ABSTRACT Divalent ions, such as Ca2+ and Mg2+ are carefully regulated in cells and body fluids, b... more ABSTRACT Divalent ions, such as Ca2+ and Mg2+ are carefully regulated in cells and body fluids, but also have wide influence on other processes in osmoregulation. For example, Ca2+ alters membrane permeability and electrical properties of epithelia, as well as the control of some ion transporters. Intracellular free Ca2+ levels are submicromolar, and extracellular fluid Ca2+ concentrations in fish are around 1.25 mM. Calcium influx, and the large inward gradient for Ca2+ must be carefully controlled, while export of Ca2+ from cells is an up-hill, energy-requiring process. The control of Ca2+ homeostasis can be considered in terms of hypercalcemic regulation and hypocalcemic regulation, dealing with control above and below the normal set point. This chapter reviews the roles of the hormones involved in hypercalcemic regulation including, prolactin, parathyroid hormone related protein (PTHrP), parathyroid hormone (PTH), and calcitriol. The roles of stanniocalcin and calcitonin in hypocalcemic regulation are also described. Knowledge gaps are identified, and potential new feedback loops in the endocrine control of Ca2+ regulation are discussed.
The peripheral conversion of the prohorm one 3,5,30,50-tetraiodothyronine (T4) to the biologicall... more The peripheral conversion of the prohorm one 3,5,30,50-tetraiodothyronine (T4) to the biologically active 3,5,30-triiodothyronine (T3), via enzymatic deiodination by deiodinases, is an im portant pathway in thyroid horm one metabolism. The aim of this study was to test if thyroid hormones and cortisol, as well as the outer ring deiodination (ORD) metabolic pathway, are involved in the osmoregulatory response of Senegalese sole (Solea senegalensis, K aup 1858). We measured osmoregulatory and endocrine parameters in imm ature juve niles S. senegalensis acclimated to seawater (SW, 38&) and that were transferred and allowed to acclimate to different salinities (5&, 15&, 38& and 55&) for 17 days. An adjustment and a chronic regulatory period were identified following acclimation. The adjustment period immediately follows the transfer, and is characterized by altered plasma osmolalities. D uring this period, plasma cortisol levels increased while plasma free T4 (fT4) levels decreased. Both hormones levels returned to norm al values on day 3 post-transfer. In the adjustment period, renal and hepatic O RD activities had increased concomitantly with the decrease in plasma fT4 levels in fishes trans ferred to extreme salinities (5& and 55&). In the chronic regulatory period, where plasma osmolality returned to norm al values, plasma cortisol had increased, whereas plasma fT4 levels decreased in animals th at were transferred to salinities other than SW. N o m ajor changes were observed in branchial O RD activity throughout the experiment. The inverse relationship between plasma cortisol and fT4 suggests an interaction between these hormones during both osmoregulatory periods while O RD pathway can be im portant in the short-term adjustment period.
We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea seneg... more We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea senegalensis), and correlate these with whole body concentrations of thyroid hormones during early development and metamorphosis. Under our rearing conditions at 19.5°C, most larvae entered metamorphosis in stage 1 at 15 days post-hatching (dph), and completed metamorphosis in stage 4 at 25 dph. The onset of metamorphosis coincided with surges in whole body T4 and T3 concentrations. Crossmon's trichrome stain colored the lumen of follicular structures brightly red, and this co-localized with a T4-immunoreactivity. Thyroid follicles were first observed in stage 0 premetamorphic larvae at 5 dph of age, and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Increases in whole body thyroid hormone levels coincided with a 2½-fold increase in the total thyroidal colloid area in stage 1 larvae (aged 15 dph) compared to stage 0 larvae (12 dph). This was preceded by an approximately 40%-increase in the follicles' epithelial cell height in stage 0 larvae at 12 dph compared to larvae at 5 dph, and by an increase in the whole body T3/T4 ratio, indicative of an increase in outer ring deiodination. We conclude that in S. senegalensis there is a clear chronology in the activation of the thyroid gland that starts in early premetamorphic larvae. General and Comparative Endocrinology 155 (2008) 686-694 P.H.M. Klaren et al. / General and Comparative Endocrinology 155 (2008) 686-694
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2015
Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in mu... more Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0μgT3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total α amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects.
