127 research outputs found
Aquaporin 4 is a ubiquitously expressed isoform in the dogfish (Squalus acanthias) shark
No full textChristopher P.Cutler is in receipt of a grant from the National Science Foundation, NSFIOS0844818.The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill > intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.Peer reviewe
Development of microarray techniques for the study of gene expression in the European eel (Anguilla anguilla) during silvering and migration to seawater
No full textElectronic version does not contain associated previously published materialThe European eel, Anguilla anguilla, has a complex life-cycle involving
migrations between the Sargasso Sea and the river systems of Europe and
North Africa. The requirement to move across large salinity gradients
presents a significant physiological challenge and the developmental stages
of the eel are closely linked to these migrations. Microarrays were created to
elucidate gene expression changes occurring during;
i. The transition from juvenile yellow to the adult sexually
maturing, migrating silver eel and;
ii. Salinity adaptation during the migration from freshwater to
seawater.
Groups (n = 6) of freshwater-acclimated yellow or silver eels were
transferred to seawater for between 6 hours and 5 months and
complementary control groups were transferred to freshwater. Brain, kidney,
intestine and gill cDNA libraries were constructed using suppression
subtractive hybridisation (SSH) techniques and a novel protocol based on
Invitrogen's Gateway cloning system. The latter technique produced a low
redundancy (~4 %) EST bank with a wide range of insert sizes (0.5 – 10 kb).
Two microarray types were produced; one comprised 5760 clones from the
two brain libraries whilst the other was a multi-tissue microarray incorporating
6144 clones from the SSH libraries. Pooled RNA samples were probed
against the microarrays to highlight differentially expressed genes. Real-time
quantitative PCR (QPCR) was used to validate the observed expression
changes of selected genes in the tissues of individual fish. Following yellow
to silver transformation of freshwater-adapted eels, the expression of tyrosine
3-mono-oxygenase/tryptophan 5-mono-oxygenase activation protein (14-3-3)
and vaccinia related kinase 3 was shown to be consistently elevated.
Prolactin expression increased in the brains of silver eels following two-day
seawater-acclimation but QPCR analysis revealed high variation amongst
freshwater-adapted eels. This is the first eel microarray study and the
expression profiles highlighted herein will provide new avenues for research
into the sexual development and salinity acclimation of A. anguill
Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs)
No full textMembers of the phosphoinositide-specific phospholipase C (PI-PLC) enzyme family
play a fundamental role in cell signalling pathways by regulating cytosolic calcium
and/or the activity of several protein kinases. This thesis reports the identification,
molecular cloning and characterisation of a potential seventh sub-class of the PI-PLC
enzyme family, the phospholipase C X-domain containing proteins (PLCXDs), which
contain only an X domain in their structure. Comparative sequence analysis has
identified at least three PLCXD isoforms in the human and mouse genomes (PLCXDs 1,
2 and 3), and at least four isoforms in the European eel (PLCXDs 1-4). Key amino acid
residues responsible for the catalytic properties of PI-PLCs were found to be conserved
in human, mouse and eel PLCXDs 1, 2 and 3, but were absent in the sequence of eel
PLCXD4. PLCXD isoforms displayed unique tissue-specific expression profiles and some
similarities between species. Interestingly, in mouse PLCXD1-3 mRNA were found to
be predominantly expressed in the brain, however this is yet to be confirmed in
humans. Analysis of in situ hybridisation data in mice revealed each PLCXD to be
localised in neurons within different brain regions, highly suggestive of unique roles in
brain function. Furthermore, the levels of PLCXD3 protein were reduced by more than
99% in cerebella samples from a mouse model of neurodegeneration (Harlequin
mouse) compared to control mice. Human PLCXD1, 2 and 3 were found to increase
phosphoinositide turnover when overexpressed in the HeLa cell line, and recombinant
PLCXD3, purified to homogeneity from E. coli, was found to interact with various
phosphoinositides including PI(4,5)P₂. ³¹P-NMR analysis of PI(4,5)P₂ and PI before and
