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Implication of mineralocorticoid receptor in rainbow trout osmoregulation
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Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S87–S96
In fish stimulation of the endogenous RAS by changes in external salinity, elevation of blood plasma ionic concentration, dehydration, blood volume depletion or lowered blood pressure act as major stimuli for the drinking. In addition, the exogenous administration of the main active components of the RAS e.g. Ang I and Ang II evoke drinking in fish. Although the role of Ang II in drinking regulation in fish is well established, little is known as to how this peptide may contribute to the absorption of the water taken up by drinking into the gut. In vitro intestinal water absorption was analysed in freshwater adapted trout in response to [Asn1, Val5]-Angiotensin II (Ang II) in isolated, everted perfused gut segments. Increasing doses of Ang II (10−13 to 10−8 mol/l) when applied to the serosal side significantly increased water absorption in a linear dosedependent manner with a threshold response of 10−12 M of Ang II. Based on the response to the receptor blockers [Sar1, Val5, Ala8]-Angiotensin II (Saralasin) and Losartan (DUP 753) our results suggest a potential role for Ang II in the regulation of water absorption in the intestine of freshwater fish possibly involving novel AT receptors. The author is funded by FCT, Ministry of Science, Portugal doi:10.1016/j.cbpa.2007.01.121
A3.4 Mouth or mitochondrion — Which the better to sip the water of life? J. Noble-Nesbitt, (University of East Anglia, United Kingdom) Many insects are able to thrive in extreme environments where few other eukaryotes are able to survive. Environmental water stress exemplifies this. Faced with high temperature and low humidity, some well adapted insects survive where other animal (and plant) species succumb. Contributing to their survival is their impervious cuticle which limits water loss and conserves existing body water. The greatest survivor species, however, are able to imbibe water, not just by drinking liquid water when available, but also when necessary directly from the vapour phase of the air down to 43% RH and at ambient temperatures of 37 °C. This emancipation from drinking in its conventional sense accounts for their remarkable survival under conditions of extreme water stress. In the greatest known exponent of this active water vapour absorption (WVA), the firebrat Thermobia domestica, its water vapour absorptive epithelium is comprised of a single layer of large cells with the highest known density of elongated mitochondria hexagonally packed very tightly alongside deep pleated infolds of an enormously hypertrophied portasome-studded apical plasma membrane (apm) in a honeycomb-like array. This absorptive apm-mitochondrial complex substitutes for drinking by the mouth when liquid water is unavailable. Current research is unravelling how this remarkable cellular system develops and matures post-embryonically in the early larval instars and continues functioning
throughout the life of the insect. Rapid mitochondrial biogenesis and apm sequestration (as portasome-studded rafts) and reformation during cell proliferation are integral to this process. doi:10.1016/j.cbpa.2007.01.122
A3.5 Molecular and cellular aspects of ion and water transport in the osmoregulatory epithelia of teleost and elasmobranch fish G. Cramb, (University of St. Andrews, United Kingdom) The physiological resilience of euryhaline teleost and elasmobranch fish to adapt to changes in external salinity has allowed these animals to successfully inhabit both marine and freshwater environments, usually at different stages of their life cycles. Salinity adaptation is the result of both the genetic and functional plasticity of key ion and water transporting proteins expressed within the major osmoregulatory tissues, the gill, gut, kidney and rectal gland. Many of these ion and water transporting proteins exhibit salinity-dependent and hormonally-regulated changes in activity and expression within these tissues that enables body fluid homeostasis irrespective of salinity changes in the external environment. The expression, asymmetrical distribution and concerted actions of these transporters within the polarised cells of both absorptive and secretory epithelia are responsible for net uptake or loss of ions and water. This presentation will summarise our current understanding of the nature of ion and water transport across absorptive and secretory epithelia and the structure, location and function of some of the transporters required to enable successful osmoregulation in both teleost and elasmobranch fish. doi:10.1016/j.cbpa.2007.01.123
A3.6 Implication of mineralocorticoid receptor in rainbow trout osmoregulation S. Milla, P. Prunet, (INRA, France) In fish, aldosterone is absent and the classical view was to suggest that cortisol has both mineralocorticoid and glucocorticoid actions. Recent studies in rainbow trout have shown that mineralocorticoid and glucocorticoid receptors (respectively rtMR, rtGR1 and rtGR2) are expressed ubiquitously. Moreover, whereas cortisol is the physiological ligand of rtGRs, 11deoxycorticosterone (DOC) was demonstrated to be the most active steroid to stimulate the rtMR transactivation activity in vitro. To clarify the role of MR in trout osmoregulation, 3 different approaches have been developed.
