Gordon Cramb

Osmoregulation is key to the survival of all organisms, from unicellular bacteria and archae to multicellular plants and animals, including mammals and man. The physiological problems faced by any organism depend on the composition of the immediate external environment and the intracellular ion and solute composition required for normal cell viability, general health, growth and successful reproduction.  All organisms, whether unicellular or multicellular, have to sustain an intracellular ion and solute composition distinct from that of the extracellular fluid, whilst sustaining water homeostasis.  In multicellular organisms, the maintenance of this dis-equilibrium of solutes across biological membranes is mediated by the activity of a variety of ion, water and solute transporters, which are often selectively expressed and located within different membrane compartments of polarized epithelial cells.  The expression, location and function of these membrane transport proteins can often be regulated, both acutely and chronically, by a myriad of extracellular signaling molecules including hormones and neurotransmitters.  Studies in my laboratory have mainly focused on investigations of the functions of many transporters, channels and enzymes expressed within the epithelial tissues of euryhaline fish, which have been selected as models for the study of the control of ion and water transport across biological membranes.  These investigations have highlighted the functional roles of many genes in many tissues and their role in successful osmoregulation when euryhaline fish move between environments of different salinity (ie freshwater to seawater).  Studies have also highlighted a role for members of the gut peptide family, called guanylins, and their actions, both locally in the GI tract as well as in the kidney, have been investigated.  The peptides mediate their response by binding and activating an integrate membrane guanylate cyclase (type C) with concomitant increases in intracellular cyclic GMP.   An additional, and novel protein involved in as yet unknown signaling pathways within epithelial cells in the eel gut, and which has a role in protein expression, has also been identified in mammalian cells and tissues.  These signaling molecules, which are members of the phospholipase C super-family, are currently being investigated at the molecular levels in cells and tissues from both teleost and mammalian origins.

Research interests are centred in three areas and these are summarised below,

1)   The molecular mechanisms of osmoregulation in aquatic vertebrates.   We are currently using molecular biological, biochemical and physiological approaches to investigate ion, water and solute homeostasis in euryhaline teleost fish such as the European Eel (Anguilla anguilla), the Atlantic salmon (Salmo salar) as well as the stenohaline marine and freshwater fishes.

A number of genes implicated in electrolyte, water and osmolyte balance, and therefore osmoregulation in teleost fish have been cloned and characterised.   Studies have concentrated mainly on euryhaline species such as the European eel (Anguilla anguilla), the Atlantic salmon (Salmo salar) and the European flounder (Pleuronectes flesus), which are all able to successfully adapt to environments of differing salinity.   This osmoregulatory flexibility is a direct consequence of the extensive plasticity in the expression of these genes that are often found in specialised epithelial cells within the gill, gut and kidney and which regulate ion and water transport.   Many other, and as yet previously unknown, osmoregulatory genes have been identified using an 18K feature eel microarray to screen for differential expression during both salinity-adaptation or sexual maturation.  So far in these studies we have identified and sequenced over 600 cDNA fragments of genes, which exhibited differential expression.   Many of these genes are known to be homologues of key mammalian solute transporters, structural proteins, membrane receptors, intracellular enzymes and transcription factors, however a number of others selected are still currently unknown.   The microarray studies also highlighted genes involved in the metabolism of the organic osmolyte inositol, implicating this cyclic alcohol as a potential osmoregulatory factor in seawater (SW)-acclimated fish.  Inositol is synthesized, transported and accumulated in epithelial tissues either directly (gill, skin and fins) or indirectly (GI tract) exposed to SW. Changes in the expression of these genes are associated with the acclimation of fish between fresh-water (0-5 mOsm/kg), sea-water (800-1020 mOsmol/kg) and in the case of eels even double-concentrated sea-water (>2000 mOsmol/kg) environments.   Using a variety of methodologies, cDNAs for over 100 different transporters, channels and enzymes involved in the synthesis, processing and distribution of a variety of ions, water, toxins and metabolites have now been cloned and sequenced from various species.  These include isoforms of both a- and b-subunits of the Na,K-ATPase, the secretory and absorptive forms of the Na,K,2Cl-cotransporter, the CFTR chloride channel, aquaporin water channels, a range of other co-transporters, exchangers, ATPases and peptide transporters.   In addition a number of genes involved in the production, distribution and accumulation of the organic osmolyte, inositol, have been show to exhibit differential expression during SW-acclimation.  These include myo-inositol phosphate (MIP) synthase, inositol monophosphatase (IMPA), as well as a number of isoforms of the sodium-dependent (SMIT) and proton-dependent (HMIT) inositol transporters.  As a result of toxicological studies with the European flounder (Pleuronectes flesus) we have also isolated complete cDNAs for two isoforms of the multi-drug resistance p-glycoprotein (MDR-A and MDR-B) and 5 partial cDNAs for the related family of multi-drug resistance proteins (MRPs 1- 5).  The roles of these proteins and resistance of fish to various environmental stressors including a wide range of natural and anthropogenic xenobiotics has also been investigated.