Uptake of T 3 was compared with that of T 4 in the embryonic heart cell line H9c2(2-1). These cel... more Uptake of T 3 was compared with that of T 4 in the embryonic heart cell line H9c2(2-1). These cells propagate as myoblasts and form differentiated myotubes upon reduction of the serum concentration, as indicated by a 31-fold increase in creatine kinase activity. Protein and DNA content per well were around 2-fold higher in myotubes than in myoblasts. T 3 and T 4 uptakes showed similar characteristics in myoblasts and myotubes, but both were 2.5-fold higher in myotubes, when expressed per well. At any time point, T 4 uptake was 2-fold higher than that of T 3 , and both uptakes were energy-, but not Na + dependent. T 3 and T 4 uptake exhibited mutual inhibition in myoblasts and myotubes: 10 µM unlabeled T 3 reduced T 4 uptake by 51-60% (P < 0.001), while 10 µM T 4 inhibited T 3 uptake by 48-51% (P < 0.001). Furthermore, T 3 and T 4 uptake in myoblasts was dose-dependently inhibited by tryptophan (maximum inhibition around 70%; P < 0.001). Exposure of the cells to T 3 or T 4 during differentiation significantly increased the fusion index (35 and 40%; P < 0.01). Finally, both myoblasts and myotubes showed a small deiodinase (D) I activity, while DII activity was undetectable. In conclusion, T 3 and T 4 share a common energy-dependent transport system in H9c2(2-1) cells, that may be important for the availability of thyroid hormone during differentiation.
We have studied the effect of ATP on Ca2+ uptake in intestinal brush border membrane vesicles (BB... more We have studied the effect of ATP on Ca2+ uptake in intestinal brush border membrane vesicles (BBMVs) of the teleost tilapia (Oreochromis mossambicus). ATP stimulated Ca2+ uptake 12-fold over the control, with a linear time course. Ionomycin and detergent treatment did not reduce BBMVs' Ca2+ content, indicating the binding of Ca2+ to a membrane component. A rank order of ATP > ADP > AMP was established for the stimulation of Ca2+ uptake. Adenosine, vanadate, adenosine 5'-[alpha, beta-methylene]triphosphate (a P2x purinoceptor agonist) and adenosine 5'-[gamma-thio]triphosphate (a P-type ATPase inhibitor) were without effect. 2-Methylthioadenosine 5'-triphosphate, a P2y purinoceptor agonist, mimicked the stimulation by ATP. As judged from a kinetic comparison, ATP hydrolysis and the stimulation by ATP of Ca2+ uptake were not compatible. The P2 purinoceptor antagonist suramin and the P2y purinoceptor antagonist Reactive Blue-2 inhibited the Ca2+ uptake stimulated ...
In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several s... more In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several species thyroid follicles are found in, for example, heart, head kidney and kidney. Such heterotopic thyroid follicles are active, and considered to work in concert with the subpharyngeal thyroid. In Mozambique tilapia (Oreochromis mossambicus) thyroid activity is, indeed, restricted to the subpharyngeal region; in common carp (Cyprinus carpio) the functional endocrine thyroid is associated with renal tissues. The subpharyngeal follicles of carp comprise only 10% of the total thyroid tissue, and these follicles neither accumulate iodide nor synthesize or secrete thyroid hormones to a significant degree. Although the shape and size of carp subpharyngeal and renal follicles vary, the epithelial cell height of the thyrocytes and thyroxine immunoreactivity do not differ, which suggests that the activity of the carp subpharyngeal thyroid follicles is dormant. Differences in thyroid physiology ...
The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host... more The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.