after the addition of PLCXD3 purified from HeLa cells and E. coli revealed no difference
in the ³¹P spectra whereas expected chemical shifts were seen following the addition
of purified bacterial PI-PLC. Significant formation of inclusion bodies was noted when
human PLCXDs 1, 2 and 3 were expressed as recombinant proteins in E. coli. Different
strategies aimed at optimising the expression of recombinant PLCXD1, 2 and 3,
including the use of different fusion proteins and screening expression in E. coli,
mammalian and insect cells had limited success, with the best soluble expression only
seen with PLCXD3 in insect cells. Attempts to scale-up the purification of PLCXD3 from
insect cells to provide sufficient protein for enzyme assays and crystal screens were
unsuccessful. The results presented herein suggest that these novel proteins possess
distinct and as yet uncharacterised tissue-specific roles in cell physiology
The effects of extracellular sodium chloride on the activity and expression of Na,K-ATPase in primary cultures of dogfish (Scyliorhinus canicula) rectal gland epithelial cells
No full textDogfish, Scyliorhinus canicula, rectal gland epithelial cells were successfully cultured using two different techniques: 1) a perfusion based technique and 2) a modified Valentich's technique. The morphology of the primary rectal gland epithelial cell cultures was investigated using light, fluorescence and electron microscopy. These studies demonstrated that the cell cultures express most of the structural features of native shark rectal gland cells, including numerous mitochondria, complex tight junctions and extensive membrane folding. The cultured cells using the perfusion technique adopted an extremely flattened morphology when grown on collagen. These cells whether grown in suspension or on collagen, displayed a striking level of vacuole formation, these vacuoles were not associated with transport epithelia. The rectal gland cell cultures were then used to investigate the effect increasing extracellular sodium chloride concentration has on rectal gland cells Na, K-ATPase activity. Increasing sodium chloride concentration in the growth medium by 50% (240 mM to 360 mM) resulted in a transient 3-4 fold increase in Na, K-ATPase activity in cell homogenates approximately 12 hours after the medium change. The response was dependent upon both sodium and chloride ions and was also inhibited by the loop diuretic bumetanide (0.1 mM within 30 minutes), indicating that entry of the ions into the cell is via the Na, K, C1 cotransporter. Incubation of cells in normal medium in the presence of the sodium ionophore monensin also resulted in a dose dependant sustained increase in Na, K-ATPase activity following a 12 hour incubation. The increase in Na, K-ATPase activity associated with increased extracellular sodium chloride concentration was only seen in cells grown on collagen and not in cells grown in suspension. Increases in activity are sensitive to the protein synthesis inhibitor cycloheximide (10 mug/ml), but not the transcriptional inhibitor actinomycin D suggesting that up-regulation of the Na, K-ATPase occurs at the level of translational regulation. Unfortunately this result could not be confirmed using Northern analysis due to unforeseen difficulties in extracting sufficient RNA from the cell cultures. Addition of bumetanide (0.1 mM) to cells grown in normal medium caused a rapid but reversible down-regulation (by 70%) of basal Na, K-ATPase activity within 30 minutes. The anti-microtubular agent colchicine (0.1 mug/ml) inhibited the bumetanide induced down-regulation of Na, K-ATPase and also the recovery of activity following bumetanide removal. The rectal gland cell cultures were used to investigate potential hormonal regulators of the shark rectal gland. The effect of the putative regulators of sodium chloride secretion scyliorhinin II and sCNP on intracellular concentrations of cAMP and cGMP was investigated. The cell cultures were shown be hormonally active as they responded with an increase in intracelllular cAMP concentration to forskolin, PGE1 and PGE2. When scyliorhinin II (10 muM) and IBMX (1 mM) was perfused through the isolated rectal gland a 2 fold increase in cAMP concentration was found in the perfusate after 8 minutes, however no increase was seen in cAMP levels when cell cultures were treated with scyliorhinin II. Shark CNP increased cGMP concentrations in the perfusates of the perfused rectal gland by up to four fold after seven minutes but there was no consistent effect on cGMP concentrations in