Molecular characterisation and expression of parathyroid hormone-related protein in the caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

Parathyroid hormone-related protein (PTHrP) is a hypercalcemic factor in fish, but the source of circulating PTHrP remains unclear. In this study investigation of the caudal neurosecretory system (CNSS), considered one of major sources of PTHrP in fish, provided valuable insights into this regulatory system. We report pthrpa and pthrpb gene cloning, characterization, expression, and responses to low salinity and hypocalcemia challenge in flounder. The pthrpa and pthrpb precursors, isolated from a European flounder CNSS library, consist of 166 and 192 amino acid residues, respectively, with an overall homology of approximately 59.2%. Both precursors contain a signal peptide and a mature peptide with cleavage and amidation sites. The flounder PTHrPA and PTHrPB peptides share only 41% sequence identity with human PTHrPA. Quantitative PCR analysis demonstrated that the bone and bladder, are respectively major sites of pthrpa and pthrpb expression in flounder. Urophysectomy confirmed the CNSS as a likely contributor to circulating PTHrP peptides. There were no significant differences in CNSS pthrpa and pthrpb mRNA expression or plasma PTHrP levels between seawater (SW) and freshwater (FW)-adapted fish, though plasma total calcium concentrations were higher in FW animals. The intraperitonial administration of EGTA rapidly induced hypocalcemia and concomitant elevation in plasma PTHrP accompanied by increases in both pthrpa and pthrpb expression in the CNSS. Together, these findings support an evolutionary conserved role for PTHrP in the endocrine regulation of calcium.

Molecular Characterization and Expression of α-Globin and ß-Globin Genes in the Euryhaline Flounder (Platichthys flesus).

In order to understand the possible role of globin genes in fish salinity adaptation, we report the molecular characterization and expression of all four subunits of haemoglobin, and their response to salinity challenge in flounder. The entire open reading frames of α1-globin and α2-globin genes were 432 and 435 bp long, respectively, whereas the ß1-globin and ß2-globin genes were both 447 bp. Although the head kidney (pronephros) is the predicted major site of haematopoiesis, real-time PCR revealed that expression of α-globin and ß-globin in kidney (mesonephros) was 1.5 times higher than in head kidney. Notably, the α1-globin and ß1-globin mRNA expression was higher than α2-globin and ß2-globin in kidney. Expression levels of all four globin subunits were higher in freshwater- (FW-) than in seawater- (SW-)adapted fish kidney. If globins do play a role in salinity adaptation, this is likely to be more important in combating the hemodilution faced by fish in FW than the dehydration and salt loading which occur in SW.

Potent cardiovascular effects of homologous urotensin II (UII)-related peptide and UII in unanesthetized eels after peripheral and central injections.

We cloned cDNAs encoding urotensin II (UII)-related peptide (URP) and UII in Japanese eel, Anguilla japonica, the former being the first such cloning in teleost fishes. Unlike the exclusive expression of UII in the urophysis, the URP gene was expressed most abundantly in the brain (medulla oblongata) followed by the urophysis. Peripheral injections of URP into eels increased blood pressure by 16.1 ± 0.8 mmHg at 0.1 nmol/kg in ventral aortic blood pressure (P(VA)) and with similar potency and efficacy to that of UII (relative potency of URP to UII = 0.83). URP/UII and ANG II preferentially acted on the branchial and systemic circulations, respectively, and the duration of effect was distinct among the three peptides in the order of UII (60 min) >URP (30 min) >ANG II (14 min) in P(VA). Urantide, a mammalian UII receptor antagonist, inhibited the URP effect (-63.6 ± 5.2%) to a greater extent than for UII (-39.9 ± 5.0%). URP and UII constricted isolated eel branchial and systemic arteries, showing their direct actions on the vascular smooth muscle. Central injection of URP increased blood pressure by 12.3 ± 0.8 mmHg at 50 pmol/eel in P(VA) and with similar efficacy but less potency (relative potency = 0.47) and shorter duration compared with UII. The central actions of URP/UII were more potent on the branchial circulation than on the systemic circulation, again opposite the effects of ANG II. The similar responses to peripheral and central injections suggest that peripheral hormones may act on the brain. Taken together, in eels, URP and UII are potent cardiovascular hormones like ANG II, acting directly on the peripheral vasculature, as well as a central vasomotor site, and their actions are mediated to different degrees by the UII receptor.

