EHC) with a tracer dilution method was performed at the end of the study. RESULTS: The present result showed that fructose-induced increases in systolic blood pressure and fasting plasma insulin levels were significantly suppressed in rats treated with celecoxib but not piroxicam. In the EHC period, celecoxib significantly reversed fructose-induced decreases in whole body glucose uptake, mainly by glucose storage. Hepatic glucose production and whole body glycolysis were not significantly changed among groups. Celecoxib but not piroxicam significantly reversed fructose-induced decreases in glycogen synthase activities in red and white quadriceps muscles and insulin-stimulated membrane GLUT4 recruitment in soleus muscles. Celecoxib and piroxicam both significantly diminished fructose-induced increases in plasma thromboxane B2 and 6-keto prostaglandin (PG) F1alpha; but only celecoxib treatment significantly attenuated a fructose-induced increase in 8-isoprostane levels. Plasma PGE metabolites were not different among groups. CONCLUSIONS: This study demonstrates that a therapeutic dose of celecoxib, but not piroxicam, could significantly attenuate fructose-induced whole body and muscular insulin resistance in rats.

Signal transduction by calcitonin Multiple ligands, receptors, and signaling pathways.

Calcitonin (CT) is a peptide hormone that is secreted by the parafollicular cells of the thyroid in response to elevated serum calcium levels. It acts to reduce serum calcium by inhibiting bone resorption and promoting renal calcium excretion. In addition to this hypocalcemie effect, calcitonin modulates the renal transport of water and several ions other than calcium and acts on the central nervous system to induce analgesia, anorexia, and gastric secretion. The CT receptor, a member of a newly described family of serpentine G protein-coupled receptors, has recently been shown to couple to multiple trimeric G proteins, thereby activating several signaling proteins, including protein kinase C, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. In kidney proximal tubule cells (LLC-PK1), the CT-activated signaling mechanisms vary in a cell cycle-dependent manner, with the receptor coupling through a G(s) protein during G(2) phase and through a G(i) protein and possibly a G(q) protein during S phase. These signaling mechanisms differentially modulate the activities of Na(+)/K(+)-ATPase and the apical Na(+)/H(+) exchanger, effector molecules that play important roles in transepithelial Na(+) transport. Cloning of CT receptors has revealed the presence of alternatively spliced cassettes, resulting in the expression of different isoforms of the receptor. The availability of these recombinant CT receptors has allowed preliminary characterization of the effects of changes in the receptor's structure on its ligand binding and signal transduction properties. Thus, the cellular and molecular biology of CT is complex, with several structurally related peptide ligands and multiple isoforms of the CT receptor that can independently activate diverse signaling pathways. As the recent exciting results in this field are extended, we can expect rapid progress in understanding the molecular basis of the diverse effects of CT and, possibly, of the CT-related peptides CGRP and amylin.

Distribution of the superficial temporal artery in the Chinese adult.

The superficial temporal artery is important in head and neck surgery. Ethnologic variation may affect surgical procedure. In this study, we evaluated the variations of the artery in Chinese adults. We measured its bifurcating location, the diameter of its vessels, and its relationship to nearby structures. A total of 26 cadavers with 52 superficial temporal arteries were examined in 3 consecutive years. The superficial temporal artery ran 1.14 cm anteriorly to the bony external auditory canal. The average diameters of the superficial temporal artery, frontal branch, and parietal branch were 2.14, 1.61, and 1.68 mm, respectively. In 45 of 52 cases (86.5 percent), bifurcation of the artery occurred well above the zygomatic arch. The present study thus demonstrated that the superficial temporal artery in the Chinese adult differs from that in the Caucasian and has provided a detailed anatomic distribution analysis of the superficial temporal artery in Chinese adults, which should benefit the clinician in dealing with operation procedures related to this artery.

Variations of the cystic artery in Chinese adults.

The origin and course of the cystic artery related to the Calot triangle were studied in 72 autopsies. The cystic artery arises from many possible origins; the right hepatic artery is the most common origin (76.6%). The Calot triangle (hepatocystic triangle), which is an important imaginary referent area for biliary surgery, is bounded by the common hepatic duct (CHD), the cystic duct, and the cystic artery. Of all the cystic arteries, 86.1% coursed through the Calot triangle, and 100% of the cystic arteries originating from the right hepatic artery coursed through the Calot triangle. However, only 54% of the cystic arteries that originated from the left, bifurcation, proper, and common hepatic arteries ran through the triangle. None of the cystic arteries that originated from the gastroduodenal, celiac, superior mesentery, or superior pancreaticoduodenal arteries passed through the triangle. Furthermore, 72.7% of the cystic arteries that originated from the right hepatic artery ran beneath the CHD as they entered the Calot triangle; the others ran anterior to the CHD. Of the cystic arteries that arose from locations other than the right hepatic artery, 29.4% ran posterior to the CHD, and 11.8% ran anterior to the CHD. The current study provides detailed information about anatomic variance in Chinese adults that may help avoid injury during open or laparoscopic cholecystectomies.