Conjugation reactions are important pathways in the peripheral metabolism of thyroid hormones. Ra... more Conjugation reactions are important pathways in the peripheral metabolism of thyroid hormones. Rat cardiac fibroblasts produce and secrete glucuronidated thyroxine (T4G) and 3,3 ,5-triiodothyronine (T3G). We here show that, compared to fibroblasts from other anatomical locations, the capacity of cardiofibroblasts to secrete T4G and T3G is highest. H9c2(2-1) myotubes, a model system for cardiomyocytes, take up T4G and T3G at a rate that is 10-15 times higher than that for the unconjugated thyroid hormones. T3 and T4, and their glucuronides, stimulate H9c2(2-1) myoblast-to-myotube differentiation. A substantial -glucuronidase activity was measured in H9c2(2-1) myotubes, and this confers a deconjugating capacity to these cells, via which native thyroid hormones can be regenerated from glucuronidated precursors. This indicates that the stimulatory effects on myoblast differentiation are exerted by the native hormones. We suggest that glucuronidation represents a mechanism to uncouple local thyroid hormone action in the heart from that in other peripheral tissues and in the systemic circulation. This could represent a mechanism for the local fine-tuning of cardiac thyroid hormone action.
We have investigated the regulation of thyroidal status and osmoregulatory capacities in juvenile... more We have investigated the regulation of thyroidal status and osmoregulatory capacities in juveniles from the teleost Solea senegalensis acclimated to different ambient temperatures. Juveniles, raised in seawater at 19°C, were acclimated for 3 weeks to temperatures of 12, 19 and 26°C. Since our preliminary observations showed that at 12°C feed intake was suppressed, our experimental design controlled for this factor. The concentration of branchial Na ? ,K ? -ATPase, estimated by measurements of enzyme activity at the optimum temperature of this enzyme (37°C), did not change. In contrast, an increase in Na ? ,K ? -ATPase activity (measured at 37°C), was observed in the kidney of 12°C-acclimated fish. In fish acclimated to 12°C, the hepatosomatic index had increased, which correlated with increased plasma levels of triglycerides and nonesterified fatty acids. Plasma cortisol levels did not differ significantly between the experimental groups. In liver and gills, the amount of iodothyronine deiodinases that exhibit thyroid hormone outer ring deiodination was up-regulated only when fish did not feed. When assayed at the acclimation temperature, kidney deiodinase activities were similar, indicating a temperature-compensation strategy. 3,5,3 0 -triiodothyronine (T3) tissue concentrations in gills and kidney did not differ significantly between experimental groups. However, at 12°C, lower T3 tissue levels were measured in plasma and liver. We conclude that S. senegalensis adjusts its osmoregulatory system to compensate for the effects of temperature on electrolyte transport capacity. The organ-specific changes in thyroid hormone metabolism at different temperatures indicate the involvement of thyroid hormones in temperature acclimation.
The effect of experimental hyperthyroidism, realized by T 4 injection, on central mediators of th... more The effect of experimental hyperthyroidism, realized by T 4 injection, on central mediators of the hypothalamo-pituitaryinterrenal axis (HPI-axis) in common carp (Cyprinus carpio L.) was studied. Our results show that hyperthyroidism evokes a marked 3 . 2-fold reduction in basal plasma cortisol levels. Corticotropin-releasing hormone-binding protein (CRH-BP) mRNA levels in the hypothalamus, measured by real-time quantitative PCR, were significantly elevated by 40%, but CRH, urotensin-I, prepro-TRH, prohormone convertase-1 (PC1), and POMC mRNA levels were unchanged. In the pituitary pars distalis, PC1, CRH receptor-1, and POMC mRNA levels were unaffected, as was ACTH content. Plasma a-MSH concentrations were significantly elevated by 30% in hyperthyroid fish, and this was reflected in PC1 and POMC mRNA levels in pituitary pars intermedia that were increased 1 . 5-and 2 . 4-fold respectively. The a-MSH content of the pars intermedia was unchanged. Hyperthyroidism has profound effects on the basal levels of a central mediator, i.e., CRH-BP, of HPI-axis function in unstressed carp in vivo, and we conclude that HPI-and hypothalamo-pituitary-thyroidaxis functions are strongly interrelated. We suggest that the changes in plasma cortisol, thyroid hormone, and a-MSH levels reflect their concerted actions on energy metabolism.
