Bradley J. Segura

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 (10−11‐10−7m) produced no change in basal amylase release from dispersed, purified acinar cells. Co‐incubation of ghrelin (10−11−10−7m) with CCK−8 (10−10m) demonstrated no inhibition of CCK‐stimulated amylase release from dispersed acini. In contrast, ghrelin (10−9−10−7m) 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.

Intercellular Calcium Waves in Cultured Enteric Glia From Neonatal Guinea Pig

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura‐2 AM microfluorimetry. Single‐cell stimulation was performed with a micromanipulator‐driven glass pipette. Data were expressed as mean ± SEM and analyzed by Students t‐test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% ± 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 μM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 μM) significantly decreased the percentage of cells responding to mechanical stimulation (6% ± 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% ± 13% vs. 60% ± 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles. GLIA 42:252–262, 2003.

No free ride? The hidden costs of delayed operative management using a spring-loaded silo for gastroschisis

PURPOSE The ideal management of gastroschisis (primary vs staged closure) has not yet been established. Despite the ease of silo placement, anecdotal experience shows that silos do not always offer benefit. The aim of this study was to highlight concerns regarding use of spring loaded silos and compare outcomes to primary closure. METHODS Thirty-seven neonates with gastroschisis treated with either primary (n = 10) or staged closure with a spring-loaded silo (n = 27) were reviewed (1998-2007). Variables included ventilator days, daily intravenous fluid, hospital days, and complication rates. SPSS (SPSS Inc, Chicago, Ill) was used to perform t test and chi(2) analyses (significance P < .05). RESULTS Survival for primary closure was 100% (10/10) compared to 89% (24/27) for staged closure (P = .548). Patients managed with silos required prolonged ventilation (16.1 +/- 4 days vs 3.6 +/- 1 days; P < or = .05) and greater intravenous fluids on days 3, 4, and 5 of life (132 +/- 25 mL/kg per day vs 104 +/- 18 mL/kg per day; P < or = .01). Although there was no difference in the complication rates between the groups, several problems were evident in the silo group: 15% (4/27) required silo replacement, 44% (12/27) required fascial defect enlargement for silo placement, and 19% (5/27) required mesh at closure. No significant differences in recovery of intestinal function were observed. Three silo patients developed ischemic complications because of vascular insufficiency at the level of the abdominal wall, leading to significant intestinal loss, ventilator and total parenteral nutrition dependence, and increased hospital stay. CONCLUSIONS Patients managed with a silo had longer ventilator requirements and greater fluid needs. This Specific technical complications leading to bowel ischemia were notable in the silo group. The silo should be carefully placed to avoid bowel twisting and the funnel effect. Larger prospective studies should be performed to provide decision-making criteria for the use of a silo vs primary closure.

Microorganisms influence the composition of honeydew produced by the silverleaf whitefly, Bemisia argentifolii

Abstract Sugars in the honeydew produced by the silverleaf whitefly, Bemisia argentifolii , and in fermentation of sucrose using homogenates of these insects were analyzed by high performance liquid chromatography. Results suggest that the unusual disaccharide, trehalulose, found in large quantity in honeydew of B. argentifolii , is produced by obligate intracellular microorganisms residing in this insects mycetomes. Some larger oligosaccharides in this honeydew may be produced by certain Bacillus spp. residing in or on the insects but these bacteria are not involved in an obligate relationship with the whitefly.

Surgical Management of Ovarian Disease in Infants, Children, and Adolescents: A 15-Year Review

BACKGROUND Despite the reported efficacy and the presumed benefits of minimally invasive surgery (MIS) for ovarian lesions in adults, questions remain as to the surgical indications, results, and outcomes for these procedures across pediatric age groups. The aim of this study was to review our experience with the management of ovarian disease in children to determine if there has been a shift in the management of these lesions from open surgery (OS) to an MIS approach in the pediatric population. METHODS An institutional review board (IRB)-approved retrospective chart review included all patients who underwent surgical management of ovarian disease from January 1, 1992 to July 10, 2007. Patients with ectopic pregnancy, known pelvic inflammatory disease, or concomitant illness requiring operative management at the time of ovarian surgery were excluded. Demographics, clinical signs and symptoms, diagnosis, surgical outcomes, and history of prior abdominal procedures were obtained. Statistical analysis included comparison of means, paired t-test, chi-squared test, and multivariate analysis, where indicated. RESULTS A total of 231 patients were evaluated in this study, with a mean age of 12.8 years (range, 3 weeks to 20 years). There were 221 (95.7%) benign lesions and 10 (4.3%) were malignant. There were 156 simple or hemorrhagic cysts (70.5%) and 46 mature teratomas (20.8%). Three complications (1.3%) occurred, which were associated with surgery and no mortalities. Abdominal pain (82.3%), nausea or vomiting (24.2%), and abdominal tenderness (10.0%) were the most common presenting symptoms or signs. Operative outcomes for benign disease (n = 221) were compared between MIS and open cases over the entire time period as well as within three consecutive 5-year time intervals. CONCLUSIONS There was a notable shift toward the management of benign ovarian disease in using MIS techniques over the course of three different 5-year intervals. This approach was also associated with shorter hospital stay, less operative blood loss, and shorter operative times, when compared to an open approach. When indicated, a laparoscopic approach should be performed for presumed benign ovarian disease in children.

