A pH-sensitive, neurogenic pathway mediates disease severity in a model of post-ERCP pancreatitis.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) has a high risk of pancreatitis although the underlying mechanisms are unclear. Transient receptor potential vanilloid 1 (TRPV1) is a cation channel expressed on C and Adelta fibres of primary sensory neurons and is activated by low pH. TRPV1 activation causes release of inflammatory mediators that produce oedema and neutrophil infiltration. We previously demonstrated that neurogenic factors contribute to the pathogenesis of pancreatitis. Resiniferatoxin (RTX) is a TRPV1 agonist that, in high doses, defunctionalises C and Adelta fibres. When we discovered that the pH of radio-opaque contrast solutions used for ERCP was 6.9, we hypothesised that low pH may contribute to the development of contrast-induced pancreatitis via activation of TRPV1. METHODS: Rats underwent equal pressure pancreatic ductal injection of contrast solutions at varying pH with or without RTX. RESULTS: Contrast solution (pH 6.9) injected into the pancreatic duct caused a significant increase in pancreatic oedema, serum amylase, neutrophil infiltration, and histological damage. Solutions of pH 7.3 injected at equal pressure caused little damage. The severity of the pancreatitis was significantly increased by injection of solutions at pH 6.0. To determine if the effects of low pH were mediated by TRPV1, RTX was added to the contrast solutions. At pH levels of 6.0 and 6.9, RTX significantly reduced the severity of pancreatitis. CONCLUSIONS: Contrast solutions with low pH contribute to the development of pancreatitis through a TRPV1-dependent mechanism. It is possible that increasing the pH of contrast solution and/or adding an agent that inhibits primary sensory nerve activation may reduce the risk of post-ERCP pancreatitis.

Prolonged two-man basic life support may result in hypocarbia in the ventilating rescuer.

OBJECTIVES: To determine whether the quality of expired air given during mouth-to-mouth ventilation differs between one- and two-person basic life support. METHODS: 15 young fit volunteers performed 15-min simulated resuscitation on a manikin. The oxygen and carbon dioxide concentration of their expired breath and the total ventilation was continuously monitored. Compression:ventilation ratios of 15:2 for one-person and 5:1 for two-person resuscitation were used. RESULTS: In two-man resuscitation, where the rescuer who is ventilating the patient is not performing chest compressions, the oxygen content of the expired breath rises (P<0.01), and the carbon dioxide content falls (P<0.01). The carbon dioxide concentration declined gradually throughout the 15-min session. Most participants complained of light-headedness on completion of the two-man session. Total ventilation did not differ between the two methods (P=0.757, 95% CI=-0.329, 0.242). CONCLUSION: Trainees in basic life support should be informed that symptoms of hypocarbia may occur in prolonged mouth-to-mouth ventilation, when acting in a two-man team. We would advise rescuers using these protocols to change places every 5 min to avoid these symptoms. These findings add further weight to the recommendations that all resuscitation should be carried out using 15:2 compression:ventilation ratio.

Capsaicin vanilloid receptor-1 mediates substance P release in experimental pancreatitis.

We examined whether the capsaicin vanilloid receptor-1 (VR1) mediates substance P (SP) release from primary sensory neurons in experimental pancreatitis. Pancreatitis was achieved by 12 hourly injections of caerulein (50 microg/kg ip) in mice. One group received capsazepine (100 micromol/kg sc), a competitive VR1 antagonist, at 4-h intervals. Neurokinin-1 receptor (NK1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK1R endocytosis. The severity of pancreatitis was assessed by measurements of serum amylase, pancreatic myeloperoxidase (MPO) activity, and histological grading. Caerulein administration caused significant elevations in serum amylase and pancreatic MPO activity, produced histological evidence of pancreatitis, and caused a dramatic increase in NK1R endocytosis. Capsazepine treatment significantly reduced the level of NK1R endocytosis, and this was associated with similar reductions in pancreatic MPO activity and histological severity of pancreatitis. These results demonstrate that repeated caerulein stimulation causes experimental pancreatitis that is mediated in part by stimulation of VR1 on primary sensory neurons, resulting in endogenous SP release.