The inhibition of Ca2--ATPase, (Na + + K~)-ATPase and Na-/Ca z+ exchange by Cd -'+ was studied in... more The inhibition of Ca2--ATPase, (Na + + K~)-ATPase and Na-/Ca z+ exchange by Cd -'+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a K,,, for Ca '-+ of 88 -+ 17 aM, and was extremely sensitive to Cd -'+ with an ICs0 of 8.2 +-3.0 pM Cd 2+, indicating an inhibition via the Ca -,+ site. (Na + + K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd > , displaying an ICs0 of 2.6 -+ 0.6/*M Cd > . Cd 2+ ions apparently compete for the Mg 2+ site of the (Na-+ K +)-ATPase. The Na +/Ca 2-exchanger was inhibited by Cd 2 + with an ICs0 of 73 --11 aM. Cd 2+ is a competitive inhibitor of the exchanger via an interaction with the Ca 2+ site (Ki = 11 aM). Bepridil, a Na + site specific inhibitor of Na-/Ca 2-exchange, induced an additional inhibition, but did not change the K~ of Cd 2+. Also, Cd 2 § is exchanged against Ca 2+, albeit to a lesser extent than Ca 2+. The exchanger is only partly blocked by the binding of Cd 2.. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca 2+ exchanger. We conclude that intracellular Cd 2-ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.
Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use ... more Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use of magnesium precipitation and differential centrifugation. The membrane preparation was enriched 17-fold in alkaline phosphatase. The membranes were 99% right-side-out oriented as indicated by the unmasking of latent glyceraldehyde-3-phosphate dehydrogenase and acetylcholine esterase activity by detergent treatment. The transport of Ca2+ in brush border membrane vesicles was analyzed. A saturable and a nonsaturable component in the uptake of Ca2+ was resolved. The saturable component is characterized by a Km much lower than the Ca2+ concentrations predicted to occur in the intestinal lumen. The nonsaturable component displays a Ca2+ permeability too high to be explained by simple diffusion. We discuss the role of the saturable component as the rate-limiting step in transmembrane Ca2+ movement, and suggest that the nonsaturable component reflects a transport mechanism operating well below its level of saturation.
ABSTRACT Divalent ions, such as Ca2+ and Mg2+ are carefully regulated in cells and body fluids, b... more ABSTRACT Divalent ions, such as Ca2+ and Mg2+ are carefully regulated in cells and body fluids, but also have wide influence on other processes in osmoregulation. For example, Ca2+ alters membrane permeability and electrical properties of epithelia, as well as the control of some ion transporters. Intracellular free Ca2+ levels are submicromolar, and extracellular fluid Ca2+ concentrations in fish are around 1.25 mM. Calcium influx, and the large inward gradient for Ca2+ must be carefully controlled, while export of Ca2+ from cells is an up-hill, energy-requiring process. The control of Ca2+ homeostasis can be considered in terms of hypercalcemic regulation and hypocalcemic regulation, dealing with control above and below the normal set point. This chapter reviews the roles of the hormones involved in hypercalcemic regulation including, prolactin, parathyroid hormone related protein (PTHrP), parathyroid hormone (PTH), and calcitriol. The roles of stanniocalcin and calcitonin in hypocalcemic regulation are also described. Knowledge gaps are identified, and potential new feedback loops in the endocrine control of Ca2+ regulation are discussed.
The peripheral conversion of the prohorm one 3,5,30,50-tetraiodothyronine (T4) to the biologicall... more The peripheral conversion of the prohorm one 3,5,30,50-tetraiodothyronine (T4) to the biologically active 3,5,30-triiodothyronine (T3), via enzymatic deiodination by deiodinases, is an im portant pathway in thyroid horm one metabolism. The aim of this study was to test if thyroid hormones and cortisol, as well as the outer ring deiodination (ORD) metabolic pathway, are involved in the osmoregulatory response of Senegalese sole (Solea senegalensis, K aup 1858). We measured osmoregulatory and endocrine parameters in imm ature juve niles S. senegalensis acclimated to seawater (SW, 38&) and that were transferred and allowed to acclimate to different salinities (5&, 15&, 38& and 55&) for 17 days. An adjustment and a chronic regulatory period were identified following acclimation. The adjustment period immediately follows the transfer, and is characterized by altered plasma osmolalities. D uring this period, plasma cortisol levels increased while plasma free T4 (fT4) levels decreased. Both hormones levels returned to norm al values on day 3 post-transfer. In the adjustment period, renal and hepatic O RD activities had increased concomitantly with the decrease in plasma fT4 levels in fishes trans ferred to extreme salinities (5& and 55&). In the chronic regulatory period, where plasma osmolality returned to norm al values, plasma cortisol had increased, whereas plasma fT4 levels decreased in animals th at were transferred to salinities other than SW. N o m ajor changes were observed in branchial O RD activity throughout the experiment. The inverse relationship between plasma cortisol and fT4 suggests an interaction between these hormones during both osmoregulatory periods while O RD pathway can be im portant in the short-term adjustment period.
