Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient salinities.

Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55ppt) for 14days, and plasma free thyroid hormones (fT3, fT4), outer ring deiodination and Na+/K+-ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40ppt, natural salinity conditions in the Bay of Cádiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit ß (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15ppt). The high salinity (55ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (dio1 and dio2) and activity did not change and were similar to controls (40ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity.

Effects of acute stress on aggression and the cortisol response in the African sharptooth catfish Clarias gariepinus: differences between day and night.

African sharptooth catfish Clarias gariepinus were housed under continuous dim light (1 lx) or 12L:12D (350-0 lx) cycles. The number of skin lesions, as indicator of aggressive acts, and plasma cortisol levels, as indicator of stress-axis activity, were measured at baseline as well as following a stressor (given in the light or dark phase). Results showed that (1) baseline plasma cortisol levels were not different between photoperiods, (2) the number of baseline skin lesions was highest for C. gariepinus housed under continuous dim light, (3) stressor-induced peak levels of plasma cortisol were highest in the light phase and (4) the number of skin lesions following a stressor was highest in the dark phase. The higher number of stressor-related skin lesions in the dark (active) phase suggests increased stressor-induced aggression while in the active phase. In addition, the data suggest that housing under continuous dim light does not result in higher stress-axis activity, as measured by baseline levels of cortisol, but does result in more stressor-induced aggression, as measured by the higher number of skin lesions. The latter may be related to the fact that the continuous dim light photoperiod has twice the number of dark-phase (active) hours in which stressor-induced aggression is stronger compared to the 12L:12D photoperiod, which has a light phase in which stressor-induced aggression is lower.

Arginase inhibition prevents inflammation and remodeling in a guinea pig model of chronic obstructive pulmonary disease.

Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.

Prednisolone induces osteoporosis-like phenotype in regenerating zebrafish scales.

UNLABELLED: We demonstrate that glucocorticoids induce an osteoporotic phenotype in regenerating scales of zebrafish. Exposure to prednisolone results in altered mineral content, enhanced matrix breakdown, and an osteoporotic gene-expression profile in osteoblasts and osteoclasts. This highlights that the zebrafish scale provides a powerful tool for preclinical osteoporosis research. INTRODUCTION: This study aims to evaluate whether glucocorticoid (prednisolone) treatment of zebrafish induces an osteoporotic phenotype in regenerating scales. Scales, a readily accessible dermal bone tissue, may provide a tool to study direct osteogenesis and its disturbance by glucocorticoids. METHODS: In adult zebrafish, treated with 25 µM prednisolone phosphate via the water, scales were removed and allowed to regenerate. During regeneration scale morphology and the molar calcium/phosphorus ratio in scales were assessed and osteoblast and osteoclast activities were monitored by time profiling of cell-specific genes; mineralization was visualized by Von Kossa staining, osteoclast activity by tartrate-resistant acid phosphatase histochemistry. RESULTS: Prednisolone (compared to controls) enhances osteoclast activity and matrix resorption and slows down the build up of the calcium/phosphorus molar ratio indicative of altered crystal maturation. Prednisolone treatment further impedes regeneration through a shift in the time profiles of osteoblast and osteoclast genes that commensurates with an osteoporosis-like imbalance in bone formation. CONCLUSIONS: A glucocorticoid-induced osteoporosis phenotype as seen in mammals was induced in regenerating scalar bone of zebrafish treated with prednisolone. An unsurpassed convenience and low cost then make the zebrafish scale a superior model for preclinical studies in osteoporosis research.

The dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats.

