Zachary M. Sellers

The evaluation and treatment of gastrointestinal disease in children with cystinosis receiving cysteamine

OBJECTIVES Cysteamine prevents organ damage in children with cystinosis, but may cause gastrointestinal (GI) symptoms. In this study we evaluated the nature of GI disease, and the value of omeprazole in controlling GI symptoms in these children. STUDY DESIGN Upper GI disease was evaluated with endoscopy, gastrin levels, and acid secretion studies after oral administration of cysteamine, before and after 16 weeks of therapy with omeprazole. A symptom score was devised. RESULTS Eleven children (mean age, 5.7 years) were studied. After cysteamine ingestion, before and after omeprazole therapy, the mean maximum acid output was significantly higher than the mean basal acid output. The maximum acid output was measured within 60 minutes of cysteamine ingestion and was reduced by omeprazole therapy (P<.01). The mean peak gastrin level was 30 minutes postcysteamine and was higher than baseline (P<.01). The initial mean symptom score (maximum score, 14) was 6.9 and fell to 0.7 (P<.0001) after 16 weeks of omeprazole therapy. At endoscopy, two children had diffuse gastric nodularity, and nearly all had cystine crystal deposits. CONCLUSIONS GI symptoms in children with cystinosis receiving cysteamine are often acid-mediated and improve with omeprazole. Cystine crystals were detected in the GI tract and may signify inadequate treatment with cysteamine.

Calcium channel γ subunits: a functionally diverse protein family

The calcium channel γ subunits comprise an eight-member protein family that share a common topology consisting of four transmembrane domains and intracellular N- and C-termini. Although the first γ subunit was identified as an auxiliary subunit of a voltage-dependent calcium channel, a review of phylogenetic, bioinformatic, and functional studies indicates that they are a functionally diverse protein family. A cluster containing γ1 and γ6 conforms to the original description of the protein family as they seem to act primarily as subunits of calcium channels expressed in muscle. Members of a second cluster (γ2, γ3, γ4, γ8) function as regulators of AMPA receptor localization and function in the brain and are collectively known as TARPs. The function of members of the third cluster (γ5, γ7) remains unclear. Our analysis shows that the members of each cluster contain conserved regulatory motifs that help to differentiate the groups. However, the physiological significance of these motifs in many cases remains to be demonstrated.

Cardiomyocytes with disrupted CFTR function require CaMKII and Ca2+-activated Cl− channel activity to maintain contraction rate

The physiological role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cardiomyocytes remains unclear. Using spontaneously beating neonatal ventricular cardiomyocytes from wild‐type (WT) or CFTR knockout (KO) mice, we examined the role of CFTR in the modulation of cardiomyocyte contraction rate. Contraction rates of spontaneously beating myocytes were captured by video imaging. Real‐time changes in intracellular ([Ca2+]i) and protein kinase A (PKA) activity were measured by fura‐2 and fluorescence resonance energy transfer, respectively. Acute inhibition of CFTR in WT cardiomyocytes using the CFTR inhibitor CFTRinh‐172 transiently inhibited the contraction rate. By contrast, cardiomyocytes from CFTR KO mice displayed normal contraction rates. Further investigation revealed that acute inhibition of CFTR activity in WT cardiomyoctyes activated L‐type Ca2+ channels, leading to a transient increase of [Ca2+]i and inhibition of PKA activity. Additionally, we found that contraction rate normalization following acute CFTR inhibition in WT cardiomyocytes or chronic deletion in cardiomyocytes from CFTR KO mice requires the activation of Ca2+/calmodulin‐dependent kinase II (CaMKII) and Ca2+‐activated Cl− channels (CaCC) because simultaneous addition of myristoylated‐autocamtide‐2‐related inhibitory peptide or niflumic acid and CFTRinh‐172 to WT cardiomyocytes or treatment of cardiomyoctes from CFTR KO mice with these agents caused sustained attenuation of contraction rates. Our results demonstrate that regulation of cardiomyocyte contraction involves CFTR. They also reveal that activation of CaMKII and CaCC compensates for loss of CFTR function. Increased dependence on CaMKII upon loss of CFTR function might leave cystic fibrosis patients at increased risk of heart dysfunction and disease.