We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea seneg... more We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea senegalensis), and correlate these with whole body concentrations of thyroid hormones during early development and metamorphosis. Under our rearing conditions at 19.5°C, most larvae entered metamorphosis in stage 1 at 15 days post-hatching (dph), and completed metamorphosis in stage 4 at 25 dph. The onset of metamorphosis coincided with surges in whole body T4 and T3 concentrations. Crossmon's trichrome stain colored the lumen of follicular structures brightly red, and this co-localized with a T4-immunoreactivity. Thyroid follicles were first observed in stage 0 premetamorphic larvae at 5 dph of age, and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Increases in whole body thyroid hormone levels coincided with a 2½-fold increase in the total thyroidal colloid area in stage 1 larvae (aged 15 dph) compared to stage 0 larvae (12 dph). This was preceded by an approximately 40%-increase in the follicles' epithelial cell height in stage 0 larvae at 12 dph compared to larvae at 5 dph, and by an increase in the whole body T3/T4 ratio, indicative of an increase in outer ring deiodination. We conclude that in S. senegalensis there is a clear chronology in the activation of the thyroid gland that starts in early premetamorphic larvae. General and Comparative Endocrinology 155 (2008) 686-694 P.H.M. Klaren et al. / General and Comparative Endocrinology 155 (2008) 686-694
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2015
Thyroid hormones, in particular 3,5,3&amp;amp;#39;-triiodothyronine or T3, are involved in mu... more Thyroid hormones, in particular 3,5,3&amp;amp;#39;-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0μgT3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total α amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects.
Uptake of T 3 was compared with that of T 4 in the embryonic heart cell line H9c2(2-1). These cel... more Uptake of T 3 was compared with that of T 4 in the embryonic heart cell line H9c2(2-1). These cells propagate as myoblasts and form differentiated myotubes upon reduction of the serum concentration, as indicated by a 31-fold increase in creatine kinase activity. Protein and DNA content per well were around 2-fold higher in myotubes than in myoblasts. T 3 and T 4 uptakes showed similar characteristics in myoblasts and myotubes, but both were 2.5-fold higher in myotubes, when expressed per well. At any time point, T 4 uptake was 2-fold higher than that of T 3 , and both uptakes were energy-, but not Na + dependent. T 3 and T 4 uptake exhibited mutual inhibition in myoblasts and myotubes: 10 µM unlabeled T 3 reduced T 4 uptake by 51-60% (P < 0.001), while 10 µM T 4 inhibited T 3 uptake by 48-51% (P < 0.001). Furthermore, T 3 and T 4 uptake in myoblasts was dose-dependently inhibited by tryptophan (maximum inhibition around 70%; P < 0.001). Exposure of the cells to T 3 or T 4 during differentiation significantly increased the fusion index (35 and 40%; P < 0.01). Finally, both myoblasts and myotubes showed a small deiodinase (D) I activity, while DII activity was undetectable. In conclusion, T 3 and T 4 share a common energy-dependent transport system in H9c2(2-1) cells, that may be important for the availability of thyroid hormone during differentiation.
