A novel transcript is up-regulated by fasting in the hypothalamus and enhances insulin signalling.

A transcript of unknown function, regulated by fasting and feeding, was identified by microarray analysis. The transcript is up-regulated in the fasting state. An 1168-bp cDNA was cloned from rat hypothalamus and sequenced. This sequence is consistent with adipogenesis down-regulating transcript 3 (AGD3) (also known as human OCC-1) mRNA. A protein sequence identical to AGD3 was determined by mass spectrometry. In the rat brain, AGD3 mRNA is distributed in the arcuate nucleus, ventromedial hypothalamus, amygdaloid nuclei, hippocampus, and somatic cortex. Double in situ hybridisation showed that AGD3 mRNA is co-localised with pro-opiomelanocortin and neuropeptide Y in arcuate nucleus neurones. AGD3 binds with insulin receptor substrate 4 and increases insulin-stimulated phospho-Akt and regulates AMP-activated protein kinase and mammalian target of rapamycin downstream target S6 kinase phosphorylation.

Induction of apoptosis by thrombin in the cultured neurons of dorsal motor nucleus of the vagus.

BACKGROUND: A previous study demonstrated the presence of protease-activated receptor (PAR) 1 and 2 in the dorsal motor nucleus of vagus (DMV). The aim of this study is to characterize the effect of thrombin on the apoptosis of DMV neurons. METHODS: The dorsal motor nucleus of vagus neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion and cultured. Apoptosis of DMV neurons were examined in cultured neurons. Apoptotic neuron was examined by TUNEL and ELISA. Data were analyzed using anova and Student's t-test. KEY RESULTS: Exposure of cultured DMV neurons to thrombin (0.1 to 10 U mL(-1)) for 24 h significantly increased apoptosis. Pretreatment of DMV neurons with hirudin attenuated the apoptotic effect of thrombin. Similar induction of apoptosis was observed for the PAR1 receptor agonist SFLLR, but not for the PAR3 agonist TFRGAP, nor for the PAR4 agonist YAPGKF. Protease-activated receptors 1 receptor antagonist Mpr(Cha) abolished the apoptotic effect of thrombin, while YPGKF, a specific antagonist for PAR4, demonstrated no effect. After administration of thrombin, phosphorylation of JNK and P38 occurred as early as 15 min, and remained elevated for up to 45 min. Pretreatment of DMV neurons with SP600125, a specific inhibitor for JNK, or SB203580, a specific inhibitor for P38, significantly inhibited apoptosis induced by thrombin. CONCLUSIONS & INFERENCES: Thrombin induces apoptosis in DMV neurons through a mechanism involving the JNK and P38 signaling pathways.

Functional protease-activated receptors in the dorsal motor nucleus of the vagus.

BACKGROUND: Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). METHODS: DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. KEY RESULT: Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca(2+)](i) expressed as DeltaF/F0 of 229 +/- 14% and 137 +/- 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum DeltaF/F0 change of 258 +/- 12% and 242 +/- 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 microm) decreased the maximal change in DeltaF/F0 induced by PAR-1 activation from 140 +/- 17% to 21 +/- 3%, while the PAR-2-mediated maximal change in DeltaF/F0 decreased from 185 +/- 21% to 19 +/- 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in DeltaF/F0 due to PAR-1 and PAR-2 activation by 72 +/- 13% and 71 +/- 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. CONCLUSIONS & INFERENCES: Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3.

Sphingosine-1-phosphate mediates calcium signaling in guinea pig enteroglial cells.

The enteric nervous system, which regulates multiple aspects of digestive activity, is composed of two major cell types, neurons and glial cells. Enteric glia, but not enteric neurons, respond to bioactive lipids with calcium signaling. The sphingomyelin metabolite sphingosine-1-phosphate (S1P) caused dose-dependent calcium (Ca(2+)) signaling using extracellular and intracellular Ca(2+). The signal transduction cascade was pertussis toxin-insensitive and involved an extracellular receptor since repetitive exposure yielded diminished responsiveness. Inhibition of either phospholipase C or the inositol 1,4,5-trisphosphate receptor abolished S1P effects. RT-PCR analysis demonstrated the presence of S1P-coupled endothelial differentiation gene (EDG) receptor mRNAs (EDG-1, EDG-3, and EDG-5) within the enteric nervous system. Immunocytochemical analysis demonstrated strong expression of both EDG-1 and EDG-3 and weak expression of EDG-5 in enteric glial cells. Other sphingomyelin cycle components, including sphingomyelin, sphingomyelinase, and sphingosine caused Ca(2+) transients in enteric glia. Related lipids lysophosphatidic acid and sphingosylphosphorylcholine also induced Ca(2+) signaling in enteric glia, suggesting that multiple lipid-activated signaling mechanisms exist in these cells.