the cultured cell monolayer. In conclusion it is believed that sCNP and scyliorhinin II mediate their actions on the regulation of sodium chloride secretion by the rectal gland at the vascular level, controlling the extent of perfusion of the gland. This study showed that high salt levels in the medium of shark rectal gland cell monolayers increased the measurable Na, K-ATPase activity and that this response was dependent on protein synthesis but not transcription. It also showed that the response is inhibited by the loop diuretic bumetanide, indicating that entry of the ions into the cell is via the Na, K, Cl cotransporter and that the increase in Na, K-ATPase activity is presumably due to an increase in intracellular sodium concentration. The hormones sCNP and scyliorhinin II appear to mediate their actions on the regulation of sodium chloride secretion by the rectal gland at the vascular level controlling the extent of perfusion of the gland. In conclusion although sodium chloride transport in the dogfish rectal gland requires much more investigation, this study has hopefully proved that dogfish epithelial cell cultures provide a good model for further investigations involving the regulation of activity and expression of the sodium pump
Biochemical actions and degradations of atrial natriuretic peptide in rat tissues
No full textAtrial natriuretic peptide (ANP) has previously been shown to increase intracellular cGMP levels in isolated rat ventricular myocytes. Using purified rat cardiac sarcolemmal membranes, a series of experiments was performed to investigate the mechanisms by which this occurs. A second series of experiments was carried out to investigate the nature of ANP degradation by preparations isolated from rat heart, lung and kidney. In rat cardiac sarcolemmal membranes, ANP produced a 1.8-fold stimulation of manganese-dependent guanylate cyclase activity, with a Km of around 1nM. This activity was attenuated by the presence of 1 nM ATP in the incubation. In the presence of magnesium, guanylate cyclase activity was reduced 20- to 40-fold, but was augmented by ATP. Similar results were obtained in the presence of ANP-PNP, a non-hydrolysable analogue of ATP. [125I]-ANP binding studies indicated the presence of two receptor/affinity states, with KD'S of less than 10 pM, and around 1 nM for the high and low affinity sites respectively. More than 90% of these receptors were of the low affinity form. Similar results were obtained with bovine adrenal cortex membranes, but with MDCK cell membranes, only high affinity binding sites could be detected. These experiments indicate that rat cardiac sarcolemmal membranes possess ANP receptors, at least a proportion of which are coupled to guanylate cyclase. Incubation of [125I]-ANP with isolated rat ventricular myocytes, or with a cytosolic fraction prepared from these cells, resulted in its rapid degradation. The proteolytic activity appeared to be due to the action of a soluble metallopeptidase. Incubation of [125I]-ANP with a cytosolic fraction prepared from rat kidney and lung indicated that similar degradative activity could be isolated from these tissues
Osmoregulation in glass eels and elvers of the European eel, 'Anguilla anguilla'
No full textGlass eels of the European eel migrate from coastal waters inland to freshwater as part of the catadromous lifecycle. The osmotic challenge faced at this time is augmented by their large surface area to volume ratio, and by the fact that the migration may only be completed after several attempts, due to the effects of tide and river flow. Glass eels and elvers developed normally when maintained in waters of differing salinity over a six month period. Drinking rates increased with environmental acclimation salinity (from 0.072 +/- 0.023 mul/g/h to 0.698 +/- 0.099 mul/g/h in FW and SW respectively), and freshwater acclimated fish exhibited a rapid drinking response upon contact with seawater. These accounts of dipsogenic behaviour are similar to those previously reported for adult eels. Results obtained from determinations of branchial Na+K+ATPase activities were more equivocal. Only after nearly five months were activities higher in SW (508.52 +/- 99.76 nmoles/Spairs gills/h) as compared to FW fish (151.65 +/- 8.9 nmoles/5pairs gills/h). Following the transfer of FW acclimated fish to SW there was a trend towards increased Na+K+ATPase activity after seven days post-transfer, which reached a significant peak after two months post-transfer. A transient increase in whole body cortisol content was noted following the transfer of fish from freshwater (388.02 + 90.38 pg/g) to seawater (6268.44 +/- 773.14 pg/g). However, it was not possible to ascertain that this was due to a direct effect of environmental salinity change. There were no clear changes in interrenal cell morphology between salinity groups, although the cells did appear reduced in size with time, regardless of environmental salinity. Total body Na+ content increased with time, and was higher in SW (58.66 +/- 1.66 mumoles/g) as compared to FW reared fish (44.85 +/-1.01 mumoles/g)