The corpuscles of Stannius, calcium-sensing receptor, and stanniocalcin: responses to calcimimetics and physiological challenges.

This study has examined whether the calcium-sensing receptor (CaSR) plays a role in control of stanniocalcin-1 (STC-1), the dominant calcium regulatory hormone of fish, comparable with that demonstrated for CaSR in the mediation of ionized calcium regulation of PTH secretion in mammals. In a previous study, we have cloned flounder STC-1 from the corpuscles of Stannius (CS). Here, we report the cloning and characterization of the CS CaSR, and the in vivo responses of this system to altered salinity, EGTA induced hypocalcemia, and calcimimetic administration. Quantitative PCR analysis demonstrated, for the first time, that the CS are major sites of CaSR expression in flounder. Immunoblot analysis of CS proteins with CaSR-specific antibodies revealed a broad band of approximately 215-300 kDa under nonreducing conditions, and bands of approximately 215-300 kDa and approximately 120-150 kDa under reducing conditions. There were no differences in CS CaSR mRNA expression or plasma STC-1 levels between seawater and freshwater (FW)-adapted fish, although CS STC-1 mRNA expression was lower in FW animals. Immunoblots showed that glycosylated monomeric forms of the CaSR migrated at a lower molecular mass in CS samples from FW animals. The ip administration of EGTA rapidly induced hypocalcemia, and a concomitant lowering of plasma STC-1. Calcimimetic administration (1 mg/kg R-568) rapidly increased plasma STC-1 levels, and reduced plasma concentrations of calcium, phosphate, and magnesium when compared with S-568-treated controls. Together, these findings support an evolutionary conserved role for the CaSR in the endocrine regulation of calcium before the appearance of parathyroid glands in tetrapods.

Enhanced renal sensitivity of the spontaneously hypertensive rat to urotensin II.

Urotensin II (UII) has been implicated widely in cardiovascular disease. The mechanism(s) through which it contributes to elevated blood pressure is unknown, but its emerging role as a regulator of mammalian renal function suggests that the kidney might be involved. The aim of this study was to determine the effect of UII on renal function in the spontaneously hypertensive rat (SHR). UII infusion (6 pmol.min(-1).100 g body wt(-1)) in anesthetized SHR and control Wistar-Kyoto (WKY) rats produced marked reductions in glomerular filtration rate (DeltaGFR WKY, n=7, -0.3+/-0.1 vs. SHR, n=7, -0.6+/-0.1 ml.min(-1).100 g body wt(-1), P=0.03), urine flow, and sodium excretion rates, which were greater in SHR by comparison with WKY rats. WKY rats also showed an increase in fractional excretion of sodium (DeltaFE(Na); +0.6+/-0.1%, P=0.02) in contrast to SHR in which no such change was observed (DeltaFE(Na) -0.6+/-0.2%). Blockade of the UII receptor (UT), and thus endogenous UII activity, with urantide evoked an increase in GFR which was greater in SHR (+0.3+/-0.1) compared with WKY rats (+0.1+/-0.1 ml.min(-1).100 g body wt(-1), P=0.04) and was accompanied by a diuresis and natriuresis. UII and UT mRNA expression were greater in the renal medulla than the cortex of both strains; however, expression levels were up to threefold higher in SHR tissue. SHR are more sensitive than WKY to UII, which acts primarily to lower GFR thus favoring salt retention in this model of hypertension.

Renal haemodynamic and tubular actions of urotensin II in the rat.

