A nonolfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features.

Adenosine receptors (ADORs) are G protein-coupled purinoceptors that have several functions including regulation of chloride secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic and expression analyses indicate that elasmobranch ADORs are nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We coexpressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N6-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A0 core that revealed unique structural features of ancestral receptors. We conclude that 1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A0 likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A0 appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.

AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

Gender Disparities in Medical Student Research Awards: A 13-Year Study From the Yale School of Medicine.

PURPOSE: The Liaison Committee on Medical Education mandates instruction in research conduct, and many U.S. medical schools require students to complete a research project. All Yale School of Medicine (YSM) graduating students submit a research thesis, and ~5% are awarded highest honors. Gender disparities exist in areas related to physician research productivity, including academic rank, research funding, and publications. The authors asked whether gender disparities exist for medical student research. METHOD: The authors conducted a retrospective review of 1,120 theses submitted by graduating medical students from 2003 to 2015 at YSM and collected data on gender, mentoring, research type, sponsoring department, and other characteristics. Multivariate logistic regression modeling examined gender differences in medical student research awards. RESULTS: Women authored 50.9% of theses, but earned only 30.9% of highest honors awards (OR 0.41; 95% CI: 0.23, 0.74). Among factors associated with increased receipt of highest honors that differed by gender, men were more likely than women to work with a mentor with a history of three or more thesis honorees, take a fifth year of study, secure competitive research funding, undertake an MD-master of health science degree, and conduct laboratory research (all P < .001). After adjustment for these factors, and for underrepresented in medicine status and sponsoring department, women remained less likely to receive highest honors (OR 0.51; 95% CI: 0.27, 0.98). CONCLUSIONS: Women YSM students were less likely to receive highest honors for medical research. Gender disparities in postgraduate biomedical research success may start during undergraduate medical education.

Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis.

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

THE SHARK RECTAL GLAND MODEL: A CHAMPION OF RECEPTOR MEDIATED CHLORIDE SECRETION THROUGH CFTR.

The dogfish shark salt gland was predicted by Smith and discovered by Burger at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine. It is an epithelial organ in the intestine composed of tubules that serve a single function: the secretion of hypertonic NaCl. Many G protein receptors are present on the basolateral surface of these tubules, including stimulatory receptors for vasoactive intestinal peptide, adenosine A2, growth hormone releasing hormone, and inhibitory receptors for somatostatin and adenosine A1. An entirely different class of stimulatory receptors is present as C-type natriuretic peptide receptors. Each stimulatory receptor evokes powerful NaCl secretion. G protein receptors bind to Gαs to activate the catalytic unit of adenylate cyclase to form cyclic adenosine monophosphate (cAMP) and protein kinase A that phosphorylates the regulatory domain of cystic fibrosis transmembrane conductance regulator, opening the channel. The C-type natriuretic peptide receptor stimulates by activating guanylate cyclase and endogenous cyclic guanosine monophosphate which inhibits type 3 phosphodiesterase, the enzyme that breaks down cAMP, thereby elevating cAMP and activating the protein kinase A pathway.

Value of Organoids from Comparative Epithelia Models.

Organoids have tremendous therapeutic potential. They were recently defined as a collection of organ-specific cell types, which self-organize through cell-sorting, develop from stem cells, and perform an organ specific function. The ability to study organoid development and growth in culture and manipulate their genetic makeup makes them particularly suitable for studying development, disease, and drug efficacy. Organoids show great promise in personalized medicine. From a single patient biopsy, investigators can make hundreds of organoids with the genetic landscape of the patient of origin. This genetic similarity makes organoids an ideal system in which to test drug efficacy. While many investigators assume human organoids are the ultimate model system, we believe that the generation of epithelial organoids of comparative model organisms has great potential. Many key transport discoveries were made using marine organisms. In this paper, we describe how deriving organoids from the spiny dogfish shark, zebrafish, and killifish can contribute to the fields of comparative biology and disease modeling with future prospects for personalized medicine.

ANP and CNP activate CFTR expressed in Xenopus laevis oocytes by direct activation of PKA.

CONTEXT: Acting through different receptors, natriuretic peptides (atrial natriuretic peptide [ANP], brain type natriuretic peptide [BNP] and C-type natriuretic peptide [CNP]) increase intracellular cGMP, which then stimulates different pathways that activate fluid secretion. OBJECTIVE: We used two-electrode voltage clamping to define the dominant pathway that is employed when natriuretic peptides activate cystic fibrosis transmembrane conductance regulator (CFTR) in the Xenopus oocyte expression system. Natriuretic peptides could activate CFTR by 1) cGMP cross-activation of protein kinase A (PKA), 2) cGMP activation of cGMP-dependent protein kinase II, 3) cGMP inhibition of phosphodiesterase type III (PDE3), or 4) direct activation of CFTR. MATERIALS AND METHODS: cRNA-microinjected Xenopus laevis oocytes were perfused with diverse compounds that examined these pathways of natriuretic peptide signaling. RESULTS AND DISCUSSION: ANP stimulated the shark CFTR (sCFTR)-mediated chloride conductance and this activation was inhibited by H-89, a specific inhibitor of PKA. After co-expression of the CNP receptor (NPR-B), sCFTR became stimulatable by CNP and was similarly inhibited by H-89, pointing to cross-activation of PKA. 8-pCPT-cGMP, a relatively cGKII-selective cGMP, failed to stimulate sCFTR. Another membrane-permeable and non-hydrolyzable analog of cGMP, 8-Br-cGMP, stimulated CFTR only at millimolar concentrations, consistent with cross-activation of PKA. The PDE inhibitors EHNA, rolipram, cilostamide, and amrinone did not significantly increase chloride conductance, arguing against a significant role for PDE2, PDE3 and PDE4 signaling in the oocyte. Sildenafil, a PDE5 inhibitor, caused a partial activation of sCFTR channels and this effect was again inhibited by H-89. CONCLUSION: From these experiments we conclude that in the Xenopus oocyte system, natriuretic peptides, 8-Br-cGMP, and PDE5 inhibitors activate CFTR by cross-activation of PKA.