We have studied the effect of ATP on Ca2+ uptake in intestinal brush border membrane vesicles (BB... more We have studied the effect of ATP on Ca2+ uptake in intestinal brush border membrane vesicles (BBMVs) of the teleost tilapia (Oreochromis mossambicus). ATP stimulated Ca2+ uptake 12-fold over the control, with a linear time course. Ionomycin and detergent treatment did not reduce BBMVs' Ca2+ content, indicating the binding of Ca2+ to a membrane component. A rank order of ATP > ADP > AMP was established for the stimulation of Ca2+ uptake. Adenosine, vanadate, adenosine 5'-[alpha, beta-methylene]triphosphate (a P2x purinoceptor agonist) and adenosine 5'-[gamma-thio]triphosphate (a P-type ATPase inhibitor) were without effect. 2-Methylthioadenosine 5'-triphosphate, a P2y purinoceptor agonist, mimicked the stimulation by ATP. As judged from a kinetic comparison, ATP hydrolysis and the stimulation by ATP of Ca2+ uptake were not compatible. The P2 purinoceptor antagonist suramin and the P2y purinoceptor antagonist Reactive Blue-2 inhibited the Ca2+ uptake stimulated ...
In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several s... more In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several species thyroid follicles are found in, for example, heart, head kidney and kidney. Such heterotopic thyroid follicles are active, and considered to work in concert with the subpharyngeal thyroid. In Mozambique tilapia (Oreochromis mossambicus) thyroid activity is, indeed, restricted to the subpharyngeal region; in common carp (Cyprinus carpio) the functional endocrine thyroid is associated with renal tissues. The subpharyngeal follicles of carp comprise only 10% of the total thyroid tissue, and these follicles neither accumulate iodide nor synthesize or secrete thyroid hormones to a significant degree. Although the shape and size of carp subpharyngeal and renal follicles vary, the epithelial cell height of the thyrocytes and thyroxine immunoreactivity do not differ, which suggests that the activity of the carp subpharyngeal thyroid follicles is dormant. Differences in thyroid physiology ...
The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host... more The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.
Conjugation reactions are important pathways in the peripheral metabolism of thyroid hormones. Ra... more Conjugation reactions are important pathways in the peripheral metabolism of thyroid hormones. Rat cardiac fibroblasts produce and secrete glucuronidated thyroxine (T4G) and 3,3 ,5-triiodothyronine (T3G). We here show that, compared to fibroblasts from other anatomical locations, the capacity of cardiofibroblasts to secrete T4G and T3G is highest. H9c2(2-1) myotubes, a model system for cardiomyocytes, take up T4G and T3G at a rate that is 10-15 times higher than that for the unconjugated thyroid hormones. T3 and T4, and their glucuronides, stimulate H9c2(2-1) myoblast-to-myotube differentiation. A substantial -glucuronidase activity was measured in H9c2(2-1) myotubes, and this confers a deconjugating capacity to these cells, via which native thyroid hormones can be regenerated from glucuronidated precursors. This indicates that the stimulatory effects on myoblast differentiation are exerted by the native hormones. We suggest that glucuronidation represents a mechanism to uncouple local thyroid hormone action in the heart from that in other peripheral tissues and in the systemic circulation. This could represent a mechanism for the local fine-tuning of cardiac thyroid hormone action.
We have investigated the regulation of thyroidal status and osmoregulatory capacities in juvenile... more We have investigated the regulation of thyroidal status and osmoregulatory capacities in juveniles from the teleost Solea senegalensis acclimated to different ambient temperatures. Juveniles, raised in seawater at 19°C, were acclimated for 3 weeks to temperatures of 12, 19 and 26°C. Since our preliminary observations showed that at 12°C feed intake was suppressed, our experimental design controlled for this factor. The concentration of branchial Na ? ,K ? -ATPase, estimated by measurements of enzyme activity at the optimum temperature of this enzyme (37°C), did not change. In contrast, an increase in Na ? ,K ? -ATPase activity (measured at 37°C), was observed in the kidney of 12°C-acclimated fish. In fish acclimated to 12°C, the hepatosomatic index had increased, which correlated with increased plasma levels of triglycerides and nonesterified fatty acids. Plasma cortisol levels did not differ significantly between the experimental groups. In liver and gills, the amount of iodothyronine deiodinases that exhibit thyroid hormone outer ring deiodination was up-regulated only when fish did not feed. When assayed at the acclimation temperature, kidney deiodinase activities were similar, indicating a temperature-compensation strategy. 3,5,3 0 -triiodothyronine (T3) tissue concentrations in gills and kidney did not differ significantly between experimental groups. However, at 12°C, lower T3 tissue levels were measured in plasma and liver. We conclude that S. senegalensis adjusts its osmoregulatory system to compensate for the effects of temperature on electrolyte transport capacity. The organ-specific changes in thyroid hormone metabolism at different temperatures indicate the involvement of thyroid hormones in temperature acclimation.