Urotensin II (UTS) is a potent vasoactive peptide that was originally identified in teleost fish. Mammalian orthologues of UTS and its receptor (UTSR) have been described in several species, including humans and rats. We have shown previously that bolus injections of UTS caused a decrease in urine flow and sodium excretion rates in parallel with marked reductions in renal blood flow (RBF) and glomerular filtration rate (GFR). The aim of this study was to determine the effect of UTS infusion at a dose that has minimal impact upon renal haemodynamics in order to identify a potential direct tubular action of UTS. Infusion of rat UTS (rUTS) at 0.6 pmol/min per 100 g body weight in male Sprague-Dawley rats, which had no effect on RBF and caused a 30% reduction in GFR, resulted in a significant increase in the fractional excretion of sodium (vehicle 2.3+/-0.6 versus rUTS 0.6 pmol 4.5+/-0.6%, P<0.05) and potassium. At the higher dose of 6 pmol/min per 100 g body weight, haemodynamic effects dominated the response. rUTS induced a marked reduction in RBF and GFR (vehicle 1.03+/-0.06 versus rUTS 6 pmol 0.31+/-0.05 ml/min per 100 g body weight, P<0.05) resulting in an anti-diuresis and anti-natriuresis, but no change in fractional excretion of sodium or potassium. Uts2d and Uts2r mRNA expression were greater in the renal medulla compared with the cortex. Together, these data support an inhibitory action of Uts2d on renal tubule sodium and potassium reabsorption in the rat, in addition to its previously described renal haemodynamic effects.

Cortisol and prolactin modulation of caudal neurosecretory system activity in the euryhaline flounder Platichthys flesus.

Previous studies have shown roles for cortisol and prolactin in osmoregulatory adaptation to seawater and freshwater, respectively, in euryhaline fish. This study of the European flounder investigated the potential for these hormones to modulate activity of the caudal neurosecretory system (CNSS), which is thought to be involved in physiological adaptation to changing external salinity. Superfusion of isolated CNSS with either cortisol or prolactin (10 microM; 15 min) led to changes in firing activity in neuroendocrine Dahlgren cells, recorded extracellularly. Cortisol evoked a modest increase in overall firing activity, with the response delayed by 4 h after treatment. The response to prolactin was short latency, continued to build up over the subsequent 4-h wash period, and comprised increased firing activity together with recruitment of previously silent Dahlgren cells. Immunoreactivity for glucocorticoid and prolactin receptors was localised to Dahlgren cells. The CNSS expression level for glucocorticoid-2 receptor mRNA, measured by Q-PCR, was significantly lower in fish fully acclimated to freshwater, compared to seawater. No differences were seen between these two states for prolactin receptor mRNA expression. These results provide evidence for a modulatory action of both hormones on the neurosecretory function of the CNSS.

Seasonal changes in peptide, receptor and ion channel mRNA expression in the caudal neurosecretory system of the European flounder (Platichthys flesus).

The caudal neurosecretory system (CNSS) of the euryhaline flounder Platichthys flesus has suggested roles in osmoregulatory, reproductive and nutritional adaptation, as fish migrate between seawater (winter) and brackish/freshwater (summer) environments. This study examined seasonal changes in mRNA expression profile of functionally important genes in the CNSS. cDNAs encoding neuropeptides, receptors and ion channels were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and screening of a flounder CNSS cDNA library. The expression profile of cloned genes was determined by real-time RT-PCR at 2-month intervals throughout the year in CNSS from seawater-adapted fish. Plasma cortisol (measured by radioimmunoassay) showed a peak in April, the time of spawning. Expression levels of mRNA for peptides urotensins I and II (UI, UII) and corticotropin releasing factor (CRF) all showed a seasonal cycle, with lowest expression in April and highest in August-October. The expression of CRF2(UI), UT(UII) and CRF1 receptors was not correlated with corresponding peptide expression. Receptors for potential neuromodulators of CNSS activity also displayed a seasonal mRNA expression profile. Glucocorticoid, 5-hydroxytryptamine, kappa-opioid and glutamate receptor expression peaked around April, suggesting that modulation of electrical activity of the neurosecretory Dahlgren cells is of particular importance at this time. Expression of mRNA for L-type Ca(2+) and Ca-activated K(+) channels was lower during the summer months. These channels underlie electrical bursting activity in Dahlgren cells. Ion channel mRNA expression was also lower in CNSS from flounder fully adapted to freshwater as opposed to seawater, consistent with previously reported observations of reduced bursting activity in Dahlgren cells from freshwater-adapted CNSS. These findings support the hypothesis that the CNSS is functionally reprogrammed to cope with changes in physiological challenge as fish migrate between sea and estuaries in winter and spring.