Monitor peptide binding sites are expressed in the rat liver and small intestine.

125I-monitor peptide binding was performed using frozen sections of the rat liver and gut and visualized using autoradiography. Saturable binding was observed in unidentified single cells in the liver and in the mucosa of the small intestine. Epidermal growth factor (EGF) and GTPgammaS did not inhibit 125I-monitor peptide binding indicating that the binding sites are not EGF receptors or G protein-coupled receptors. The liver binding site exhibited an affinity 3.7-4.4-fold higher than those in the small intestine. It has been established that intraluminal monitor peptide releases cholecystokinin from the small intestine. The present results indicate that monitor peptide may also have liver associated functions.

Time to onset of effect of fluticasone propionate in patients with asthma.

BACKGROUND: The effectiveness of inhaled glucocorticoids in the treatment of asthma is well documented; however, times to onset and maximal treatment effects of these agents have been poorly described. OBJECTIVE: We sought to determine the time to onset of effect and the time to the best observed effect of inhaled fluticasone propionate (FP) in patients with asthma. METHODS: Data from 8 randomized, double-blind, placebo-controlled clinical trials of at least 8 weeks' duration were analyzed. Corticosteroid-naive patients (n = 1461) were treated with either FP (25 micrograms to 500 micrograms) or placebo twice daily. Efficacy evaluations included morning peak expiratory flow (PEF), asthma symptom scores, supplemental albuterol use, and FEV1. RESULTS: Statistically significant improvements in PEF, asthma symptom scores, and supplemental albuterol use were observed beginning on day 1 of treatment in the FP group versus the placebo group (P <.001); significant increases in FEV1 were observed at the first measurement at week 1 (P <.001). The best observed effect occurred within 3 weeks of the start of FP treatment for PEF (+36 L/min) and FEV1 (+0.52 L) and within 2 weeks for reduction in supplemental albuterol use and asthma symptom scores. Patients with the most severe airflow obstruction had the greatest change in PEF (+56 L/min) and fastest time to 50% of best observed effect (3 days) compared with patients with mild or moderate airflow obstruction; however, time to best observed effect was similar in the 3 groups (20 to 27 days). CONCLUSION: In patients with asthma, the onset of significant benefit of FP on PEF, symptoms, and rescue albuterol use occurred within 1 day of the start of therapy. FEV1 improved within 1 week of the start of therapy (the first measurement after randomization). There was no effect of sex, age, or dose of FP on the time to response. The best observed response in PEF varies with the degree of baseline airflow obstruction; however, the degree of airflow obstruction has no effect on the time to best observed response.

Activation of calcium channels by cAMP in STC-1 cells is dependent upon Ca2+ calmodulin-dependent protein kinase II.

Activation of L-type calcium channels in the neuroendocrine, cholecytstokinin-secreting cell line, STC-1, is vital for secretion of CCK. In the present study, the regulation of L-type Ca2+ channels by cAMP and Ca2+ calmodulin dependent protein kinase II (CaM-KII) in STC-1 cells was investigated. Exposure to 3-isobutyl-1-methylxanthine (IBMX) increased intracellular cAMP levels, whole cell Ca2+ currents and activated Ca2+ channels in cell-attached membrane patches. Furthermore, in Fura-2AM loaded cells, cytosolic Ca2+ levels increased upon exposure to IBMX. By contrast, pretreatment of cells with the CaM-KII inhibitor KN-62, prevented IBMX activation of Ca2+ channels in cell-attached patches or increases in cytosolic Ca2+ levels. Inclusion of the synthetic peptide fragment 290-309 of CaM-KII, a CaM-KII antagonist, in the pipette solution, blocked the activation of whole cell Ca2+ currents upon addition of IBMX. These results indicate a unique mechanism of L-type Ca2+ channel activation involving two phosphorylation events.