Lysophosphatidic acid stimulates calcium transients in enteric glia.

The enteric nervous system plays an integral role in the gastrointestinal tract. Within this intricate network, enteric glia are crucial in the maintenance of normal bowel function, yet their signaling mechanisms are poorly understood. Enteric glia, and not enteric neurons, selectively responded to lysophosphatidic acid (LPA), a product of phosphatidylcholine metabolism, with dose-dependent calcium (Ca(2+)) signaling over a range from 100 pM to 10 microM. The elicited calcium transients involved both the mobilization of intracellular Ca(2+) stores and the influx of extracellular Ca(2+) as LPA signals were obliterated following the depletion of intracellular Ca(2+) and attenuated by the removal of Ca(2+) from the perfusion buffer. Pretreatment with pertussis toxin (100 ng/ml) reduced the magnitude of LPA Ca(2+) transients (95+/-20 nM vs 168+/-17 nM for controls). Repetitive exposure yielded diminished responsiveness, with a 25% reduction in [Ca(2+)](i) between first and second exposures. Inhibition of the inositol 1,4,5-trisphosphate (IP(3)) receptor with 200 microM 2-aminoethoxydiphenylborate (2APB) abolished LPA signals. RT-PCR analysis demonstrated the presence of two LPA-coupled endothelial differentiation gene (EDG) receptor mRNAs (EDG-2 and EDG-7) in myenteric plexus primary cultures. EDG-2 expression in glial cells of the ENS was confirmed immunocytochemically.

Inhibition of pancreatic protein secretion by ghrelin in the rat.

1. The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. 2. In vivo, pancreatic protein output stimulated by CCK-8 (400 pmol kg(-1) h(-1)) was dose-dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg(-1) h(-1)) by 45 +/- 8 and 84 +/- 7 %, respectively. 3. In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg(-1) h(-1)) significantly inhibited CCK-stimulated pancreatic protein secretion by 75 +/- 18 %. 4. Infusion of ghrelin (12 nmol kg(-1) h(-1)) abolished pancreatic protein secretion caused by the central vagal stimulant 2-deoxy-D-glucose (75 mg kg(-1)), whereas bethanechol-stimulated pancreatic protein output was inhibited by only 59 +/- 7 %. 5. In vitro, ghrelin (10(-11)-10(-7) M) produced no change in basal amylase release from dispersed, purified acinar cells. Co-incubation of ghrelin (10(-11)-10(-7) M) with CCK-8 (10(-10) M) demonstrated no inhibition of CCK-stimulated amylase release from dispersed acini. In contrast, ghrelin (10(-9)-10(-7) M) dose-dependently inhibited amylase release from pancreatic lobules exposed to 75 mM potassium. 6. Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons.

Doctor-patient communication in surgery: attitudes and expectations of general surgery patients about the involvement and education of surgical residents.

BACKGROUND: Education is a major function of academic medical centers. At these teaching institutions residents provide a substantial amount of care on medical and surgical services. The attitudes of patients about the training of surgical residents and the impact of residents on patients' perceptions of care in a surgical setting are unknown. STUDY DESIGN: Patients admitted to the gastrointestinal surgery service completed a 30-item survey designed for this study. Patients included in the study underwent operations and had a postoperative inpatient hospital stay. We analyzed patients' answers to determine frequency and correlations among answers. RESULTS: Two hundred patients participated in the study during a 7-month period between July 1999 and January 2000. A majority of patients were comfortable having residents involved in their care (86%) and felt it was important to help educate future surgeons (91%). Most did not feel inconvenienced by being at a teaching hospital (71%) and felt they received extra attention there (74%). Patients were more willing to participate in resident education if they expected to have several physicians involved in their care, felt that they received extra attention, or if the teaching atmosphere did not inconvenience them. Despite the stated willingness of patients to help with surgical resident education, 32% answered that they would not want residents doing any of their operation. CONCLUSIONS: Surgical resident education is well received and considered important by patients. Patient orientation to the resident education process is vital to patients' perceptions of care and may render patients more willing to participate in educational activities.