Drugs that are able to shift effort-related decision making in intact rats towards high-effort response options are largely unknown. Here, we examined the effects of two candidate drugs, MRZ-9547 and its l-enantiomer MRZ-9546 on progressive ratio (PR) responding using two different tasks, a standard PR task that involves increasing ratio requirements and a PR/chow feeding choice task in which animals can lever press for preferred food pellets under a PR schedule or approach freely available less preferred lab chow. Furthermore, we assessed the mechanisms of action of both drugs using in vitro-assay methods and in vivo-microdialysis. Results reveal that MRZ-9547 is a selective dopamine transporter (DAT) inhibitor that moderately stimulated striatal dopamine release. MRZ-9546 was a much less potent DAT inhibitor. Furthermore, MRZ-9547 dose dependently increased the tendency to work for food reinforcement both in the standard PR task and the PR/chow feeding choice task, MRZ-9546 was considerably less active. Relative to MRZ-9547, other DAT-interfering drugs had only moderate (methylphenidate) or marginal (modafinil, d-amphetamine) stimulant effects on PR responding in either task. Collectively, our data demonstrate that the DAT inhibitor MRZ-9547 can markedly stimulate PR responding and shift effort-related decision making in intact rats towards high-effort response options. An analysis of effort-related decision making in rodents could provide an animal model for motivational dysfunctions related to effort expenditure such as fatigue, e.g. in Parkinson's disease or major depression. Our findings suggest that DAT inhibitors such as MRZ-9547 could be potentially useful for treating energy-related symptoms in neurological or neuropsychiatric disorders.

Deficient serotonin neurotransmission and depression-like serotonin biomarker alterations in tryptophan hydroxylase 2 (Tph2) loss-of-function mice.

Probably the foremost hypothesis of depression is the 5-hydroxytryptamine (5-HT, serotonin) deficiency hypothesis. Accordingly, anomalies in putative 5-HT biomarkers have repeatedly been reported in depression patients. However, whether such anomalies in fact reflect deficient central 5-HT neurotransmission remains unresolved. We employed a naturalistic model of 5-HT deficiency, the tryptophan hydroxylase 2 (Tph2) R439H knockin mouse, to address this question. We report that Tph2 knockin mice have reduced basal and stimulated levels of extracellular 5-HT (5-HT(Ext)). Interestingly, cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) and fenfluramine-induced plasma prolactin levels are markedly diminished in the Tph2 knockin mice. These data seemingly confirm that low CSF 5-HIAA and fenfluramine-induced plasma prolactin reflects chronic, endogenous central nervous system (CNS) 5-HT deficiency. Moreover, 5-HT(1A) receptor agonist-induced hypothermia is blunted and frontal cortex 5-HT(2A) receptors are increased in the Tph2 knockin mice. These data likewise parallel core findings in depression, but are usually attributed to anomalies in the respective receptors rather than resulting from CNS 5-HT deficiency. Further, 5-HT(2A) receptor function is enhanced in the Tph2 knockin mice. In contrast, 5-HT(1A) receptor levels and G-protein coupling is normal in Tph2 knockin mice, indicating that the blunted hypothermic response relates directly to the low 5-HT(Ext). Thus, we show that not only low CSF 5-HIAA and a blunted fenfluramine-induced prolactin response, but also blunted 5-HT(1A) agonist-induced hypothermia and increased 5-HT(2A) receptor levels are bona fide biomarkers of chronic, endogenous 5-HT deficiency. Potentially, some of these biomarkers could identify patients likely to have 5-HT deficiency. This could have clinical research utility or even guide pharmacotherapy.

Aluminum exposure impacts brain plasticity and behavior in Atlantic salmon (Salmo salar).