Evidence for nitric oxide role in the caudal neurosecretory system of the European flounder, Platichthys flesus.

A neuromodulatory role for nitric oxide has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined its potential as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder. Immunocytochemistry using an antibody raised against human neuronal nitric oxide synthase (NOS) indicated the presence of NOS in the Dahlgren cells. Quantitative RT-PCR, using a flounder-specific probe, revealed NOS mRNA expression in the CNSS. In July, though not in September, NOS mRNA expression was significantly higher in fish fully adapted to seawater, compared to freshwater-adapted fish. Following acute transfer of fish from freshwater to seawater, NOS mRNA expression was elevated at 8h and then recovered by 24h. In pharmacological experiments in vitro, application of NO donors (SNAP, SNP) caused an increase in electrical activity (firing frequency) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The NOS substrate, l-arginine, led to increased firing frequency, cell recruitment and enhanced bursting activity. However, this effect was not blocked by the NOS inhibitor L-NAME. These findings suggest that NO acts as a modulator within the CNSS, potentially enhancing electrical activity and hence secretory output. A role in supporting adaptation to hyperosmotic conditions is also indicated.

Fish caudal neurosecretory system: a model for the study of neuroendocrine secretion.

The caudal neurosecretory system (CNSS) is unique to fish and has suggested homeostatic roles in osmoregulation and reproduction. Magnocellular neuroendocrine Dahlgren cells, located in the terminal segments of the spinal cord, project to a neurohaemal organ, the urophysis, from which neuropeptides are released. In the euryhaline flounder Platichthys flesus Dahlgren cells synthesise at least four peptides, including urotensins I and II and CRF. These peptides are differentially expressed with co-localisation of up to three in a single cell. Dahlgren cells display a range of electrical firing patterns, including characteristic bursting activity, which is dependent on L-type Ca(2+) and Ca-activated K(+)channels. Activity is modulated by a range of extrinsic and intrinsic neuromodulators. This includes autoregulation by the secreted peptides themselves, leading to enhanced bursting. Electrophysiological and mRNA expression studies have examined changes in response to altered physiological demands. Bursting activity is more robust and more Dahlgren cells are recruited in seawater compared to freshwater adapted fish and this is mirrored by a reduction in mRNA expression for L-type Ca(2+) and Ca-activated K(+) channels. Acute seawater/freshwater transfer experiments support a role for UII in adaptation to hyperosmotic conditions. Responses to stress suggest a shared role for CRF and UI, released from the CNSS. We hypothesise that the Dahlgren cell population is reprogrammed, both in anticipation of and in response to changed physiological demands, and this is seen as changes in gene expression profile and electrical activity. The CNSS shows striking parallels with the hypothalamic-neurohypophysial system, providing a highly accessible system for studies of neuroendocrine mechanisms. Furthermore, the presence of homologues of urotensins throughout the vertebrates has sparked new interest in these peptides and their functional evolution.

Molecular characterization and expression of urotensin II and its receptor in the flounder (Platichthys flesus): a hormone system supporting body fluid homeostasis in euryhaline fish.

Urotensin II (UII) is a potent vasoconstrictor in mammals, but the source of circulating UII remains unclear. Investigations of the caudal neurosecretory system (CNSS), considered the major source of UII in fish, alongside target tissue expression of UII receptor (UT), can provide valuable insights into this highly conserved regulatory system. We report UII gene characterization, expression of the first fish UT, and responses to salinity challenge in flounder. The 12-aa UII peptide shares 73% sequence identity with pig and human UII. Flounder UT receptor shares 56.7% identity with rat. Although the CNSS is the major site of UII expression, RT-PCR revealed expression of UII and UT in all tissues tested. Around 30-40% of large CNSS Dahlgren cells expressed UII, alone or in combination with urotensin I and/or corticotrophin releasing hormone. Immunolocalization of UT in osmoregulatory tissues (gill, kidney) was associated with vascular elements. There were no consistent differences in CNSS UII expression or plasma UII between seawater (SW)- and freshwater (FW)-adapted fish, although gill and kidney UT expression was lower in FW animals. After acute transfer from SW to FW, plasma UII and kidney and gill UT expression were reduced, whereas UT expression in kidney was increased after reverse transfer. UII appears to be more important to combat dehydration and salt-loading in SW than the hemodilution faced in FW. Potentially, altered target tissue sensitivity through changes in UT expression, is an important physiological controlling mechanism, not only relevant for migratory fish but also likely conserved in mammals.