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S87–S96
We first checked rtMR localization in the main osmoregulatory organs: gill, kidney and intestine. In addition, after an osmotic challenge (direct SW–FW and FW–SW transfers), we measured significant variations of rtMR transcripts in these organs. However, no variations of plasma DOC and cortisol levels were observed and, for both hormones, plasma level fluctuates within the same range of concentration (5– 15 ng/ml). A second strategy aims to analyze in vitro the effect of DOC on Na+K+-ATPase activity using the incubated gill test developed by McCormick and Bern (1989). We confirmed a stimulatory effect of cortisol on Na+ K+ -ATPase activity whereas DOC had an inhibitory effect. The effects of cortisol and DOC on other transport protein gene expression will also be presented. Finally, we have developed a transcriptome approach using high density array spotted with 9000 annotated trout EST. This study allowed us to characterize a list of genes regulated by MR. All these data will be discussed in the context of a probable role of MR in freshwater osmoregulation. doi:10.1016/j.cbpa.2007.01.124
A3.7 Aquaporin channels in teleost and elasmobranch fish C. Cutler, (Georgia Southern University, United States) Information concerning the role of aquaporin water and small solute channels in fish has until recently been sparse. Sequence data has become available regarding aquaporin isoforms in various teleost fish, with around 17 isoforms present in each of Pufferfish (Fugu rubripes) and Zebrafish (Danio rerio), and with at least 11 isoforms also expressed in the European eel (Anguilla anguilla). Research into the role of these teleost isoforms has so far focused on five aquaporins, with many of these studies occurring in eels. Four of these isoforms, comprising two homologues of mammalian aquaporin 1 (called AQP1 and AQP1 duplicate), one homologue of mammalian aquaporin 3 (AQP3) and a novel aqua-glyceroporin called AQPe are expressed in eel “osmoregulatory tissues”, the gill, the gastro-intestinal tract and the kidney. AQP1 was found in most tissues although expression predominated in the intestine and kidney, AQP1 duplicate was more sporadically expressed, but with high levels found in the oesophagus and kidney, AQP3 expression was found in the intestine, oesophagus and gill with small amounts in kidney, and AQPe was found in the intestine and kidney. Recent work in elasmobranches has also revealed the presence of several aquaporin homologues in this more evolutionarily ancient lineage. Homology comparisons suggest that at least 5 aquaporin isoforms exist in elasmobranches. Recently obtained data concerning all of these aquaporin isoforms will be presented. doi:10.1016/j.cbpa.2007.01.125
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A3.8 Towards understanding the function and regulation of fish neuropeptide hormones: An integrative approach to study osmoregulation W. Lu, C. McCrohan, R. Balment, (The University of Manchester, United Kingdom) Recent studies have revealed the importance of stress related peptide hormones to human endocrinology. These peptides include corticotropin-releasing hormone (CRH), urocortins (human urotensin-I), and urotensin-II. Recently we have demonstrated that the caudal neurosecretory system (CNSS) is the major site of expression of UI and UII genes as well as CRH. In situ hybridisation and immunocytochemistry revealed that, these peptides are differentially expressed in different cells in the same CNSS, including cellular colocalisation of two or three peptides. Further, EM immunogold studies have shown co-localisation of peptides in the same neuron terminal and, indeed, in the same granule. Together with gene expression and electrophysiology findings, we conclude that the CNSS in response to environmental salinity change involves differential reprogramming of Dahlgren cells leading to changes in the peptide synthetic and secretory capacity of the whole system, and suggests an early, possibly shared role for these peptides in controlling physiological function. These observations imply an unforeseen role for these ancient fish hormone in the physiological and perhaps pathophysiological regulation of body fluids in higher vertebrates. doi:10.1016/j.cbpa.2007.01.126
A3.9 Endocrine regulation of blood flow and secretion rate in the salt gland of the estuarine crocodile, Crocodylus porosus W. Anderson, (University of Manitoba, Canada); I. DeVries, R. Cramp, C. Franklin, (University of Queensland, Australia) The estuarine crocodile, Crocodylus porosus, is found in a range of habitats that cover fresh water, estuarine and marine environments. Plasma analysis from field samples show that C. porosus maintains its osmolality within a very narrow range between 299–305 mOsm kg−1 regardless of the salinity of their environment. This demonstrates a particularly plastic capacity to maintain homeostatic regulation of salt and water balance in C. porosus. Salt balance in these animals is achieved by both renal and extra-renal processes. The extra-renal processes involve post renal modification of the urine by the cloaca and also secretion of a highly concentrated salt solution from the lingual salt gland. The evidence suggests that the crocodilian salt gland bares all the hallmarks of similar glands found in birds, and elasmobranch fishes as it is highly vascularised and has numerous tubular structures that are modified in response to changes in environmental salinity. To date few investigations have examined the