Neurohumoral control of the exocrine pancreas.

Recent advances in the study of pancreatic exocrine secretion are reviewed, with an emphasis on neurohumoral mechanisms. In the past year, cDNA for the human pancreatic sodium-bicarbonate cotransporter was cloned, and the expressed protein was localized to pancreatic acini and ductal cells. Recent information suggests that the cholecystokinin B receptor has a role in pancreatic amylase release. Further evidence supports the concept of a protease-sensitive negative feedback mechanism regulating pancreatic exocrine secretion. Study of the expression of the receptors responsible for the regulation of pancreatic function has proven fruitful in the determination of the molecular mechanisms of hormone signal transduction and desensitization. Studies of peptide 1, pituitary adenylate cyclase-activating peptide, and gastrin-releasing peptide have shown how these peptides participate in the regulation of pancreatic secretion and have provided information on intracellular signaling pathways obtained using rat pancreatic tumor cells. Neural regulation via cholinergic receptors in isolated pancreatic acini and the mechanisms responsible for other neurotransmitters, such as calcitonin gene-related peptide, histamine, and dopamine, are reviewed. This review highlights recent discoveries in the neurohumoral regulation of pancreatic exocrine secretion.

Inhibition of Na+/H+ exchange stimulates CCK secretion in STC-1 cells.

It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.

Efficacy of salmeterol xinafoate powder in children with chronic persistent asthma.

BACKGROUND: The efficacy and safety of salmeterol powder have not previously been evaluated in children with asthma in the United States. OBJECTIVE: The efficacy and safety of salmeterol powder versus placebo were compared in children between the ages of 4 and 11 years with chronic persistent asthma. METHODS: A randomized, double-blind, placebo-controlled, parallel group trial was performed at 11 clinical centers. Two hundred seven patients were randomly assigned to receive 50 microg salmeterol powder or placebo (and albuterol as needed) twice daily via a breath-actuated device for 12 weeks. Twelve-hour serial pulmonary function assessments were conducted on day 1 and at week 12. Daily recordings of morning and evening peak expiratory flow (PEF), supplemental albuterol use, asthma symptoms, and nocturnal awakenings were assessed. RESULTS: On day 1 and at week 12, weighted mean percent of predicted PEF (P < .001, day 1 and P=.008, week 12) and weighted mean forced expiratory volume in one second (P < .001, day 1 and week 12) were significantly higher at all timepoints evaluated over the 12-hour postdosing period in patients treated with salmeterol powder compared with placebo. Overall reductions in supplemental albuterol use and mean asthma symptom scores were also significantly greater in children administered salmeterol compared with placebo (P=.004 and P=.006, respectively). The frequency of adverse events was similar in the two treatment groups. CONCLUSION: Salmeterol powder (50 microg twice daily) is effective and safe in producing bronchodilation and relieving symptoms in children with chronic persistent asthma during 12 weeks of treatment.

Investigation into voltage breakdown in glyceryl trinitrate patches.

There are clinical reports of explosions occurring in glyceryl trinitrate (GTN) patches left on patients who are defibrillated and it is widely recommended that such patches are removed before attempting defibrillation. We devised an experimental set up that simulated the electrical conditions under which human defibrillation is conducted, thus making possible accurate measurements of defibrillator wave form. No problems occurred with GTN patches that incorporated a plastic backing. However, with GTN patches that incorporated a metal mesh backing, an explosive effect was consistently observed when current from the defibrillator electrode flowed through the metallic backing. The GTN paste in the patches was unaffected and therefore, we conclude that the apparent explosions reported are solely due to voltage breakdown as the paddle voltage is close to 3 kV resulting from the 360 J of energy. Our results emphasise the need for good technique during defibrillation to avoid current flow through such patches. In addition, our results have implications for the design of patches used for the transcutaneous delivery of drugs.