Heat-stable enterotoxin of Escherichia coli (STa) can stimulate duodenal HCO3− secretion via a novel GC-C- and CFTR-independent pathway

The heat‐stable enterotoxin of Esche richia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC‐C) has been shown to be the primary receptor involved in mediating this response. However, numer ous studies have suggested the existence of an alternative STa‐binding receptor. The aims of this study were to determine whether a non‐GC‐C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. 125I‐STa‐binding experiments were performed with intestinal muco sae from GC‐C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa‐binding receptor was explored by exam ining STa‐, uroguanylin‐, and guanylin‐stimulated duo denal bicarbonate secretion (DBS) in GC‐C KO mice in vitro and in vivo. Significant 125I‐STa‐binding occurred in the proximal small intestines of GC‐C KO and WT mice. Analysis of binding coefficients and pH depen dence showed that 125I‐STa‐binding in GC‐C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimu lated a significant increase in DBS in GC‐C KO mice. Uroguanylin‐ and guanylin‐stimulated DBS were signif icantly inhibited by glibenclamide, but not by 4,4 ′‐diisothiocyanato‐stilbene‐2,2 ′‐disulfonic acid (DIDS). However, STa‐stimulated DBS was unaffected by glib enclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non‐GC‐C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin‐ and guanylin‐stimulated DBS are cystic fibrosis transmembrane con ductance regulator (CFTR) dependent, STa‐stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intesti nal bicarbonate secretion in cystic fibrosis.—Sellers, Z. M., Mann, E., Smith, A., Ko, K. H., Giannella, R., Cohen, M. B., Barrett, K. E., Dong, H. Heat‐stable enterotoxin of Escherichia coli (STa) can stimulate du odenal HCO3− secretion via a novel GC‐C‐ and CFTR‐ independent pathway. FASEB J. 22, 1306–1316 (2008)

MRP4 and CFTR in the regulation of cAMP and β-adrenergic contraction in cardiac myocytes.

Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of β-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl(-) channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and β-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, β(1)-, β(2)-, and β(1)/β(2)-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via β(1)-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in β-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes.

A critical GxxxA motif in the γ6 calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current

The eight members of the calcium channel γ subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of γ2, γ3, γ4 and γ8 (the TARPs) modulate AMPA receptor localization and function, the γ1 and γ6 subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the γ6 subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of γ6 subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of γ6 is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix–helix interactions in its effects. Results from co‐immunoprecipitation experiments confirm a physical association of γ6 with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of γ6 reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the γ6 calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density.

Left ventricular and aortic dysfunction in cystic fibrosis mice

BACKGROUND Left ventricular (LV) abnormalities have been reported in cystic fibrosis (CF); however, it remains unclear if loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes heart defects independent of lung disease. METHODS Using gut-corrected F508del CFTR mutant mice (ΔF508), which do not develop human lung disease, we examined in vivo heart and aortic function via 2D transthoracic echocardiography and LV catheterization. RESULTS ΔF508 mouse hearts showed LV concentric remodeling along with enhanced inotropy (increased +dP/dt, fractional shortening, decreased isovolumetric contraction time) and greater lusitropy (-dP/dt, Tau). Aortas displayed increased stiffness and altered diastolic flow. β-adrenergic stimulation revealed diminished cardiac reserve (attenuated +dP/dt,-dP/dt, LV pressure). CONCLUSIONS In a mouse model of CF, CFTR mutation leads to LV remodeling with alteration of cardiac and aortic functions in the absence of lung disease. As CF patients live longer, more active lives, their risk for cardiovascular disease should be considered.

A role for CagA/VacA in Helicobacter pylori inhibition of murine duodenal mucosal bicarbonate secretion.

Duodenal mucosal bicarbonate secretion is diminished in patients with Helicobacter pylori (HP)-associated duodenal ulcer disease. We examined whether HP water extracts inhibit murine duodenal mucosal bicarbonate secretion {in vitro}, and the mechanisms involved. Murine duodenal mucosae were mounted in Ussing chambers. Short-circuit current and bicarbonate secretion was measured. CagA/VacA-positive HP water extract (HPWE+/plus;) markedly inhibited PGE2-, carbachol-, or the calcium ionophore A23187-stimulated bicarbonate secretion in a dose-dependent manner. While 3-isobutyl-1-methylxanthine-stimulated bicarbonate secretion was not affected by HPWE+/plus;, HPWE+/plus; did diminish forskolin-stimulated bicarbonate secretion. HPWE+/plus; markedly diminished PGE2-induced increases in duodenal mucosal cAMP. CagA/VacA of HP decreases Ca2+-mediated bicarbonate secretiondownstream of increases in intracellular Ca2+. Dimunition of PGE2-stimulated bicarbonate secretion occurs, in part, by inhibition of adenylate cyclase, which leads to decreased cAMP levels. The ability of virulent HP strains to inhibit duodenal bicarbonate secretion through multiple intracellular pathways likely contributes to the pathogenesis of HP-associated duodenal ulcer disease.