Functional overlap of IP(3)- and cADP-ribose-sensitive calcium stores in guinea pig myenteric neurons.

In myenteric neurons two different receptor subtypes govern the intracellular Ca(2+) stores: the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) and the ryanodine receptor (RyR). Their degree of functional overlap was determined by examining Ca(2+) release in these cells through both superfusion techniques and intracellular microinjection. Microinjection of IP(3) (50 microM) and cADP-ribose (cADPr, 50 microM), specific ligands for the IP(3)R and RyR, respectively, demonstrated mobilization of intracellular Ca(2+) stores. Perfusion with cinnarizine (50 microM) or dantrolene (10 microM), antagonists of the IP(3)R and RyR, respectively, eliminated the Ca(2+) response to microinjected IP(3) and cADPr. Superfusion of the neurons with 100 microM ATP, an IP(3)-mediated Ca(2+)-mobilizing agonist, caused intracellular Ca(2+) increments, which were antagonized by cinnarizine, and the RyR antagonists dantrolene, procaine (5 mM), and ryanodine (1 microM). Caffeine (10 mM) was applied repetitively in Ca(2+)-free conditions to deplete RyR-sensitive stores; subsequent perfusion with ATP demonstrated a Ca(2+) response. Conversely, caffeine caused a Ca(2+) response after repetitive ATP exposures. The internal Ca(2+) stores of myenteric neurons are governed by two receptor subtypes, IP(3)R and RyR, which share partial functional overlap.

Surgical management of peptic ulcer disease in the Helicobacter era--management of bleeding peptic ulcer.

Bleeding continues to be a significant cause of morbidity and mortality for patients with peptic ulcer disease. Recent advances have changed the management of this disease. Upper endoscopy with or without endoscopic therapy is the preferred procedure during the initial evaluation of upper gastrointestinal bleeding. With its excellent success rates, many patients are being cured with endoscopic therapy followed by eradication of Helicobacter pylori. H. pylori is now thought to have an important role in the pathogenesis of a majority of gastric and duodenal ulcers. This finding has led to the recommendation that patients with peptic ulcer disease be treated with regimens effective against this organism. Currently, patients who are older and who have more severe underlying medical conditions present a challenge. This review will address the options for treatment of peptic ulcer bleeding. In addition, knowledge gained regarding H. pylori infection and use of nonsteroidal anti-inflammatory drugs will be discussed.

Stimulation of rat pancreatic exocrine secretion by cocaine- and amphetamine-regulated transcript peptide.

Cocaine- and amphetamine-regulated transcript (CART) peptide is a recently described neuropeptide that has been localized to areas of the central and peripheral nervous systems. CART has been shown to be involved in feeding behavior when injected centrally, however, its effects upon peripheral tissues have not been studied. This report describes the effects of CART peptide on rat pancreatic exocrine secretion. Infusion of CART peptide caused four-fold increases in amylase secretion from anesthetized rats that had been fashioned with a bile-pancreatic duct cannula. CART peptide-induced increases in pancreatic secretion appear to involve pathways that are sensitive to both acetylcholine (ACh) and cholecystokinin (CCK) since pre-treatment with atropine (ACh receptor antagonist) or L-364,718 (CCK-A receptor antagonist) inhibited the effects of CART peptide on amylase secretion. Pre-treatment with a combination of atropine and L-364,718 abolished the effects of CART peptide. When isolated rat pancreatic acini were exposed to varying doses of CART peptide, no increase in amylase secretion was observed. The results of the present study suggest that CART peptide has stimulatory effects upon pancreatic exocrine secretion. CART peptide-induced increases in pancreatic secretion appear to be indirectly mediated as no direct effect upon pancreatic acini was shown. CART peptide likely acts upon either peripheral or central regulators of pancreatic secretory function that are distant from the acinar unit.