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S87–S96
In fish stimulation of the endogenous RAS by changes in external salinity, elevation of blood plasma ionic concentration, dehydration, blood volume depletion or lowered blood pressure act as major stimuli for the drinking. In addition, the exogenous administration of the main active components of the RAS e.g. Ang I and Ang II evoke drinking in fish. Although the role of Ang II in drinking regulation in fish is well established, little is known as to how this peptide may contribute to the absorption of the water taken up by drinking into the gut. In vitro intestinal water absorption was analysed in freshwater adapted trout in response to [Asn1, Val5]-Angiotensin II (Ang II) in isolated, everted perfused gut segments. Increasing doses of Ang II (10−13 to 10−8 mol/l) when applied to the serosal side significantly increased water absorption in a linear dosedependent manner with a threshold response of 10−12 M of Ang II. Based on the response to the receptor blockers [Sar1, Val5, Ala8]-Angiotensin II (Saralasin) and Losartan (DUP 753) our results suggest a potential role for Ang II in the regulation of water absorption in the intestine of freshwater fish possibly involving novel AT receptors. The author is funded by FCT, Ministry of Science, Portugal doi:10.1016/j.cbpa.2007.01.121
A3.4 Mouth or mitochondrion — Which the better to sip the water of life? J. Noble-Nesbitt, (University of East Anglia, United Kingdom) Many insects are able to thrive in extreme environments where few other eukaryotes are able to survive. Environmental water stress exemplifies this. Faced with high temperature and low humidity, some well adapted insects survive where other animal (and plant) species succumb. Contributing to their survival is their impervious cuticle which limits water loss and conserves existing body water. The greatest survivor species, however, are able to imbibe water, not just by drinking liquid water when available, but also when necessary directly from the vapour phase of the air down to 43% RH and at ambient temperatures of 37 °C. This emancipation from drinking in its conventional sense accounts for their remarkable survival under conditions of extreme water stress. In the greatest known exponent of this active water vapour absorption (WVA), the firebrat Thermobia domestica, its water vapour absorptive epithelium is comprised of a single layer of large cells with the highest known density of elongated mitochondria hexagonally packed very tightly alongside deep pleated infolds of an enormously hypertrophied portasome-studded apical plasma membrane (apm) in a honeycomb-like array. This absorptive apm-mitochondrial complex substitutes for drinking by the mouth when liquid water is unavailable. Current research is unravelling how this remarkable cellular system develops and matures post-embryonically in the early larval instars and continues functioning
throughout the life of the insect. Rapid mitochondrial biogenesis and apm sequestration (as portasome-studded rafts) and reformation during cell proliferation are integral to this process. doi:10.1016/j.cbpa.2007.01.122
A3.5 Molecular and cellular aspects of ion and water transport in the osmoregulatory epithelia of teleost and elasmobranch fish G. Cramb, (University of St. Andrews, United Kingdom) The physiological resilience of euryhaline teleost and elasmobranch fish to adapt to changes in external salinity has allowed these animals to successfully inhabit both marine and freshwater environments, usually at different stages of their life cycles. Salinity adaptation is the result of both the genetic and functional plasticity of key ion and water transporting proteins expressed within the major osmoregulatory tissues, the gill, gut, kidney and rectal gland. Many of these ion and water transporting proteins exhibit salinity-dependent and hormonally-regulated changes in activity and expression within these tissues that enables body fluid homeostasis irrespective of salinity changes in the external environment. The expression, asymmetrical distribution and concerted actions of these transporters within the polarised cells of both absorptive and secretory epithelia are responsible for net uptake or loss of ions and water. This presentation will summarise our current understanding of the nature of ion and water transport across absorptive and secretory epithelia and the structure, location and function of some of the transporters required to enable successful osmoregulation in both teleost and elasmobranch fish. doi:10.1016/j.cbpa.2007.01.123
A3.6 Implication of mineralocorticoid receptor in rainbow trout osmoregulation S. Milla, P. Prunet, (INRA, France) In fish, aldosterone is absent and the classical view was to suggest that cortisol has both mineralocorticoid and glucocorticoid actions. Recent studies in rainbow trout have shown that mineralocorticoid and glucocorticoid receptors (respectively rtMR, rtGR1 and rtGR2) are expressed ubiquitously. Moreover, whereas cortisol is the physiological ligand of rtGRs, 11deoxycorticosterone (DOC) was demonstrated to be the most active steroid to stimulate the rtMR transactivation activity in vitro. To clarify the role of MR in trout osmoregulation, 3 different approaches have been developed.