Aluminum (Al) toxicity occurs frequently in natural aquatic ecosystems as a result of acid deposition and natural weathering processes. Detrimental effects of Al toxicity on aquatic organisms are well known and can have consequences for survival. Fish exposed to Al in low pH waters will experience physiological and neuroendocrine changes that disrupt homeostasis and alter behavior. To investigate the effects of Al exposure on both the brain and behavior, Atlantic salmon (Salmo salar) kept in water treated with Al (pH 5.7, 0.37±0.04 µmol 1(-1) Al) for 2 weeks were compared with fish kept in under control conditions (pH 6.7, <0.04 µmol 1(-1) Al). Fish exposed to Al and acidic conditions had increased Al accumulation in the gills and decreased gill Na(+), K(+)-ATPase activity, which impaired osmoregulatory capacity and caused physiological stress, indicated by elevated plasma cortisol and glucose levels. Here we show for the first time that exposure to Al in acidic conditions also impaired learning performance in a maze task. Al toxicity also reduced the expression of NeuroD1 transcript levels in the forebrain of exposed fish. As in mammals, these data show that exposure to chronic stress, such as acidified Al, can reduce neural plasticity during behavioral challenges in salmon, and may impair the ability to cope with new environments.

Increased arginase activity contributes to airway remodelling in chronic allergic asthma.

Airway remodelling, characterised by increased airway smooth muscle (ASM) mass, subepithelial fibrosis, goblet cell hyperplasia and mucus gland hypertrophy, is a feature of chronic asthma. Increased arginase activity could contribute to these features via increased formation of polyamines and l-proline downstream of the arginase product l-ornithine, and via reduced nitric oxide synthesis. Using the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH), we studied the role of arginase in airway remodelling using a guinea pig model of chronic asthma. Ovalbumin-sensitised guinea pigs were treated with ABH or PBS via inhalation before each of 12 weekly allergen or saline challenges, and indices of arginase activity, and airway remodelling, inflammation and responsiveness were studied 24 h after the final challenge. Pulmonary arginase activity of repeatedly allergen-challenged guinea pigs was increased. Allergen challenge also increased ASM mass and maximal contraction of denuded tracheal rings, which were prevented by ABH. ABH also attenuated allergen-induced pulmonary hydroxyproline (fibrosis) and putrescine, mucus gland hypertrophy, goblet cell hyperplasia, airway eosinophilia and interleukin-13, whereas an increased l-ornithine/l-citrulline ratio in the lung was normalised. Moreover, allergen-induced hyperresponsiveness of perfused tracheae was fully abrogated by ABH. These findings demonstrate that arginase is prominently involved in allergen-induced airway remodelling, inflammation and hyperresponsiveness in chronic asthma.

The isoelectric point, a key to understanding a variety of biochemical problems: a minireview.

We address the importance of the isoelectric point (IEP) of proteins and membrane components such as phospholipids for our understanding and interpretation of isoforms and opposite charge interactions in the formation of complexes. Five examples drawn from the literature are newly approached from the IEP point of view to clarify general principles.

The vitamin D receptor and its ligand 1alpha,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar).

Seaward migration of Salmo salar is preceded by preparatory physiological adaptations (parr-smolt transformation) to allow for a switch from freshwater (FW) to seawater (SW), which also means a switch in ambient calcium from hypocalcic (<1 mM Ca(2+)) to the plasma (~1.25 mM Ca(2+)) and to strongly hypercalcic (8-12 mM Ca(2+)). Uptake, storage (skeleton, scales) and excretion of calcium need careful regulation. In fish, the vitamin D endocrine system plays a rather enigmatic role in calcium physiology. Here, we give direct evidence for calcitriol involvement in SW migration. We report the full sequence of the nuclear vitamin D receptor (sVDR0) and two alternatively spliced variants resulting from intron retention (sVDR1 and sVDR2). In FW parr, SW adapting smolts, and in SW adults, plasma concentrations of 25(OH)D(3) and 24,25(OH)(2)D(3) did not change significantly. Plasma calcitriol concentrations were lowest in FW parr, doubled during smoltification and remained elevated in SW adults. Increased calcitriol coincided with a twofold decrease in sVDR mRNA levels in gill, intestine, and kidney of FW smolts and SW adults, when compared with parr. Clearly, there was a negative feedback and dynamic response of the vitamin D endocrine system during parr-smolt transformation. The onset of these dynamic changes in FW parr warrants a further search for the endocrines that initiate these changes. We speculate that the vitamin D system plays a crucial role in calcium and phosphorus handling in Atlantic salmon.