Inhibition of gastric emptying in response to intestinal lipid is dependent on chylomicron formation.

Lipid in the intestine initiates feedback inhibition of proximal gastrointestinal function and food intake. In rats and humans, inhibition of gastric emptying is mediated, at least in part, by cholecystokinin (CCK)-A receptors, and in rats there is evidence for involvement of an intestinal vagal afferent pathway. The mechanism by which luminal lipid acts to release CCK or activate vagal afferent nerve terminals is unclear. The role of chylomicron formation in this sensory transduction pathway has been investigated using the hydrophobic surfactant Pluronic L-81 that inhibits chylomicron formation. Gastric emptying of liquids was measured in awake rats fitted with a Thomas gastric fistula and a duodenal cannula. Intestinal perfusion of lipid induced a dose-dependent inhibition of gastric emptying (6, 12, and 39% inhibition for 25, 50, and 100 mg lipid, respectively). Perfusion of lipid with Pluronic L-81 (2.8% wt/vol) reversed the lipid-induced inhibition of gastric emptying. Pluronic L-63, a chemically similar surfactant that has no effect on chylomicron formation, had no effect on lipid-induced inhibition of gastric emptying. Perfusion of the intestine with lipid (100 mg) increased plasma levels of CCK from 1.9 +/- 0.8 to 6. 5 +/- 1 pM. This increase was blocked by Pluronic L-81 but unaffected by L-63. These results provide evidence that chylomicron formation is important in the signaling of lipid in the intestinal lumen to CCK endocrine cells and to producing feedback inhibition of gastric emptying.

Dose-related response to inhaled fluticasone propionate in patients with methacholine-induced bronchial hyperresponsiveness: a double-blind, placebo-controlled study.

Dose-response relationships with inhaled corticosteroids in the treatment of asthma have been difficult to establish. A multicenter, double-blind, parallel-group study was conducted to evaluate the clinical efficacy and safety of low doses of inhaled fluticasone propionate (FP) in patients with mild to moderate asthma. Methacholine challenge testing was conducted in addition to measurement of traditional efficacy variables. After a single-blind screening period, 138 patients > or = 12 years of age were randomly assigned to receive placebo, FP 50 microg, or FP 100 microg, twice daily for 8 weeks. The results of methacholine challenge testing averaged over all visits favored FP 200 microg/day over placebo and FP 100 microg/day (p < 0.05); there were no significant differences between placebo and FP 100 microg/day. Mean changes from baseline to endpoint favored each dose of FP over placebo based on forced expiratory volume in 1 sec (FEV1), patient-measured peak expiratory flow (PEF), total symptom scores, and rescue bronchodilator use (p < 0.05); there were no differences in these parameters between the two doses of FP. The addition of methacholine challenge testing allowed definition of a dose-response relationship that was not apparent with traditional efficacy variables.

Salmeterol powder compared with albuterol aerosol as maintenance therapy for asthma in adolescent and adult patients.

Two multicenter, randomized, double-masked, placebo-controlled studies involving 451 adolescent and adult patients with mild-to-moderate asthma compared the efficacy and safety of salmeterol powder 50 micrograms twice daily with albuterol 180 micrograms four times daily or placebo (with albuterol as needed) for 12 weeks. Patients had forced expiratory volume in 1 second (FEV1) of 50% to 80%. Throughout the 12-week treatment period, the mean change from baseline in percentage of predicted FEV1 was significantly greater with salmeterol than with placebo; mean area under the curve for FEV1 was significantly greater with salmeterol than with albuterol or placebo. Significant improvements in morning and evening peak expiratory flow, percentage of nights without awakening, and asthma symptoms were observed with salmeterol. Salmeterol was well tolerated, and no clinically significant changes in electrocardiographic activity were observed.

Bioactivity of intraduodenally and intravenously infused fragments of luminal cholecystokinin releasing factor (LCRF).