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S87–S96
We first checked rtMR localization in the main osmoregulatory organs: gill, kidney and intestine. In addition, after an osmotic challenge (direct SW–FW and FW–SW transfers), we measured significant variations of rtMR transcripts in these organs. However, no variations of plasma DOC and cortisol levels were observed and, for both hormones, plasma level fluctuates within the same range of concentration (5– 15 ng/ml). A second strategy aims to analyze in vitro the effect of DOC on Na+K+-ATPase activity using the incubated gill test developed by McCormick and Bern (1989). We confirmed a stimulatory effect of cortisol on Na+ K+ -ATPase activity whereas DOC had an inhibitory effect. The effects of cortisol and DOC on other transport protein gene expression will also be presented. Finally, we have developed a transcriptome approach using high density array spotted with 9000 annotated trout EST. This study allowed us to characterize a list of genes regulated by MR. All these data will be discussed in the context of a probable role of MR in freshwater osmoregulation. doi:10.1016/j.cbpa.2007.01.124
A3.7 Aquaporin channels in teleost and elasmobranch fish C. Cutler, (Georgia Southern University, United States) Information concerning the role of aquaporin water and small solute channels in fish has until recently been sparse. Sequence data has become available regarding aquaporin isoforms in various teleost fish, with around 17 isoforms present in each of Pufferfish (Fugu rubripes) and Zebrafish (Danio rerio), and with at least 11 isoforms also expressed in the European eel (Anguilla anguilla). Research into the role of these teleost isoforms has so far focused on five aquaporins, with many of these studies occurring in eels. Four of these isoforms, comprising two homologues of mammalian aquaporin 1 (called AQP1 and AQP1 duplicate), one homologue of mammalian aquaporin 3 (AQP3) and a novel aqua-glyceroporin called AQPe are expressed in eel “osmoregulatory tissues”, the gill, the gastro-intestinal tract and the kidney. AQP1 was found in most tissues although expression predominated in the intestine and kidney, AQP1 duplicate was more sporadically expressed, but with high levels found in the oesophagus and kidney, AQP3 expression was found in the intestine, oesophagus and gill with small amounts in kidney, and AQPe was found in the intestine and kidney. Recent work in elasmobranches has also revealed the presence of several aquaporin homologues in this more evolutionarily ancient lineage. Homology comparisons suggest that at least 5 aquaporin isoforms exist in elasmobranches. Recently obtained data concerning all of these aquaporin isoforms will be presented. doi:10.1016/j.cbpa.2007.01.125
S89
A3.8 Towards understanding the function and regulation of fish neuropeptide hormones: An integrative approach to study osmoregulation W. Lu, C. McCrohan, R. Balment, (The University of Manchester, United Kingdom) Recent studies have revealed the importance of stress related peptide hormones to human endocrinology. These peptides include corticotropin-releasing hormone (CRH), urocortins (human urotensin-I), and urotensin-II. Recently we have demonstrated that the caudal neurosecretory system (CNSS) is the major site of expression of UI and UII genes as well as CRH. In situ hybridisation and immunocytochemistry revealed that, these peptides are differentially expressed in different cells in the same CNSS, including cellular colocalisation of two or three peptides. Further, EM immunogold studies have shown co-localisation of peptides in the same neuron terminal and, indeed, in the same granule. Together with gene expression and electrophysiology findings, we conclude that the CNSS in response to environmental salinity change involves differential reprogramming of Dahlgren cells leading to changes in the peptide synthetic and secretory capacity of the whole system, and suggests an early, possibly shared role for these peptides in controlling physiological function. These observations imply an unforeseen role for these ancient fish hormone in the physiological and perhaps pathophysiological regulation of body fluids in higher vertebrates. doi:10.1016/j.cbpa.2007.01.126
A3.9 Endocrine regulation of blood flow and secretion rate in the salt gland of the estuarine crocodile, Crocodylus porosus W. Anderson, (University of Manitoba, Canada); I. DeVries, R. Cramp, C. Franklin, (University of Queensland, Australia) The estuarine crocodile, Crocodylus porosus, is found in a range of habitats that cover fresh water, estuarine and marine environments. Plasma analysis from field samples show that C. porosus maintains its osmolality within a very narrow range between 299–305 mOsm kg−1 regardless of the salinity of their environment. This demonstrates a particularly plastic capacity to maintain homeostatic regulation of salt and water balance in C. porosus. Salt balance in these animals is achieved by both renal and extra-renal processes. The extra-renal processes involve post renal modification of the urine by the cloaca and also secretion of a highly concentrated salt solution from the lingual salt gland. The evidence suggests that the crocodilian salt gland bares all the hallmarks of similar glands found in birds, and elasmobranch fishes as it is highly vascularised and has numerous tubular structures that are modified in response to changes in environmental salinity. To date few investigations have examined the