Parathyroid hormone-related protein and calcium regulation in vitamin D-deficient sea bream (Sparus auratus).

Gilthead sea bream (Sparus auratus L.) were fed a vitamin D-deficient diet for 22 weeks. Growth rate, whole body mineral pools and calcium balance were determined. Plasma parathyroid hormone-related protein (PTHrP) and calcitriol levels were assessed. Expression of mRNA for pthrp and pth1r was quantified in gills and hypophysis. Fish on vitamin D-deficient diet (D- fish) showed reduced growth and lower calcium turnover (calcium influx, efflux and accumulation rates decreased) and unaltered plasma calcium levels. Plasma calcitriol levels became undetectable, PTHrP levels decreased in the D- fish. In controls, a significant increase in plasma PTHrP level over time was seen, i.e. it increased with body mass. Relationships were found between plasma PTHrP and the whole body pools of calcium, phosphorus and magnesium, indicative of a role for PTHrP in bone development. Expression of pthrp and pth1r mRNA was down-regulated in the hypophysis of D-fish, whereas in gill tissue, pthrp and pth1r mRNA were up-regulated. We conclude that lower pthrp mRNA expression and plasma values in D- fish reflect lower turnover of PTHrP under conditions of hampered growth; up-regulation of pthrp mRNA in gills indicate compensatory paracrine activity of PTHrP during calcitriol deficiency to guarantee well-regulated branchial calcium uptake. This is the first report to document a relation between PTHrP and calcitriol in fish.

α-MSH and melanocortin receptors at early ontogeny in European sea bass (Dicentrarchus labrax, L.).

Temporal patterns of whole-body α-MSH concentrations and of transcripts of melanocortin receptors during early development as well as the endocrine response (α-MSH, cortisol, MCR mRNAs) to stress at the end of the larval period were characterized in Dicentrarchus labrax. Immunohistochemistry showed α-MSH positive cells in the pituitary pars intermedia in all stages examined. As development proceeds, α-MSH content gradually increases; mRNA levels of mc2r and mc4r remain low until first feeding where peak values are observed. Mc1r expression was constant during development, pomc mRNA levels remain low until the stage of flexion after which a significant increase is observed. At the stage of the formation of all fins, whole-body cortisol and α-MSH concentrations responded with peak values at 2 h post stress. Additionally, the stress challenge resulted in elevated transcript levels of pomc, mc2r and mc4r but not in mc1r, with a pattern characterized by peak values at 1 h post stress and a strong correlation with whole body α-MSH concentrations was found. Our data provide for the first time a view on the importance of the α-MSH stress response in early development of European sea bass, an additional and relatively poorly understood signal involved in the stress response in teleosts.

Cloning and sequence analysis of a hypothalamic cDNA encoding a D1c dopamine receptor in tilapia.

Physiological and pharmacological studies have indicated that during acid stress a D1-like dopamine receptor becomes functional on intermediate pituitary melanocyte-stimulating hormone cells of tilapia (Oreochromis mossambicus). As a first step towards physiological expression studies we isolated a D1-like dopamine receptor from a tilapia hypothalamus cDNA library. Construction of a phylogenetic tree of most of the D1-like receptors known in human, rat, Xenopus, goldfish and Drosophila revealed that the here presented clone is most likely the tilapia equivalent of the Xenopus D1c dopamine receptor.

Measurement of PTHrP, PTHR1, and CaSR expression levels in tissues of sea bream (Sparus aurata) using quantitative PCR.