The effect of experimental hyperthyroidism, realized by T 4 injection, on central mediators of th... more The effect of experimental hyperthyroidism, realized by T 4 injection, on central mediators of the hypothalamo-pituitaryinterrenal axis (HPI-axis) in common carp (Cyprinus carpio L.) was studied. Our results show that hyperthyroidism evokes a marked 3 . 2-fold reduction in basal plasma cortisol levels. Corticotropin-releasing hormone-binding protein (CRH-BP) mRNA levels in the hypothalamus, measured by real-time quantitative PCR, were significantly elevated by 40%, but CRH, urotensin-I, prepro-TRH, prohormone convertase-1 (PC1), and POMC mRNA levels were unchanged. In the pituitary pars distalis, PC1, CRH receptor-1, and POMC mRNA levels were unaffected, as was ACTH content. Plasma a-MSH concentrations were significantly elevated by 30% in hyperthyroid fish, and this was reflected in PC1 and POMC mRNA levels in pituitary pars intermedia that were increased 1 . 5-and 2 . 4-fold respectively. The a-MSH content of the pars intermedia was unchanged. Hyperthyroidism has profound effects on the basal levels of a central mediator, i.e., CRH-BP, of HPI-axis function in unstressed carp in vivo, and we conclude that HPI-and hypothalamo-pituitary-thyroidaxis functions are strongly interrelated. We suggest that the changes in plasma cortisol, thyroid hormone, and a-MSH levels reflect their concerted actions on energy metabolism.
The inhibition of Ca2--ATPase, (Na + + K~)-ATPase and Na-/Ca z+ exchange by Cd -'+ was studied in... more The inhibition of Ca2--ATPase, (Na + + K~)-ATPase and Na-/Ca z+ exchange by Cd -'+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a K,,, for Ca '-+ of 88 -+ 17 aM, and was extremely sensitive to Cd -'+ with an ICs0 of 8.2 +-3.0 pM Cd 2+, indicating an inhibition via the Ca -,+ site. (Na + + K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd > , displaying an ICs0 of 2.6 -+ 0.6/*M Cd > . Cd 2+ ions apparently compete for the Mg 2+ site of the (Na-+ K +)-ATPase. The Na +/Ca 2-exchanger was inhibited by Cd 2 + with an ICs0 of 73 --11 aM. Cd 2+ is a competitive inhibitor of the exchanger via an interaction with the Ca 2+ site (Ki = 11 aM). Bepridil, a Na + site specific inhibitor of Na-/Ca 2-exchange, induced an additional inhibition, but did not change the K~ of Cd 2+. Also, Cd 2 § is exchanged against Ca 2+, albeit to a lesser extent than Ca 2+. The exchanger is only partly blocked by the binding of Cd 2.. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca 2+ exchanger. We conclude that intracellular Cd 2-ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.
Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use ... more Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use of magnesium precipitation and differential centrifugation. The membrane preparation was enriched 17-fold in alkaline phosphatase. The membranes were 99% right-side-out oriented as indicated by the unmasking of latent glyceraldehyde-3-phosphate dehydrogenase and acetylcholine esterase activity by detergent treatment. The transport of Ca2+ in brush border membrane vesicles was analyzed. A saturable and a nonsaturable component in the uptake of Ca2+ was resolved. The saturable component is characterized by a Km much lower than the Ca2+ concentrations predicted to occur in the intestinal lumen. The nonsaturable component displays a Ca2+ permeability too high to be explained by simple diffusion. We discuss the role of the saturable component as the rate-limiting step in transmembrane Ca2+ movement, and suggest that the nonsaturable component reflects a transport mechanism operating well below its level of saturation.
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Papers by Peter Klaren