Endocrine control of osmoregulation in the euryhaline eel, 'Anguilla anguilla'
No full text1. Groups of eels, Anguilla anguilla, were adapted from freshwater (FW) to seawater, (SW) for periods of 90 - 300 mins. maximum (acute transfer), 0 - 7 days (chronic transfer), or for more than 14 days (longterm seawater transfer). 2. Acute SW transfer led to a decline in blood pressure, an elevation in plasma osmolality and chloride concentration, an immediate "reflex" drinking response and a non-significant increase in plasma angiotensin II (AH) concentration. 3. Administration of papaverine to FW adapted eel caused hypotension, with subsequent recovery of blood pressure, elevation in the drinking rate and plasma All concentration, and a decline in plasma osmolality. Captopril alone had no effect on blood pressure, drinking rate, osmolality or All concentration, but was successful in partially blocking the papaverine-induced blood pressure recovery and increase in AH concentration, with complete inhibition of the drinking. 4. Administration of papaverine to SW adapted eel caused hypotension, with partial recovery of blood pressure, increased drinking rate, plasma All concentration and plasma osmolality. Captopril alone caused a sustained decrease in blood pressure, inhibition of basal SW drinking and a reduction in plasma All concentration, with change in plasma osmolality. Administration of captopril prior to papaverine was successful in partially blocking the papaverine-induced recovery in blood pressure, increase in drinking, plasma All concentration, and plasma osmolality. 5. Chronic SW transfer led to a general decline in blood pressure, increase in plasma electrolyte concentration, elevation in drinking rate after 4-5 days, an increase in plasma All concentration, and a rise in Na+-K+-ATPase, all leading to long term SW values. 6. Plasma arginine vasotocin concentrations were unchanged in long term-FW and SW adapted fish, with a small transitory rise after 4 days in SW. 7. Cortisol plasma concentrations were similar in both long term- FW and SW- adapted fish, with a rise observed 1 day after transfer to SW. 8. Metabolic clearance rates (MCR) and blood production rates (BPR) were significantly elevated in long term SW adapted fish and during chronic SW adaptation, compared to the FW levels. Binding of 125I-AII to gill (filaments and lamellae), brain (cerebellum and medulla oblongata), kidney (head and caudal), and liver was observed in long term FW and SW adapted fish and 6 day SW transfer animals, with significant increase observed in binding in the caudal kidney and cerebellum and medulla oblongata between the FW group and 6 day SW transfer group
A new method for culturing dogfish shark (Scyliorhinus canicula) rectal gland epithelial cells
No full text1. Dogfish, Scyliorhinus canicula, rectal gland epithelial cells were successfully cultured using two different techniques: 1) a perfusion based technique and 2) a modified Valentich's technique (Valentich, 1991). 2. Growth stages of cultures were monitored, showing attachment of tubules at approximately two days with a complete monolayer formed between seven and ten days. Cultures were able to be maintained for up to twenty days. Photos were taken illustrating epithelial cell migration and cell viability. 3. Administration of Ca+2 and Mg+2 free Ringer + 2 mM ethylenediamine tetra-acetic acid (EDTA) + 1% trypsin successfully reduced cultures growing in 96-well plates to single cells after a time course of 20 min to allow for accurate cell counts of approximately 22,000 cells per well. 4. 10-6 M shark C-type natriuretic peptide (sCNP) induced stimulation of cGMP in cultured rectal gland epithelial cells over a time course of 240 sec with maximal stimulation occurring at 180 sec. Limited experiments with scyliorhinin II and rectin showed little effects in stimulating cGMP
The effect of feeding on ion transport in the rectal gland of the European dogfish (Scyliorhinus canicula)
