Biomedical subjects

John N Forrest

Publications and source records attributed to John N Forrest.

6 recordsLinked to original sources

PubMed2006

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels.

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.

Amino Acid Sequence↗

PubMed2006

The comparative toxicogenomics database: a cross-species resource for building chemical-gene interaction networks.

Chemicals in the environment play a critical role in the etiology of many human diseases. Despite their prevalence, the molecular mechanisms of action and the effects of chemicals on susceptibility to disease are not well understood. To promote understanding of these mechanisms, the Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org/) presents scientifically reviewed and curated information on chemicals, relevant genes and proteins, and their interactions in vertebrates and invertebrates. CTD integrates sequence, reference, species, microarray, and general toxicology information to provide a unique centralized resource for toxicogenomic research. The database also provides visualization capabilities that enable cross-species comparisons of gene and protein sequences. These comparisons will facilitate understanding of structure-function correlations and the genetic basis of susceptibility. Manual curation and integration of cross-species chemical-gene and chemical-protein interactions from the literature are now underway. These data will provide information for building complex interaction networks. New CTD features include (1) cross-species gene, rather than sequence, query and visualization capabilities; (2) integrated cross-links to microarray data from chemicals, genes, and sequences in CTD; (3) a reference set related to chemical-gene and protein interactions identified by an information retrieval system; and (4) a "Chemicals in the News" initiative that provides links from CTD chemicals to environmental health articles from the popular press. Here we describe these new features and our novel cross-species curation of chemical-gene and chemical-protein interactions.

Animals↗

PubMed2006

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.

1-Methyl-3-isobutylxanthine↗

PubMed2005

Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102.

The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perfusing with 10 microM forskolin (FOR) and 1 mM IBMX, and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl(-) conductance was inhibited 69% by 1 microM mercuric chloride and 78% by 5 microM mercuric chloride (IC(50) of 0.8 microM). Despite comparable stimulation of conductance, hCFTR was insensitive to 1 microM HgCl(2) and maximum inhibition was 15% at the highest concentration used (5 microM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50-200 microM) also reversibly inhibited sCFTR (40-75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site-specific mutations introducing the first but not the second shark unique cysteine in hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark vs. human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue.

4-Chloromercuribenzenesulfonate↗

PubMed2003

The Comparative Toxicogenomics Database (CTD).

The Mount Desert Island Biological Laboratory in Salsbury Cove, Maine, USA, is developing the Comparative Toxicogenomics Database (CTD), a community-supported genomic resource devoted to genes and proteins of human toxicologic significance. CTD will be the first publicly available database to a) provide annotated associations among genes, proteins, references, and toxic agents, with a focus on annotating data from aquatic and mammalian organisms; b) include nucleotide and protein sequences from diverse species; c) offer a range of analysis tools for customized comparative studies; and d) provide information to investigators on available molecular reagents. This combination of features will facilitate cross-species comparisons of toxicologically significant genes and proteins. These comparisons will promote understanding of molecular evolution, the significance of conserved sequences, the genetic basis of variable sensitivity to environmental agents, and the complex interactions between the environment and human health. CTD is currently under development, and the planned scope and functions of the database are described herein. The intent of this report is to invite community participation in the development of CTD to ensure that it will be a valuable resource for environmental health, molecular biology, and toxicology research.

Animals↗

PubMed2002

Intracellular accumulation of mercury enhances P450 CYP1A1 expression and Cl- currents in cultured shark rectal gland cells.

The effects of acute and subchronic exposure to mercury on the Cl- current (ICl) were investigated in cultured shark rectal gland (SRG) cells. The effects of intracellular accumulation of mercury on cytochrome P450 (P450) were also assessed. Bath perfusion of a cocktail solution containing forskolin, 1-isobutyl-3-methylxanthine, and 8-bromoadenosine monophosphate enhanced ICl. Addition of 10 microM HgCl2 significantly inhibited the cAMP-activated ICl (p < 0.05, n = 11). Intracellular dialysis with ATP gamma S did not prevent the inhibitory effect of mercury on ICl. In contrast, incubation of SRG cells with 10 microM HgCl2 for 48 hrs markedly increased ICl (p < 0.01, n = 12). Dephosphorylation of the channel by intracellular dialysis with phosphatase I and II abolished the mercury-incubated increase in ICl. The P450-mediated metabolite of arachidonic acid, 11,12-epoxyeicosatrienoic acid (11,12-EET), significantly increased ICl. However, application of 11,12-dihydroxyeicosatrienoic acid (11,12-DHT) did not alter ICl. Mercury incubation for 48 hrs did not alter the protein expression of Cl- channels, but caused an induction of CYP1A1 in cultured SRG cells. In addition, co-incubation of SRG cells with mercury and the P450 inhibitor clotrimazole prevented the mercury-incubated increase in ICl. Our results demonstrate that acute and subchronic application of mercury has opposing effects on ICl in cultured SRG cells. The acute effect of mercury on ICl may result from mercury blockade of Cl- channels. The subchronic effect of mercury on ICl may be due to an induction of P450 CYP1A1 and its mediated metabolites, but not due to an over-expression of Cl- channels.

Adenosine Triphosphate↗