Day-night specific binding of 2-[125I]iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species.

Some of melatonin's (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[(125)I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K (d)) and maximal binding densities (B (max)) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K (d) values in the tissues in the picomolar range indicated the high affinity. K (d) and B (max) values were tissue- and species-dependent. The GTP analogue [Guanosine 5'-O-(3-thiotriphosphate)] treatment significantly reduced the B (max) value, indicating that the 2-[(125)I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K (d) and B (max) values were observed. These are the first studies of the presence of 2-[(125)I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.

Increased renal tubular reabsorption of calcium and magnesium by the offspring of diabetic rat pregnancy.

Diabetic pregnancy has a marked influence on offspring calcium and magnesium homeostasis. Urinary excretion of calcium and magnesium is reduced, yet offspring of diabetic pregnancy exhibit hypomagnesemia and hypocalcemia. The aim of this study was to measure renal hemodynamic and tubular function in the offspring of diabetic (OD) and control, nondiabetic (OC) rats at 4 and 8 wk of age to determine the glomerular and tubular mechanisms through which renal calcium and magnesium handling are programmed in utero. The fraction of filtered calcium that was excreted was significantly lower in OD at both 4 and 8 wk of age [8 wk: OC (n = 6), 11.8 +/- 2.9 versus OD (n = 5), 4.3 +/- 0.6%; p < 0.05] and that of magnesium was lower at 8 wk of age [OC (n = 6), 42.4 +/- 7.5 versus OD (n = 5), 13.0 +/- 1.7%; p < 0.01]. This increased reabsorption occurred despite an elevated GFR in OD. These findings clearly indicate that tubular reabsorptive mechanisms for calcium and magnesium are increased markedly in OD. Serum PTH concentration was reduced in 8-wk-old OD [OC (n = 7), 539.4 +/- 142.1 versus OD (n = 9), 174.3 +/- 69.4 pg/ml; p < 0.05], consistent with previous reports in human infants. Taken together, these observations suggest that the basis for the altered renal magnesium and calcium handling in OD involves increased tubular transport activity and possibly increased sensitivity of these mechanisms to PTH.

Rapid natriuretic action of aldosterone in the rat.

Rapid, nongenomic actions of aldosterone have been demonstrated in a number of cell types in vitro, including renal cell lines, but there remains little direct evidence that it is able to exert rapid effects on the kidney in the whole animal. Accordingly, the aim of this study was to determine whether aldosterone induces rapid changes in the renal handling of electrolytes or acid-base balance in the anesthetized rat. With the use of a servo-controlled fluid replacement system, spontaneous urine output by anesthetized male Sprague-Dawley rats was replaced with 2.5% dextrose. After a 3-h equilibration and a 1-h control period, rats were infused with aldosterone (42 pmol/min) or vehicle for 1 h. Aldosterone infusion induced a rapid (within 15 min) increase in sodium excretion that peaked at 0.24 +/- 0.08 compared with 0.04 +/- 0.01 micromol x min(-1) 100 x body weight(-1) (P = 0.041) in the vehicle-infused rats. This natriuresis was not associated with changes in glomerular filtration rate; urine flow rate; potassium, chloride, or bicarbonate excretion; or urine pH. The mechanisms involved are unclear, but because we have previously shown that aldosterone stimulates a rapid (4 min) increase in cAMP generation in the rat inner medullary collecting duct (IMCD) (Sheader EA, Wargent ET, Ashton N, and Balment RJ. J Endocrinol 175: 343-347, 2002), they could involve cAMP-mediated activation of the cystic fibrosis transmembrane conductance regulator chloride channel, which drives sodium secretion in the IMCD.