Gastric inhibitory peptide, serotonin, and glucagon are unexpected chloride secretagogues in the rectal gland of the skate (Leucoraja erinacea).

Since the discovery of the rectal gland of the dogfish shark 50 years ago, experiments with this tissue have greatly aided our understanding of secondary active chloride secretion and the secretagogues responsible for this function. In contrast, very little is known about the rectal gland of skates. In the present experiments, we performed the first studies in the perfused rectal gland of the little skate (Leucoraja erinacea), an organ weighing less than one-tenth of the shark rectal gland. Our results indicate that the skate gland can be studied by modified perfusion techniques and in primary culture monolayers, and that secretion is blocked by the inhibitors of membrane proteins required for secondary active chloride secretion. Our major finding is that three G protein-coupled receptor agonists, the incretin gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide, as well as glucagon and serotonin, are unexpected potent chloride secretagogues in the skate but not the shark. Glucagon stimulated chloride secretion to a mean value of 1,661 ± 587 µeq·h(-1)·g(-1) and serotonin stimulated to 2,893 ± 699 µeq·h(-1)·g(-1). GIP stimulated chloride secretion to 3,733 ± 679 µeq·h(-1)·g(-1) and significantly increased tissue cAMP content compared with basal conditions. This is the first report of GIP functioning as a chloride secretagogue in any species or tissue.

cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland.

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101.

Comparison of diverse orthologs is a powerful tool to study the structure and function of channel proteins. We investigated the response of human, killifish, pig, and shark cystic fibrosis transmembrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTR(inh)-172, glibenclamide, and GlyH-101. In three systems, including organ perfusion of the shark rectal gland, primary cultures of shark rectal gland tubules, and expression studies of each ortholog in cRNA microinjected Xenopus laevis oocytes, we observed fundamental differences in the sensitivity to inhibition by these channel blockers. In organ perfusion studies, shark CFTR was insensitive to inhibition by CFTR(inh)-172. This insensitivity was also seen in short-circuit current experiments with cultured rectal gland tubular epithelial cells (maximum inhibition 4 ± 1.3%). In oocyte expression studies, shark CFTR was again insensitive to CFTR(inh)-172 (maximum inhibition 10.3 ± 2.5% at 25 µM), pig CFTR was insensitive to glibenclamide (maximum inhibition 18.4 ± 4.4% at 250 µM), and all orthologs were sensitive to GlyH-101. The amino acid residues considered responsible by previous site-directed mutagenesis for binding of the three inhibitors are conserved in the four CFTR isoforms studied. These experiments demonstrate a profound difference in the sensitivity of different orthologs of CFTR proteins to inhibition by CFTR blockers that cannot be explained by mutagenesis of single amino acids. We believe that the potency of the inhibitors CFTR(inh)-172, glibenclamide, and GlyH-101 on the CFTR chloride channel protein is likely dictated by the local environment and the three-dimensional structure of additional residues that form the vestibules, the chloride pore, and regulatory regions of the channel.

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.

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.

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.

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.

Marine organism cell biology and regulatory sequence discoveryin comparative functional genomics.

The use of bioinformatics to integrate phenotypic and genomic data from mammalian models is well established as a means of understanding human biology and disease. Beyond direct biomedical applications of these approaches in predicting structure-function relationships between coding sequences and protein activities, comparative studies also promote understanding of molecular evolution and the relationship between genomic sequence and morphological and physiological specialization. Recently recognized is the potential of comparative studies to identify functionally significant regulatory regions and to generate experimentally testable hypotheses that contribute to understanding mechanisms that regulate gene expression, including transcriptional activity, alternative splicing and transcript stability. Functional tests of hypotheses generated by computational approaches require experimentally tractable in vitro systems, including cell cultures. Comparative sequence analysis strategies that use genomic sequences from a variety of evolutionarily diverse organisms are critical for identifying conserved regulatory motifs in the 5'-upstream, 3'-downstream and introns of genes. Genomic sequences and gene orthologues in the first aquatic vertebrate and protovertebrate organisms to be fully sequenced (Fugu rubripes, Ciona intestinalis, Tetraodon nigroviridis, Danio rerio) as well as in the elasmobranchs, spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea), and marine invertebrate models such as the sea urchin (Strongylocentrotus purpuratus) are valuable in the prediction of putative genomic regulatory regions. Cell cultures have been derived for these and other model species. Data and tools resulting from these kinds of studies will contribute to understanding transcriptional regulation of biomedically important genes and provide new avenues for medical therapeutics and disease prevention.

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.

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.