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.

A review of laparoscopic Nissen fundoplication in children weighing less than 5 kg

PURPOSE Minimally invasive procedures in small infants and neonates are being performed in increasing numbers. In this study, we describe our institutions experience with laparoscopic Nissen fundoplications (LNFs) in children weighing less than 5 kg. METHODS All cases of LNF attempted in children weighing less than 5 kg since January 2003 at a tertiary-care pediatric hospital were reviewed after Institutional Review Board approval. RESULTS One hundred twenty-two children weighing less than 5 kg underwent LNF during the study period. They ranged from 2 weeks to 3 years of age (mean, 94 +/- 61.3 days) and weighed 1.94 to 4.99 kg (mean, 3.68 +/- 0.77 kg). Twenty-nine percent (n = 35) were neurologically impaired. Eighty-eight percent (n = 107) had concurrent gastrostomy tube placement. Eight (7%) were converted to laparotomy. The average operative time was 112 +/- 46 minutes. Seventy-one percent (n = 87) required intensive care unit use for an average of 14.3 +/- 17.4 days. The average time to start enteral feeds was 2.6 +/- 2.6 days. Thirty-one percent (n = 38) required postoperative mechanical ventilation for an average of 12.0 +/- 20.6 days. The average hospital length of stay was 36.6 +/- 36.0 days (range, 3-175 days). Six patients (5%) had a complication or recurrent gastroesophageal reflux. Three patients had recurrent reflux, one of which underwent another LNF. One patient had a gastric perforation. Another required a redo LNF after a disrupted wrap was noted at a recurrent hiatal hernia repair. Lastly, one patient had bleeding from an accessory hepatic artery with liver retractor placement. CONCLUSIONS Laparoscopic Nissen fundoplication can safely and effectively be performed in small children (<5 kg) with similar outcomes and rates of complication as previously published reports in larger children. These children, however, do have prolonged intensive care unit and mechanical ventilation use associated with their prematurity and significant comorbidities.

Presence of functionally active protease-activated receptors 1 and 2 in myenteric glia

Protease‐activated receptors (PARs) belong to the family of membrane receptors coupled to G‐proteins; their presence is reported in a wide variety of cells. The object of this study was to demonstrate the presence of PAR‐1 and PAR‐2 in myenteric glia of the guinea pig, and to elucidate the cellular mechanisms that are triggered upon receptor activation. Thrombin and PAR‐1 agonist peptide (PARP‐1) activate PAR‐1 with a maximum mean ± SEM change in intracellular calcium concentration with respect to basal level (Δ[Ca2+]i) of 183 ± 18 nm and 169 ± 6 nm, respectively. Trypsin and PAR‐2 agonist peptide (PARP‐2) activate PAR‐2 with a maximum Δ[Ca2+]i of 364 ± 28 nm and 239 ± 19 nm, respectively. Inhibition of phospholipase C by U73312 (1 µm) decreased the Δ[Ca2+]i due to PAR‐1 activation from 167 ± 10 nm to 87 ± 6 nm. The PAR‐2‐mediated Δ[Ca2+]i decreased from 193 ± 10 nm to 124 ± 8 nm when phospholipase C activity was inhibited. Blockade of sphingosine kinase with dimethylsphingosine (1 µm) decreased the Δ[Ca2+]i due to PAR‐2 activation from 149 ± 19 nm to 67 ± 1 nm, but did not influence the PAR‐1‐mediated Δ[Ca2+]i. PAR‐1 and PAR‐2 were localized in myenteric glia by immunolabeling. Our results indicate that PAR‐1 and PAR‐2 are present in myenteric glia of the guinea pig, and their activation leads to increases in intracellular calcium via different signal transduction mechanisms that involve activation of phospholipase C and sphingosine kinase.

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.