Biochemical characterization and immunohistochemical localization of urotensin II in the human brainstem and spinal cord.

The human urotensin II (UII) precursor encompasses several potential cleavage sites and thus, processing of pro-UII may generate various forms of mature UII including the peptides of 11 (UII11), 16 (UII16) and 19 (UII19) residues. Until now, the native form of human UII had not been characterized. Here, we show that the major UII peptide occurring in the human spinal cord corresponds to UII11. In contrast, neither the UII16 nor the UII19 forms could be detected. In 50% of the brainstem and in all the spinal cord extracts analysed, a second minor UII-immunoreactive peptide was resolved. Immunohistochemical labelling of the cervical segment of the human spinal cord revealed that the UII-immunoreactive material was confined to a subset of ventral horn motoneurones. These data provide the first evidence that in the human, the UII precursor, expressed in motoneurones, is processed at the tribasic KKR93 cleavage site to generate a mature form of UII of 11 amino acids. The absence of N-terminally elongated forms of UII of 16 and 19 residues indicates that pro-UII is not cleaved at the R85 or K88 monobasic sites. Finally, the minor UII-immunoreactive peptide detected in several tissue extracts might correspond to an extended form of UII resulting from the processing of the UII precursor at the basic RK50 or RK66 doublets.

Coexpression of corticotropin-releasing hormone and urotensin i precursor genes in the caudal neurosecretory system of the euryhaline flounder (Platichthys flesus): a possible shared role in peripheral regulation.

CRH and urotensin I (UI) are neuroendocrine peptides that belong to the superfamily of corticotropin-releasing factors. In mammals, these peptides regulate the stress response and other central nervous system functions, whereas in fish an involvement for UI in osmoregulation has also been suggested. We have identified, characterized, and localized the genes encoding these peptides in a unique fish neuroendocrine organ, the caudal neurosecretory system (CNSS). The CRH and UI precursors, isolated from a European flounder CNSS library, consist of 168 and 147 amino acid residues, respectively, with an overall homology of approximately 50%. Both precursors contain a signal peptide, a divergent cryptic region and a 41-amino acid mature peptide with cleavage and amidation sites. Genomic organization showed that whole CRH and UI coding sequences are contained in a single exon. Northern blot analysis and quantitative PCR of a range of tissues confirmed the CNSS as a major site of expression of both CRH and UI and thus serves as a likely source of circulating peptides. In situ hybridization demonstrated that CRH and UI colocalize to the same cells of the CNSS. Our findings suggest that, in euryhaline fish, the CNSS is a major site of production of CRH and probably contributes to the high circulating levels observed in response to specific environmental challenges. Furthermore, the localization of CRH and UI within the same cell population suggests an early, possibly shared role for these peptides in controlling stress-mediated adaptive plasticity.

Renal action of acute chloroquine and paracetamol administration in the anesthetized, fluid-balanced rat.

Chloroquine induces diuresis, natriuresis, and an increase in glomerular filtration rate (GFR) in the rat. These responses are modified in rats with analgesic nephropathy induced by long-term paracetamol (acetaminophen) administration. Here, the effects of acute paracetamol treatment on renal function and the response to chloroquine are reported. Under intraval anesthesia (100 mg kg-1) male Sprague-Dawley rats (n = 6/group) were infused with 2.5% dextrose for 3 h. After a control hour, they received either vehicle, chloroquine (0.04 mg h-1), paracetamol (priming dose of 210 mg kg-1 followed by 110 mg kg-1h-1) or chloroquine and paracetamol over the next hour. Compared with vehicle, chloroquine infusion resulted in increases in GFR (2.4 +/- 0.3 versus 4.8 +/- 0.6 ml min-1), urine flow (4.2 +/- 0.3 versus 10.4 +/- 0.7 ml h-1), and sodium excretion (47.7 +/- 4.1 versus 171.2 +/- 18.6 micromol h-1) and a reduction in urine osmolality (223.2 +/- 5.9 versus 121.7 +/- 23.9 mOsM kg-1). Paracetamol reduced sodium excretion but had no effect on urine flow, GFR, or urine osmolality. When combined, paracetamol blocked the chloroquine-induced diuresis (3.9 +/- 0.7 ml h-1) and natriuresis (22.6 +/- 8.5 micromol h-1), attenuated the increase in glomerular filtration rate (3.5 +/- 0.2 ml min-1), and raised urine osmolality (280.0 +/- 22.8 mOsM kg-1). The differing effects of acute and long-term paracetamol treatment on basal and chloroquine-mediated renal function suggest that the length of prior exposure to paracetamol, and thus the presence of analgesic nephropathy, is an important determinant of the renal response to chloroquine.