Collaborative ventures associated with this work include scientists working at the Universities of Exeter, (Professor J. A. Brown: expression of MDR/MRPs in flounder), Manchester (Professor R. Balment; microarrays for the identification of differentially expressed genes in flounder and eel), Liverpool (Professor A. Cossins, microarray manufacture), Aarhus (Drs F. Cornelius and Y. Mahmmoud; the role of FXYD proteins in the regulation of Na,K-ATPase), Odense (Dr S. Madsen; cloning, expression and function of ion transporters in salmonid fish), Laval at Quebec and Leuven, Belgium (Professors L Bernatchez and G. Maes; characterization of differential gene expression in eel leptocephali in the Sargasso sea), Montpellier (Dr G Charmantier; cloning and function of aquaporins in the sea bass), IFREMER Research Institute, Brest (Professor G. Boeuf and Dr C. Ferec; CFTR genes in teleost fish), Queensland (Dr Craig Franklin; osmoregulation in the euryhaline bullshark), Ottawa (Professor S.F. Perry; Ca-ATPases and V-type ATPases in salmonids), Georgia (Dr Chris Cutler; ion and water transporters in the eel).

2)    The molecular mechanisms and physiological actions of the guanylin/guanylate cyclase signaling system.

Guanylin, uroguanylin, and in teleost fish renoguanylin, are members of a family of gut peptides which act to promote fluid secretion within the intestine and the kidney. The gut peptides mediate their actions by binding and activating a specific isoform (GC-C) of the membrane guanylate cyclase family.   Other guanylate cyclase isoforms (GC-A and GC-B) are the endogenous receptors for the related family of natriuretic peptides (ANP, BNP and CNP), which we have also investigated in both mammalian and fish species.

Full-length cDNAs for guanylin, uroguanylin and renoguanylin and two isoforms of their receptor, GC-C, have been cloned and sequenced from various teleost, elasmobranch and amphibian species.   Our most recent work has concentrated on the characterisation of these peptides and their mechanisms of action in the euryhaline eel.  These genes have been found to exhibit differential tissue expression and seawater acclimation specifically up-regulates mRNA expression for uroguanylin in the intestine and one GC-C receptor isoform in the intestine and to a lesser extent the kidney.   We are currently using bacterial expression systems for the preparation of the peptide prohormones to investigate the processing of these precursor proteins into the active peptides and to also produce workable amounts of active peptides for functional studies on secretion in both intestine and kidney.    One of the GC-C isoforms is currently being expressed in various animal and insect cell lines for structural and functional studies.  Antibodies to these peptides/receptor proteins have also been prepared to examine tissue/cell expression and distribution at the protein level within the eel.   These antibodies and nucleic acid probes are being used together with various pharmacological, biochemical and physiological techniques to characterise the expression, regulation and cross-talk between the guanylate cyclase receptors and other tissue signaling systems to elucidate the secretory functions of these peptides. 

 Collaborative ventures associated with this work include scientists working at the Universities of Missouri (Professor Leonard Forte; guanylin peptides in fish) and Cleveland (Professor Focco van den Akker; the crystal structure of eel guanylate cyclases). 

3). Structural and functional characterization of phospholipase C X-domain (PLCXD) containing genes in teleost fish and mammals.

Microarray studies highlighted a 90% reduction in the expression of a PLCXD gene, which was highly expressed in the intestine of sexually immature freshwater “yellow” eels, when the fish metamorphosed into sexually maturing “silver” eels.  Preliminary work indicates that loss of expression is associated with general atrophy of the gut in non-feeding silver eels as they migrate back to the Sargasso sea to breed.  The precise functional role of this gene in the eel is under current investigation.  

Recent comparative studies in both mouse and humans have revealed that three different isoforms of PLCXD (XDs 1-3) exhibit differential expression in variety of tissues.  In addition at least one of these isoforms exhibits a number of C-terminal spliceoforms in mammals.  The physiological functions of these isoforms/spliceoforms are currently unknown but a number of related investigations have been initiated using a number of animal cell lines as well as the use of zebrafish to study tissue expression during development and the effects of gene knock-out.  

Collaborative ventures associated with this work include scientists working at the Universities of Aarhus (Dr P. Morth; bacterial expression, structure and enzymatic activities) and Newcastle (Dr J Sayer: expression and development in zebrafish).

RNA/DNA isolation, RT-PCR, gene cloning and expression, microarray techniques, northern blotting, western blotting, immunohistochemistry, primary cell preparation and cell and tissue culture, HPLC, RIA, RRA, cell fractionation, protein purification, 2D-PAGE.

pharmaceutical industry

aquaculture industry

Epithelia; ion transport; water transport; solute transport; osmoregulation; hormonal regulation; transcriptomics; quantitative proteomics