No full textPlasma sodium, chloride and urea concentrations and plasma osmolalities were determined after dogfish were dietary adapted to both acute and chronic feeding regimes. Following single feeding events (20g squid / kg body wt) plasma sodium and chloride concentrations dropped transiently by a maximum of 27% and 18% respectively, two days after feeding. There was however no significant change in the plasma urea concentration or plasma osmolality. Determination of blood vessel calibre by histological examination of rectal glands before, and 12 hours after, a single feeding event suggested an increase in the blood supply to the gland. Repeated feeding events (2.5g pellets / kg body wt) over a period of 1 month were conducted with pelleted diets containing either 1% or 6% w / w NaCl and blood samples were taken weekly for analysis. Plasma sodium concentrations were significantly reduced by approximately 10% after 1 and 3 weeks of feeding and plasma osmolalities fell again by approximately 10% at 1, 2 and 3 weeks when fish were fed on the 1% w / w NaCl pellet diet. However both parameters returned to normal values by the end of the study period. Likewise when fish were fed the high salt (6% w / w NaCl) pellet diet, plasma sodium concentrations and plasma osmolalities again fell by 10-15% after 1, 2 and 3 weeks of repeated feeding. Plasma sodium recovered to normal values within 28 days however the plasma osmolality remained significantly lower than controls. Total RNA was extracted from the rectal gland and homologous and heterologous cDNA probes for the alpha1, beta1 subunits of the Na, K-ATPase and the sCFTR and Na-K-Cl cotransporter were used in Northern and dot blot analyses to identify and quantify the levels of mRNA expression of these major ion transporter proteins during dietary adaptation. Maximal Na, K-ATPase activities in rectal gland homogenates increased transiently by over 40-fold, 9 hours after a single feeding episode (20g squid / kg body wt). There was no concomitant increase in either alpha1 or beta1 subunit of the Na, K-ATPase mRNAs over the same time period however the abundance of both al and (31 Na, K-ATPase subunit mRNAs slowly increased by 75% and 39% respectively two days after feeding and were maintained at that level for either 10 days or 5 days respectively. In chronic dietary adaptations to a squid diet, a 40% increase in beta1 Na, K-ATPase subunit mRNA was found after 4 weeks of repeated feeding however there was no significant increase in al subunit mRNA expression or Na, K-ATPase activity. In contrast chronic dietary adaptation to the 6% w / w NaCl pellet diet resulted in a 3-fold increase in Na, K-ATPase activity however there was no concomitant increase in either the alpha1 or beta1 subunit of the Na, K-ATPase mRNAs. After a single feeding event with the squid diet, mRNAs for both the sCFTR and Na-K-Cl cotransporter proteins were significantly increased, by 55% and 65% respectively, five days after the feeding episode compared to controls
Expression and localization of Aquaporin 1a in the sea-bass (Dicentrarchus labrax) during ontogeny
No full textThe successful establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. In order to understand this process we have studied the adaptation of a euryhaline fish, the sea-bass Dicentrarchus labrax, to various salinities during its ontogeny. The expression and localization of Aquaporin 1a (AQP1a) mRNA and protein were determined in different osmoregulatory tissues. In larvae, the sites of AQP1a expression are variable and they shift according to age, implying functional changes. In juveniles after metamorphosis (D32–D48 post-hatch, 15–25mm) and in pre-adults, an increase in AQP1a transcript abundance was noted in the digestive tract, and the AQP1a location was observed in the intestine. In juveniles (D87–D100 post-hatch, 38–48 mm), the transcript levels of AQP1a in the digestive tract and in the kidney were higher in sea water (SW) than at lower salinity. These observations, in agreement with existing models, suggest that in SW-acclimated fish, the imbibed water is absorbed via AQP1a through the digestive tract, particularly the intestine and the rectum. In addition, AQP1a may play a role in water reabsorption in the kidney. These mechanisms compensate dehydration in SW, and they contribute to the adaptation of juveniles to salinity changes during sea-lagoon migrations. These results contribute to the interpretation of the adaptation of populations to habitats where salinity varies.Peer reviewe
- …