Renal function in a rat model of analgesic nephropathy: effect of chloroquine.

The antimalaria drug chloroquine is often taken against a background of analgesic nephropathy caused by nonsteroidal anti-inflammatory drugs such as paracetamol (acetaminophen). Chloroquine has marked effects on the normal kidney and stimulates an increase in plasma vasopressin via nitric oxide. The aim of this study was to determine the renal action of chloroquine in a model of analgesic nephropathy. Sprague-Dawley rats (n = 6-8/group) were treated with paracetamol (500 mg kg(-1) day(-1)) for 30 days in drinking water to induce analgesic nephropathy; control rats received normal tap water. Under intraval anesthesia (100 mg kg(-1)) rats were infused with 2.5% dextrose for 3 h to equilibrate and after a control hour they received either vehicle, chloroquine (0.04 mg h(-1)), N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric-oxide synthase inhibitor, 60 micro g kg(-1) h(-1)) or combined chloroquine and L-NAME over the next hour. Plasma was collected from a parallel group of animals for vasopressin radioimmunoassay. Long-term paracetamol treatment resulted in a decrease in glomerular filtration rate (p < 0.05), sodium excretion (p < 0.001), and urine osmolality (p < 0.001), but no change in urine flow rate compared with untreated animals. Chloroquine administration in paracetamol treated rats induced a significant reduction (p < 0.05) in urine flow rate and a significant increase in plasma vasopressin (p < 0.001). These effects were blocked by coadministration of L-NAME and thus seem to be mediated by a pathway involving nitric oxide. However, these responses contrast with the chloroquine-induced diuresis previously observed in untreated rats, possibly reflecting paracetamol inhibition of renal prostaglandin synthesis and consequent moderation of vasopressin's action.

Isolation, synthesis, and biological activity of flounder [Asn1,Ile5,Thr9] angiotensin I.

A novel angiotensin I (ANG I) has been isolated from incubates of plasma and kidney extracts of the flounder, Platichthys flesus, using ion-exchange, gel-permeation, and reverse-phase high performance liquid chromatography (HPLC). Its sequence was determined as H-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-Thr-Leu-OH by sequence analysis and mass spectrometry. No vasopressor activity was detected at the elution position of [Asp(1)] ANG I in ion-exchange HPLC. The sequence was confirmed by identity of the elution position with the synthetic peptide in two different HPLC systems. When compared with ANG I isolated from other teleost fish, flounder ANG I uniquely has an isoleucine at position 5 rather than valine. Injection of angiotensin II (ANG II) into chronically cannulated flounder resulted in a dose-dependent pressor response, native [Asn(1),Ile(5)] ANG II, was found to elicit pressor responses comparable with those seen when teleost [Asn(1),Val(5)] ANG II and human [Asp(1),Ile(5)] ANG II were injected into flounder over the dose range 0.02-1.00 nmol/kg(-1). Plasma concentrations of the neurohypophysial peptide AVT were measured in chronically cannulated flounder following the injection of ANG II to examine the effect of ANG II on circulating AVT concentration. The injection of [Asn(1),Ile(5)] ANG II (1 nmolkg(-1)) or [Asp(1),Ile(5)] ANG II (2.5 nmolkg(-1)) resulted in a significant fall in the circulating levels of AVT suggesting that ANG II either directly or indirectly negatively influences AVT secretion.