Strain rate echocardiography uncovers subclinical left ventricular dysfunction in cystic fibrosis.

BACKGROUND CFTR is expressed in cardiac myocytes. In mice, lack of CFTR alters cardiomyocyte contraction and Ca2+ signaling, and decreases cardiac reserve. We undertook a pilot study evaluating left ventricular (LV) function in CF patients using strain and strain rate echocardiography. METHODS Echocardiography with tissue Doppler and strain and strain rate imaging were performed in 8 CF adults following pulmonary function tests. Results were compared to literature values obtained in healthy subjects. RESULTS All CF individuals had normal LV ejection fractions. In contrast, 50% of men and 100% of women with CF had decreased LV systolic strain. Strain rates were significantly decreased in 100% of CF individuals. RV function was normal and LV function did not correlate with lung function. CONCLUSIONS Strain and strain rate echocardiography identified LV systolic abnormalities in CF individuals not detected by conventional echocardiography. We propose that this echocardiography modality may identify subclinical cardiac dysfunction in CF.

Management of Acute Pancreatitis in the Pediatric Population: A Clinical Report From the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition Pancreas Committee

BACKGROUND While the incidence of acute pancreatitis (AP) in children is increasing, management recommendations rely on adult published guidelines. Pediatric-specific recommendations are needed. METHODS The NASPGHAN Pancreas committee performed a MEDLINE review using several pre-selected key terms relating to management considerations in adult and pediatric AP. The literature was summarized, quality of evidence reviewed, and statements of recommendations developed. The authorship met to discuss the evidence, statements, and voted on recommendations. A consensus of at least 75% was required to approve a recommendation. RESULTS The diagnosis of pediatric AP should follow the published INSPPIRE definitions (by meeting at least two out of three criteria: (1) abdominal pain compatible with AP, (2) serum amylase and/or lipase values ≥3 times upper limits of normal, (3) imaging findings consistent with AP). Adequate fluid resuscitation with crystalloid appears key especially within the first 24 h. Analgesia may include opioid medications when opioid-sparing measures are inadequate. Pulmonary, cardiovascular, and renal status should be closely monitored particularly within the first 48 hours. Enteral nutrition should be started as early as tolerated, whether through oral, gastric, or jejunal route. Little evidence supports the use of prophylactic antibiotics, anti-oxidants, probiotics, and protease inhibitors. Esophago-gastro-duodenoscopy, endoscopic retrograde cholangiopancreatography and endoscopic ultrasonography have limited roles in diagnosis and management. Children should be carefully followed for development of early or late complications as well as recurrent attacks of AP. CONCLUSIONS This clinical report represents the first English-language recommendations for the management of pediatric AP. Future aims should include prospective multi-center pediatric studies to further validate these recommendations and optimize care for children with AP.Background: Although the incidence of acute pancreatitis (AP) in children is increasing, management recommendations rely on adult published guidelines. Pediatric-specific recommendations are needed. Methods: The North American Society for Pediatric Gastroenterology, Hepatology and Nutrition Pancreas committee performed a MEDLINE review using several preselected key terms relating to management considerations in adult and pediatric AP. The literature was summarized, quality of evidence reviewed, and statements of recommendations developed. The authorship met to discuss the evidence, statements, and voted on recommendations. A consensus of at least 75% was required to approve a recommendation. Results: The diagnosis of pediatric AP should follow the published INternational Study Group of Pediatric Pancreatitis: In Search for a CuRE definitions (by meeting at least 2 out of 3 criteria: (1) abdominal pain compatible with AP, (2) serum amylase and/or lipase values ≥3 times upper limits of normal, (3) imaging findings consistent with AP). Adequate fluid resuscitation with crystalloid appears key especially within the first 24 hours. Analgesia may include opioid medications when opioid-sparing measures are inadequate. Pulmonary, cardiovascular, and renal status should be closely monitored particularly within the first 48 hours. Enteral nutrition should be started as early as tolerated, whether through oral, gastric, or jejunal route. Little evidence supports the use of prophylactic antibiotics, antioxidants, probiotics, and protease inhibitors. Esophago-gastro-duodenoscopy, endoscopic retrograde cholangiopancreatography, and endoscopic ultrasonography have limited roles in diagnosis and management. Children should be carefully followed for development of early or late complications and recurrent attacks of AP. Conclusions: This clinical report represents the first English-language recommendations for the management of pediatric AP. Future aims should include prospective multicenter pediatric studies to further validate these recommendations and optimize care for children with AP.