A luminal cholecystokinin releasing factor (LCRF), has been purified from intestinal secretion and found to have a mass of 8136 daltons. The amino-terminal 41 residues have been sequenced. Previous studies showed that intraduodenal infusion of the synthetic amino-terminal 35 amino acid peptide, LCRF1-35 significantly stimulated pancreatic protein and fluid secretion in conscious rats, but the peptide did not stimulate amylase release from isolated, dispersed pancreatic acini. In the present study, several fragments of LCRF were synthesized and tested for CCK-releasing activity (pancreatic protein secretion) to determine whether shorter fragments of LCRF exhibit the characteristic biological activity of native LCRF and synthetic LCRF1-35. Compounds tested were LCRF1-41, LCRF1-35, LCRF1-65 and LCRF11-25. Of the fragments shorter than LCRF1-35, only LCRF11-25 but not LCRF1-6 had significant CCK releasing activity. LCRF1-41 was equivalent to LCRF1-35 in potency and efficacy. Intravenous and intraduodenal infusion of LCRF1-35 elicited nearly identical dose-response curves.

Distribution and localization of a novel cholecystokinin-releasing factor in the rat gastrointestinal tract.

The purpose of this study was to examine the distribution and localization of an intestinal cholecystokinin (CCK)-releasing factor, called luminal CCK-releasing factor (LCRF), in the gastrointestinal tract and pancreas of the rat. RIA analysis indicates that LCRF immunoreactivity is found throughout the gut including the pancreas, stomach, duodenum, jejunum, ileum, and colon with the highest levels in the small intestine. Immunohistochemistry analysis shows LCRF immunoreactivity staining in intestinal villi, Brunner's glands of the duodenum, the duodenal myenteric plexus, gastric pits, pancreatic ductules, and pancreatic islets. These results indicate potential sources for secretagogue-stimulated release of luminal LCRF and support the hypothesis that LCRF is secreted into the intestinal lumen to stimulate CCK release from mucosal CCK cells.

Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes.

To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 microM) to block Na+ channels, exposure of the apical membrane to ATP significantly increased the short-circuit current (Isc) from 18.2 +/- 5.9 to 52.8 +/- 12.7 microA (n = 18). The response to ATP is mediated by basolateral-to-apical Cl- transport because it is inhibited by 1) the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 microM) in the apical chamber, 2) the K+ channel blocker Ba2+ (5 mM), or 3) the Na(+)-K(+)-2Cl- cotransport inhibitor bumetanide (200 microM) in the basolateral chamber. Other nucleotides stimulated an increase in Isc with a rank order potency of UTP = ATP = adenosine 5'-O-(3)-thiotriphosphate, consistent with P2u purinergic receptors. ADP, AMP, 2-methylthioadenosine 5'-triphosphate, and adenosine had no effect. A cDNA encoding a rat P2u receptor (rP2uR) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl- secretion through activation of P2u receptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

An amino-terminal fragment of LCRF, LCRF-(1-35), has the same activity as the natural peptide.

A cholecystokinin (CCK)-releasing peptide, luminal CCK-releasing factor (LCRF), has been purified from rat jejunal secretion. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8,136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for the amino-terminal 41 residues. To determine the biologically active region of the molecule, a peptide was synthesized consisting of the amino-terminal 35 amino acids of LCRF. In this study, intraduodenal infusion of LCRF-(1-35) significantly stimulated pancreatic secretion in conscious rats. The dose-response curves to LCRF-(1-35) and to monitor peptide were similar and biphasic, with higher doses producing submaximal pancreatic secretory responses. The CCK-A receptor antagonist MK-329 abolished the pancreatic secretory response to intraduodenally infused LCRF-(1-35). These results demonstrate that LCRF biological activity is contained within the amino-terminal 35-amino acid portion of LCRF, and this fragment may be useful for investigating the role of LCRF in gastrointestinal function.