Andrew P. Morris

Age-Dependent Diarrhea Induced by a Rotaviral Nonstructural Glycoprotein

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114–135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

NSP4 elicits age-dependent diarrhea and Ca2+mediated I− influx into intestinal crypts of CF mice

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4114-135 peptide, can overcome these gastrointestinal complications in CF (CFTRm3Bay null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR+/+), heterozygous (CFTR+/-), or homozygous (CFTR-/-) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca2+ mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I-influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR-/- background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca2+or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca2+-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4114-135 to counteract chronic constipation/obstructive bowel syndrome in CF patients.Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4(114-135) peptide, can overcome these gastrointestinal complications in CF (CFTR(m3Bay) null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR(+/+)), heterozygous (CFTR(+/-)), or homozygous (CFTR(-/-)) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca(2+) mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I(-) influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR(-/-) background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca(2+) or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca(2+)-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4(114-135) to counteract chronic constipation/obstructive bowel syndrome in CF patients.

Natriuretic Peptides and Nitric Oxide Stimulate cGMP Synthesis in Different Cellular Compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05–10 nM), whereas SNAP (0.01–100 μM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 μM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 μM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 μM SNAP; whereas 100 μM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Junctional adhesion molecules (JAMs) are differentially expressed in fibroblasts and co-localize with ZO-1 to adherens-like junctions

Junctional Adhesion Molecules (JAMs) are components and regulators of the well-characterized epithelial and endothelial tight junction. Since the molecular components of native fibroblast adherens-like junctions remain poorly described we determined JAM expression profiles in fibroblasts. We found JAM-C on human dermal, lung, and corneal primary fibroblast cultures. Within murine lines, JAM-A was found in L-cells, JAM-C in 3T3 L1 cells, and both JAM-A and JAM-C were co-expressed in NIH 3T3 fibroblasts. In primary dermal fibroblasts, JAM-C concentrated at zipper-like junctions that formed between apposing cells. Dual immunostaining showed JAM-C co-localization with the ZO-1 intracellular scaffolding molecule at cell contacts that ranged from 7 μm to over 25 μm in length. JAM-C also labeled similar zipper-like junctions detected with N-Cadherin and Cadherin-11 antibodies. We conclude that endogenous JAM-C is an integral component of the dermal fibroblast adherens-like junction, and our data extend the expression and potential function of JAMs into mesenchymal tissues.

Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

SEVERE PHENOTYPE IN MICE WITH TERMINATION MUTATION IN EXON 2 OF CYSTIC FIBROSIS GENE

Mice with a termination codon mutation in exon 2 of the cystic fibrosis (CF) gene were generated using homologous recombination in embryonic stem cells. Animals homozygous for the mutant allele display a severe intestinal phenotype similar to that previously reported for CF mutant mice. The null nature of this allele was demonstrated by the absence of detectable wild-type mRNA, by the absence of detectable CFTR in the serous gland collecting ducts of salivary tissues, and by the lack of cAMP-mediated short-circuit current responses in colonic epithelium of mutant animals.

AF17 Competes with AF9 for Binding to Dot1a to Up-regulate Transcription of Epithelial Na+ Channel α

We previously reported that Dot1a·AF9 complex represses transcription of the epithelial Na+ channel subunit α (α-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na+ currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an α-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na+ currents. AF17 overexpression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a·AF9-mediated repression of α-ENaC and other target genes.

The Regulation of Epithelial Cell cAMP- and Calcium-Dependent Chloride Channels

This chapter has focused on two types of chloride conductance found in epithelial cells. The leap from the Ussing chamber to patch-clamp studies has identified yet other conductances present which have also been electrophysiologically characterized. In the case of the swelling activated wholecell chloride current, a physiological function is apparent and a single-channel basis found, but its genetic identity remains unknown (see reviews by Frizzell and Morris, 1994; and Strange et al., 1996). The outwardly rectified chloride channel has been the subject of considerable electrophysiological interest over the past 10 years and is well characterized at the single-channel level, but its physiological function remains controversial (reviewed by Frizzell and Morris, 1994; Devidas and Guggino, 1997). Yet other conductances related to the CLC gene family also appear to be present in epithelial cells of the kidney (reviewed by Jentsch, 1996; Jentsch and Gunter, 1997) where physiological functions for some isoforms are emerging. Clearly, there remain many unknowns. Chief among these is the molecular basis of GCa2+Cl and many of other the conductances. As sequences become available it is expected that the wealth of information gained by investigation into CFTR function will provide a conceptual blueprint for similar studies in these later channel clones.

Chapter 8 Chloride Conductances of Salt-Secreting Epithelial Cells

Publisher Summary There has been much interest in apical membrane receptors for regulatory substances released from immune cells. Chloride conductance pathways at the apical membranes of epithelial cells play a well-defined role in determining the rate of salt secretion across the epithelia. Salt secretion is regulated on the seconds–minutes time scale by a variety of endocrine substances, neurotransmitters, and immune cell products, whose receptors lie predominantly at the plasma-facing, basolateral membranes. Secretion can be activated also by bacterial enterotoxins, acting from the lumen, for example, those inducing intestinal secretion and diarrhea. It is evident that Cl conductance in secretory cells has been identified at a variety of levels of resolution. The role of secondary-active Cl transport in salt secretion has been identified, from transepithelial measurements, and the location of Cl conductances at the apical membranes is resolved, using microelectrode recordings. The introduction of single-channel recording techniques generated candidates for the apical membrane secretory Cl channel. However, the leap from the Ussing chamber to the patch pipette left in its wake considerable uncertainty, as to the single-channel basis of the regulated Cl conductance pathways of secretory epithelial cells. Ultimately, whole-cell recording techniques bridged this gap and identified three regulated Cl current components, two of which are controlled by secretory agonists, the other by cell volume. The primary signal transduction events that control the activities of the secretory Cl conductance have emerged, but the understanding of their detailed modulation is incomplete.