A quantitative PCR (Q-PCR) method has been established to measure the mRNA expression levels of parathyroid hormone-related protein (PTHrP), parathyroid hormone receptor type 1 (PTHR1), and calcium-sensing receptor (CaSR) in sea bream (Sparus aurata), using the housekeeping gene, beta-actin, as endogenous control. TaqMan primers and probes were designed using the Primer Express program, according to the published/unpublished sequences of the three target genes and beta-actin of sea bream. Different tissues including gill, kidney, duodenum, hindgut, rectum, liver, heart, brain, pituitary, skin, muscle, and gonad were removed and immediately snap-frozen from three juvenile sea bream (100-150 g) cultured in sea water. The mRNAs were extracted and reverse-transcribed into cDNAs, which were subsequently examined by the ABI 5700 system using an optimized Q-PCR method. Triplicate measures of each sample indicated consistency of the technique. However, the mRNA expression levels for each transcript in these tissues were variable between fish and also relatively low. Nevertheless, this methodology can be used in the future studies of factors that may alter gene expression in these tissues.

Differences in inhibitory avoidance, cortisol and brain gene expression in TL and AB zebrafish.

Recently, we established an inhibitory avoidance paradigm in Tupfel Long-Fin (TL) zebrafish. Here, we compared task performance of TL fish and fish from the AB strain; another widely used strain and shown to differ genetically and behaviourally from TL fish. Whole-body cortisol and telencephalic gene expression related to stress, anxiety and fear were measured before and 2 h post-task. Inhibitory avoidance was assessed in a 3-day paradigm: fish learn to avoid swimming from a white to a black compartment where a 3V-shock is given: day 1 (first shock), day 2 (second shock) and day 3 (no shock, sampling). Tupfel Long-Fin fish rapidly learned to avoid the black compartment and showed an increase in avoidance-related spatial behaviour in the white compartment across days. In contrast, AB fish showed no inhibitory avoidance learning. AB fish had higher basal cortisol levels and expression levels of stress-axis related genes than TL fish. Tupfel Long-Fin fish showed post-task learning-related changes in cortisol and gene expression levels, but these responses were not seen in AB fish. We conclude that AB fish show higher cortisol levels and no inhibitory avoidance than TL fish. The differential learning responses of these Danio strains may unmask genetically defined risks for stress-related disorders.

The effect of chronic apomorphine treatment on the ultrastructure of the prolactin cells and on plasma prolactin levels in young and aged male Wistar rats.

Effects of two doses of apomorphine on the plasma prolactin (PRL) levels and on the ultrastructure of PRL cells in young and aged male Wistar rats were investigated. In young and aged control rats no significant differences were found between the plasma PRL levels. Immunocytochemical staining with anti-r-PRL revealed significant differences between young and aged control rats; in young rats the number of PRL cells with polymorphic granules exceeded the number of cells with round granules, whereas in aged rats almost exclusively cells with round granules were found. In young rats, chronic treatment with a low dose (0.01 mg/kg/day) of apomorphine did not result in a significant change in plasma PRL level or cell morphology. However, high dose (0.25 mg/kg/day) of apomorphine resulted in a significant decrease in plasma PRL levels, a decrease of number of cells with polymorphic granules and an increase of cells with round granules. The occurrence of PRL cells with round granules and plasma levels was negatively correlated. In aged rats, apomorphine (0.01 or 0.25 mg/kg/day) treatment did not affect plasma levels nor did it affect the distribution of the cell types. We conclude that in young rats PRL cells are sensitive to apomorphine and that their ultrastructure reflects a phase of the secretory cycle. In aged rats, the cells appear to have lost their sensitivity to apomorphine. The fact, that the distribution over the different cell types in control aged rats is similar to that of the apomorphine-treated young rats, suggests a strong influence of endogenous dopamine on PRL cell physiology in the aged rat.

Structural characterisation of a cyprinid (Cyprinus carpio L.) CRH, CRH-BP and CRH-R1, and the role of these proteins in the acute stress response.