The deletion of 14 amino acids in the seventh transmembrane domain of a naturally occurring calcitonin receptor isoform alters ligand binding and selectively abolishes coupling to phospholipase C.

The cDNA that encodes the rabbit calcitonin receptor was cloned by screening a rabbit osteoclast library. Reverse transcription-polymerase chain reaction amplification of calcitonin receptor sequences from rabbit osteoclast RNA yielded cDNAs that encode two isoforms of the calcitonin receptor. One isoform is homologous to the C1a isoform previously identified in multiple cell types and species, while the second, designated CTRDeltae13, is a previously unidentified isoform that is apparently generated by alternative splicing during mRNA processing that deletes exon 13, resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Expression of mRNA transcripts encoding the two isoforms varies in a tissue-specific manner, with CTRDeltae13 accounting for less than 15% of the total calcitonin receptor mRNA in osteoclasts, kidney, and brain, but comprising at least 50% of the transcripts in skeletal muscle and lung. The two isoforms were expressed, and the ligand binding and signal transduction properties were characterized. Deletion of the residues in the seventh transmembrane domain in CTRDeltae13 reduced the binding affinity for salmon and human calcitonin by more than 10-fold and approximately 2-fold, respectively, resulting in a receptor that failed to discriminate between the two forms of calcitonin. Both isoforms activated adenylyl cyclase, with EC50 values consistent with the difference in ligand affinities. In contrast, only the C1a isoform, but not the CTRDeltae13 isoform, activated phospholipase C. Thus, while the CTRDeltae13 remains active despite the deletion of a significant portion of its seventh transmembrane domain, it has significantly altered ligand recognition and signal transduction properties.

Protein kinase C antagonizes pertussis-toxin-sensitive coupling of the calcitonin receptor to adenylyl cyclase.

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis-toxin-sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin-dependent activation of adenylyl cyclase in HEK-293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor-dependent activation of Gi. The possibility that protein kinase C could be antagonizing Galphai-adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin-induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK-293 cells, that stably express a myc-tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Galphai subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Galphai is negatively regulated by protein kinase C.

The calcitonin receptor stimulates Shc tyrosine phosphorylation and Erk1/2 activation. Involvement of Gi, protein kinase C, and calcium.

While it is well established that adenylyl cyclase and phospholipase C-beta are two proximal signal effectors for the calcitonin receptor, the more distal signaling pathways are less well characterized. G protein-coupled receptors can activate Erk1/2 by Gs-, Gi-, or Gq-dependent signaling pathways, depending on the specific receptor and cell type examined. Since the calcitonin receptor can couple to all three of these G proteins, the ability of calcitonin to activate Erk1/2 was investigated. Calcitonin induced time- and concentration-dependent increases in Shc tyrosine phosphorylation, Shc-Grb2 association and Erk1/2 phosphorylation and activation in a HEK 293 cell line that stably expresses the rabbit calcitonin receptor C1a isoform. Pertussis toxin, which inactivates Gi, and calphostin C, a protein kinase C inhibitor, each partially inhibited calcitonin-induced Shc tyrosine phosphorylation, Shc-Grb2 association, and Erk1/2 phosphorylation. In contrast, neither forskolin nor H89, a protein kinase A inhibitor, had a significant effect on basal or calcitonin-stimulated Erk1/2 phosphorylation. Our results suggest that the calcitonin receptor induces Shc phosphorylation and Erk1/2 activation in HEK293 cells by parallel Gi- and PKC-dependent mechanisms. The calcitonin-induced elevation of cytosolic free Ca2+ was required for Erk1/2 phosphorylation, since preventing any change in cytosolic free Ca2+ by chelating both cytosolic and extracellular Ca2+ abolished the response. However, the change in Ca2+ that is induced by calcitonin is not sufficient to account for the calcitonin-induced Erk1/2 phosphorylation, since treatment with 100 nM ionomycin or 10 microM thapsigargin, each of which induced elevations of Ca2+ comparable to those induced by calcitonin, induced significantly less Erk1/2 phosphorylation than that induced by calcitonin. Erk1/2 may have important roles as downstream effectors mediating cellular responses to calcitonin stimulation.