Cholinergic stimulation of rat acinar cells increases c-fos and c-jun expression via a mitogen-activated protein kinase-dependent pathway.

Acetylcholine release from cholinergic neurons regulates pancreatic exocrine function through pathways that are still under investigation. Pancreatic AR42J acinar cells were studied to determine intracellular calcium ([Ca(2+)](i)) release, enzyme activation, and gene expression in response to the acetylcholine analog carbachol (CCh). CCh stimulated dose-dependent increases in [Ca(2+)](i) that were inhibited by atropine and by specific inhibitors to the muscarinic receptor subtypes m1 and m3. Polymerase chain reaction analysis was performed, which sequenced products corresponding to the m1 and m3 receptor subtypes but not the m2 subtype. CCh also stimulated mitogen-activated protein kinase activity. CCh induced time-and dose-dependent increases in the c-fos and c-jun early-response genes, which were blocked by m1 and m3 inhibition but not by m2 inhibition.

Cholinergic intrapancreatic neurons induce Ca²+ signaling and early-response gene expression in pancreatic acinar cells.

Pancreatic exocrine function has been demonstrated to be under neuronal regulation. The pathways responsible for this effect, and the long-term consequences of such interactions, are incompletely described. The effects of neuronal depolarization on pancreatic acinar cells were studied to determine whether calcium signaling and c-fos expression were activated. In pancreatic lobules, which contain both neurons and acinar cells, agonists that selectively stimulated neurons increased intracellular calcium in acinar cells. Depolarization also led to the expression of c-fos protein in 24% +/- 4% of the acinar cells. In AR42J pancreatic acinar cells, cholinergic stimulation demonstrated an average increase of 398 +/- 19 nmol/L in intracellular calcium levels, and induced c-fos expression that was time and dose dependent. The data indicate that intrapancreatic neurons induce Ca²+ signaling and early-response gene expression in pancreatic acinar cells.

ATP induces c-fos expression in C6 glioma cells by activation of P(2Y) receptors.

BACKGROUND: Extracellular ATP functions in the enteric nervous system as a neurotransmitter, and recent evidence suggests ATP may regulate development through effects on cellular proliferation. METHODS: The action of ATP at purinoceptors and the role of second messenger pathways in c-fos mRNA expression in C6 glioma cells were investigated using the techniques of Northern and Western blotting. RESULTS: Treatment of C6 cells with ATP caused a time- and dose-dependent increase in c-fos expression. The rank order of agonist potency was ATP = ADP > gammasATP > alphabetaATP > betagammaATP > AMP = UTP. The ATP-induced c-fos increment was inhibited by three P(2Y) receptor antagonists-suramin, reactive blue, and DIDS-by 99+/-3, 89+/-7, and 61+/-14%, respectively. The ATP-stimulated c-fos expression was attenuated by phospholipase C inhibitor (U73122), protein kinase C (PKC) down-regulation (4alpha-phorbol 12-myristate 13-acetate and chelerythrine), mitogen-activated protein (MAP) kinase inhibition (apigenin), an inhibitor of MAP kinase kinase (PD98059), down-regulation of adenylate cyclase (SQ22536), and inhibition of type II protein kinase A (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate), but was not affected by inhibition of type I protein kinase A (8-bromoadenosine-3',5'-cyclic monophosphorothioate) and inhibitors of calmodulin kinase (KN93 and KN62). Phosphorylated MAP kinase was increased in cells exposed to ATP. This effect was suppressed by chelerythrine. CONCLUSIONS: These studies demonstrate that ATP-induced c-fos mRNA expression is under multifactorial regulation.

Perceived obstacles to career success for women in academic surgery.