We elucidated the structure of the principle factors regulating the initiation of the acute stress response in common carp: corticotrophin-releasing hormone (CRH), CRH-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP). Phylogenetic analyses reveal that these proteins are evolutionarily well conserved in vertebrates. CRH and CRH-BP expression are not co-localised in the same hypothalamic perikarya. On the contrary, CRH-BP expression is limited to the perimeter of the nucleus preopticus (NPO), but is abundant in other regions, including an area directly rostral from, and in close proximity to, the NPO. Despite the lack of co-expression, the nerve fibres projecting onto both the rostral pars distalis (rPD) as well as the large fibre bundles projecting onto the pars intermedia (PI) contain CRH as well as CRH-BP, suggesting that both ACTH release from the rPD as well as the release of PI melanotrope content is regulated via CRH and CRH-BP. Finally, we show via real-time quantitative PCR that expression of hypothalamic CRH and CRH-BP following a 24 h restraint significantly increases, whereas PD CRH-R1 expression decreases; this reflects desensitisation of the PD for hypothalamic CRH output. We conclude that these factors are actively involved in the regulation of acute stress responses in the teleost fish.

CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.

An algorithm is presented for the calculation of metal ion concentrations from given total metal concentrations (and vice versa) in physiological media containing metal-chelating compounds. In such media, conditions differ from those used for stability constant determination of metal-chelator equilibria; therefore calculated metal ion concentrations are incorrect. We recompute stability constants to reflect the effects of ionic strength and temperature of physiological solutions. Twelve different equilibria can be considered per metal-chelator pair. The computer program also calculates the contribution of ionized species of metals, chelator, complexes and pH buffers to ionic strength. Measurements with a Ca-selective electrode and with fura-2 show that calculated ionic Ca2+ concentrations are correct from 10 nM up to the millimolar range. The importance of the correct calculation of metal ion concentrations in physiological experiments is demonstrated by data, and derived kinetic parameters, on Na+/Ca2+ exchange and the ATP-dependent Ca2+ pump of enterocyte plasma membrane vesicles. The program is written in Turbo Pascal and will run on IBM-compatible computers. It is menu-driven and supports the use of a Microsoft mouse.

Cortisol inhibits apoptosis in carp neutrophilic granulocytes.

The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding studies with radioactive cortisol, a single class of glucocorticoid receptors was detected with high affinity (Kd = 2.6 nM) and low capacity (497 receptors/cell) for cortisol binding. Both in vitro and in vivo cortisol treatment did not affect neutrophil respiratory burst activity. These data indicate that cortisol can augment the supply of functional neutrophilic granulocytes in conditions of acute stress, which may be essential for survival, since phagocytes form the first line of defence against micro-organisms.

Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L..

In mammalian T and B cells glucocorticoids (GS) regulate development and selection through induction of apoptosis; more recently GS-induced apoptosis has also been implicated in the removal of circulating, activated T and B cells following an immune response. In an earlier report we have given the first evidence for cortisol-induced apoptosis as an immune regulator in an aquatic vertebrate, the common carp. Here we report on subpopulation-specific sensitivity of carp peripheral blood leukocytes (PBL) to cortisol-induced apoptosis. B cells, the most abundant leukocyte subpopulation in fish blood, are sensitised to cortisol-induced apoptosis by activation with the mitogens LPS or PHA. Cortisol-induced apoptosis in B cells is receptor mediated as it is blocked by the synthetic GS receptor blocker RU486. In contrast to what is known for mammalian lymphocytes, apoptosis in carp T cells is hardly affected by cortisol, both in unstimulated and in PHA-stimulated cell cultures. A culture supernatant of PHA-prestimulated PBL, containing IL-2-like activity, decreased spontaneous apoptosis in both T and B cells, but did not affect cortisol-induced apoptosis in B cells. Apoptosis in thrombocytes was unaffected by either mitogens, cortisol, or lymphocyte supernatant. The difference between mammalian and fish leukocyte sensitivity to cortisol is discussed in the light of differences in the immune response of mammals and fish.