HYPOTHESIS: We conducted this study to determine whether concerns expressed by male and female surgeons at 1 academic center are generally reflective of broader concerns for academic surgery and academic medicine. We reviewed published studies concerning women in academic surgery within the context of reporting the results of a survey of both male and female surgeons at 1 academic center. DATA SOURCES: We developed a survey that included demographic information, work experience, and social issues. The survey was distributed to the entire faculty. For key questions, we compared answers between male and female faculty. Additional data came from the published literature. STUDY SELECTION: We reviewed all available studies identified by a MEDLINE search with key words women and academic and medicine or physician. Included studies contained either data collection or editorial comment concerning women in academic medicine. DATA EXTRACTION: Data and opinions from all included studies paralleling survey questions were extracted from each article. DATA SYNTHESIS: Male and female faculty members reported different experiences and perceptions, specifically relating to relationships between family and professional life and perceptions of subtle sex-related biases. Both men and women reported insufficient mentoring and difficulties in balancing personal and professional responsibilities. CONCLUSIONS: Attitudes, behaviors, and traditions surrounding how we structure work and evaluate participation in academic surgery are more difficult to change than just addressing obvious inequities in support for female surgeons. However, attempting the deeper changes is worthwhile, because addressing obstacles faced by female faculty, many of which also affect men, will allow progress toward environments that attract and retain the best physicians, regardless of sex.

Endothelin-1 stimulates c-fos mRNA expression in C6 glioma cells via MAP kinase pathway.

Exposure of C6 glioma cells to endothelin-1 (ET-1) caused dose-dependent (10(-11) M to 10(-7) M) increments in intracellular calcium concentration ([Ca2+]i) and c-fos mRNA expression (4.5-fold) that were abolished by the endothelinA receptor antagonist, BQ610, and by inhibition of phospholipase C with U73122. ET-1 stimulated c-fos mRNA expression was also inhibited by protein kinase C inhibition (chelerythrine) and by the MAP kinase kinase inhibitor PD98059, but not by inhibitors of tyrosine kinases, protein kinase A type I or II, calmodulin kinase II, or calcium channel blockade. C6 cells treated with ET-1 demonstrated a significant increase in MAP kinase activity as evidenced by Western blotting. These results indicate a mechanism of long-term signaling by ET-1 involving an ET(A) receptor-mediated, phospholipase C(beta)-linked pathway that is dependent on protein kinase C and MAP kinase activation.

Calcium signaling induced by angiotensin II in the pancreatic acinar cell line AR42J.

The purpose of this study was to characterize the nature and mechanisms of angiotensin II-evoked calcium signaling in AR42J cells. Cytosolic calcium concentrations were determined using fura-2-based microfluorimetry. Angiotensin II causes elevations in free cytosolic calcium ([Ca2+]i) in the rat pancreatic acinar cell line AR42J. The mechanisms of angiotensin II-evoked calcium signaling were examined using fura-2-based fluorescent digital microscopy. Angiotensin II caused dose-dependent increments in [Ca2+]i over a concentration range of 0.1-1,000 nM, with an average increment of 243 +/- 16 nM at an angiotensin II concentration of 1,000 nM. Dup753, an AT1-specific antagonist, inhibited angiotensin II-evoked signaling, whereas the AT2 antagonist PD123,319 had no effect. Preincubation with the phospholipase C inhibitor U73122 reduced the response in [Ca2+]i to 25% of that of the control. Thapsigargin abolished angiotensin II-evoked calcium signaling. The inositol 1,4,5-trisphosphate receptor antagonist heparin introduced by radiofrequency electroporation inhibited responses to 46 +/- 6% of controls. Angiotensin II-evoked signals were reduced in magnitude and duration by elimination of Ca2+ from the extracellular buffer. Preincubation with pertussis toxin (100 ng/ml) had no effect. Angiotensin II did not stimulate cyclic AMP or suppress vasoactive intestinal peptide stimulated cyclic AMP production over the concentration range that caused Ca2+ signaling.

Calcium signaling pathways in the enteric nervous system.

The intracellular calcium level is an important regulator of cellular functioning, and its tight regulation occurs at several levels. Influx of extracellular calcium occurs via voltage-gated channels in response to a depolarization, and "capacitative" extracellular calcium occurs with cellular calcium depletion. Intracellular release of calcium also occurs through both the inositol trisphosphate- and ryanodine-sensitive stores of calcium. The following will detail these events and recent discoveries in Ca2+ signaling, and will specifically focus on their role in the functioning of the enteric nervous system.