Effect of acute restraint on hypothalamic pro-vasotocin mRNA expression in flounder, Platichthys flesus.

Arginine vasotocin (AVT) stimulates release of adenocorticotrophin hormone (ACTH) in trout. However, AVT's role in fish hypothalamic-pituitary-interrenal-axis (HPIA) is not fully understood. Here, we examined distribution of AVT and glucocorticoid receptor (GR) in the magnocellular preoptic nucleus (PM) and the AVT/cortisol response to acute restraint in flounder. The GR/AVT distribution in the PM was determined using double immunohistochemistry (IHC). Flounder were confined in nets, immersed in water for 30m, with plasma and tissue samples taken prior to, 3, 24 and 48h post-confinement. Plasma osmolality, Na(+), Cl(-) and cortisol were taken as indicators of HPIA activation. Plasma AVT was measured proVT mRNA expression in the PM was detected using in situ hybridisation (ISH) with a S35 labelled oligoprobe for homologous flounder proVT. Double IHC showed the presence of GR in AVT synthesising neurones of the PM. Plasma Na(+), Cl(-), osmolality and cortisol (1.0+/-0.9 to 183.6+/-3.1mM; p<0.001) increased significantly 3h post-restraint: recovering to control levels after 48h. Plasma AVT levels did not change. However, a concomitant increase in proVT mRNA expression in the magnocellular (PMm) and gigantocellular (PMg) neurones of the PM was observed (11.1+/-1.8 to 55.2+/-9.1% 24h post-restraint; p<0.001) and levels still remained significantly elevated at 48h (p<0.01). This suggests that PMm and PMg AVT neurones are associated with HPIA activation following acute restraint, including potential cortisol negative feedback. The extended elevation of hypothalamus proAVT mRNA expression following a single acute stressor affords a possible mechanism to moderate sensitivity of the HPIA to subsequent challenges.

Salinity adaptation and gene profiling analysis in the European eel (Anguilla anguilla) using microarray technology.

The life cycle of the European eel (Anguilla anguilla) includes two long migratory periods, when the newly hatched leptocephali larvae drift on ocean currents from the Sargasso Sea to the shores of Western Europe and then again up to 30 years later when adult eels swim back to their place of birth for reproductive purposes. Prior to the migration from fresh water (FW) to sea water (SW) adult yellow eels undergo various anatomical and physiological adaptations (silvering) which promote sexual development and aid the transition to increased environmental salinities. The aim of this study was to identify and characterise changes in gene expression within the major osmoregulatory tissues of the eel which enable these fish to make the physiological adaptations required for transfer to SW environments. In particular, changes in the expression of the FW-adapting hormone prolactin were correlated with differential expression of known osmoregulatory important genes within the gill, intestine and kidney following the acclimation of eels to SW. Various tissues were sampled from individual fish at selected intervals over a 5-month period following FW/SW transfer and RNA was isolated. Suppressive subtractive hybridization (SSH) was used for enrichment of differentially expressed genes. Microarrays comprising 6144 cDNAs spotted in triplicate, from brain, gill, intestine and kidney libraries (1536 randomly selected clones per tissue library), were hybridized with appropriate targets and analysed. Microarray results were validated using known genes implicated in osmoregulation, such as prolactin, growth hormone, Na, K-ATPase and some unknown genes, the role of which in osmoregulation needs to be elucidated.

Urotensin II: ancient hormone with new functions in vertebrate body fluid regulation.

Urotensin II (UII), described in many fish species, is secreted by the caudal neurosecretory system, a unique fish neuroendocrine structure. We have examined UII secretion and its control in euryhaline fish, supporting a proposed role in osmoregulation. However, it is now apparent that UII is present in other vertebrates, including mammals. The 12-amino-acid peptide has been highly conserved and the key cyclic region is common from fish to humans. Our UII radioimmunoassay for flounder, directed to this cyclic region, has shown circulating UII levels in humans and rats comparable with those in fish. In mammals, UII cardiovascular effects vary between species, with vasoconstriction only evident in specific vascular beds. The kidney expresses UII receptors and responds to UII administration by a reduction in glomerular filtration rate, urine flow, and excretion of the major ions. Interestingly, plasma levels of UII are chronically elevated in rat models of hypertension. These observations imply an unforeseen role for this ancient fish hormone in the physiological and perhaps pathophysiological regulation of body fluids in higher vertebrates, including humans.

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.

Electrical activity of caudal neurosecretory neurons in seawater- and freshwater-adapted flounder: responses to cholinergic agonists.

The caudal neurosecretory system (CNSS) of the euryhaline flounder is involved in osmoregulatory responses underlying adaptation to seawater and freshwater. This study compared electrophysiological activity and responses to cholinergic agonists in the neuroendocrine Dahlgren cells in an in vitro preparation taken from fully seawater- (SWA) or freshwater-adapted (FWA) fish. Resting membrane and action potential parameters showed few differences between SWA and FWA cells. The hyperpolarisation-activated sag potential and depolarising afterpotential were present under both conditions; however, amplitude of the latter was significantly greater in SWA cells. The proportions of cells within the population exhibiting different firing patterns were similar in both adaptation states. However, bursting parameters were more variable in FWA cells, suggesting that bursting activity was less robust. The muscarinic agonist, oxotremorine, was largely inhibitory in Dahlgren cells, but increased activity in a non-Dahlgren cell population, alpha neurons. Nicotine promoted bursting activity in SWA Dahlgren cells, whereas it inhibited over half of FWA cells.

Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1-34 N-terminal peptide.

A specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1-34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1-35(Tyr)) was used for iodination. Human (1-34) parathyroid hormone (PTH), human (1-34) PTHrP, and rat (1-34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1-34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1-34) PTHrP in plasma was 5.2+/-0.44 ng/ml (mean+/-SEM, n=20) for flounder and 2.5+/-0.29 ng/ml (n=64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1-34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7+/-6.1 ng/g wet tissue and 2.3+/-0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.

Rapid stimulation of cyclic AMP production by aldosterone in rat inner medullary collecting ducts.

Aldosterone stimulates sodium transport in the inner medullary collecting duct (IMCD) via the classic genomic pathway, but it is not known whether it also acts via a rapid, non-conventional pathway in this part of the nephron. The IMCD regulates the final sodium content of urine and expresses vasopressin receptors coupled to adenylate cyclase. The recently reported rapid, non-genomic actions of aldosterone have been associated mainly with an increase in intracellular Ca(2+); however, it has also been shown to stimulate camp generation. Thus the aim of this study was to determine whether aldosterone stimulates rapid generation of cAMP in isolated IMCD segments. IMCD segments were microdissected from Sprague-Dawley rat kidneys and incubated at 37 degrees C for 4 min with aldosterone (10(-12) to 10(-6) M), vasopressin (10(-12) to 10(-6) M), or a combination of hormones in the presence of a phosphodiesterase inhibitor. cAMP was measured by radioimmunoassay. While corticosterone and dexamethasone were ineffective, aldosterone stimulated a dose-dependent increase in cAMP within 4 min (P<0.05). This action of aldosterone was not inhibited by the MR antagonist spironolactone. Co-incubation of aldosterone with vasopressin resulted in a further increase in cAMP generation above that induced by the neurohypophysial hormone alone. Aldosterone-mediated cAMP generation was not inhibited by a vasopressin V(1) or V(2) receptor antagonist. These data support a novel and rapid, non-genomic effect of aldosterone in IMCD. Aldosterone does not apparently interact with the vasopressin receptor to stimulate cAMP generation.

Calcium-sensing receptors and parathyroid hormone-related protein in the caudal neurosecretory system of the flounder (Platichthys flesus).

The caudal neurosecretory system of the flounder (Platichthys flesus) has been examined by immunocytochemistry and in situ hybridization for the expression of parathyroid hormone-related protein (PTHrP) and calcium-sensing receptors (CaSR). The N-terminus nucleotide and deduced amino acid sequences of flounder PTHrP were determined and used to prepare oligonucleotide probes and homologous antiserum. The Dahlgren cells of the posterior spinal cord and their axons contained PTHrP protein which was also detected around the capillaries of the urophysis. PTHrP gene expression was abundant in the Dahlgren perikarya and axons in the spinal cord, but it was absent from nerve endings in the urophysis. Calcium-sensing receptor protein was present in the Dahlgren perikarya and axons, also with abundant gene expression, but there was neither protein nor mRNA in the urophysis. There were no apparent differences between freshwater- and seawater-adapted fish in either CaSR or PTHrP expression in the caudal neurosecretory system. These observations suggest that Dahlgren cells produce PTHrP which may be released from axons abutting capillaries in the urophysis. However, the sensing of ionic calcium appears to be confined to the perikarya of the Dahlgren cells in the spinal cord neuropil, suggesting that they are responsive to calcium in the central nervous system rather than the general circulation.

Neurohypophysial hormones and renal function in fish and mammals.

The two major basic neurohypophysial peptides, arginine vasopressin (AVP) of mammals and arginine vasotocin (AVT) of all non-mammalian vertebrates, share common structure and major roles in regulating renal function. In this review the complexity of AVP actions within the mammalian kidney is discussed and comparisons are made with the emerging picture of AVT's renal effects in fish. It has become apparent that the antidiuretic action of the neurohypophysial hormones is an ancient phylogenetic phenomenon, although this is based upon reduced glomerular filtration in fish by comparison with predominant tubular effects in mammals. Nonetheless, there appears to be retention of AVP effects upon the functional heterogeneity of nephron populations in mammals. Preliminary evidence for the possible existence of V(2)-type (tubular) neurohypophysial hormone receptors in fish, implies possible AVT actions which parallel those in mammals on tubular ion transport. Further insight from recent mammalian tubule microperfusion studies suggests that in teleost fish both apical (tubular lumen) and basolateral (blood borne) AVT have the potential to modulate renal function, though this remains to be examined.

The caudal neurosecretory system: control and function of a novel neuroendocrine system in fish.

The caudal neurosecretory system (CNSS) of fish was first defined over 70 years ago yet despite much investigation, a clear physiological role has yet to be elucidated. Although the CNSS structure is as yet thought to be confined to piscine species, the secreted peptides, urotensins I and II (UI and UII), have been detected in a number of vertebrate species, most recently illustrated by the isolation of UII in humans. The apparent importance of these peptides, suggested by their relative phylogenetic conservation, is further supported by the complex control mechanisms associated with their secretion. The CNSS in teleosts is known to receive extensive and diverse innervation from the higher central nervous system, with evidence for the presence of cholinergic, noradrenergic, serotonergic, and peptidergic descending inputs. Recent observations also suggest the presence of glucocorticoid receptors in the flounder CNSS, supporting previous evidence for a possible role as a pituitary-independent mechanism controlling cortisol secretion. The most convincing evidence as to a physiological role for the CNSS in fish has stemmed from the direct and indirect influence of the urotensins on osmoregulatory function. Recent advances allowing the measurement of circulating levels of UII in the flounder have supported this. In addition, there is evidence to suggest some seasonal variation in peptide levels supporting the notion that the CNSS may have an integrative role in the control of coordinated changes in the reproductive, osmoregulatory and nutritional systems of migratory euryhaline species.

Chloroquine inhibits arginine vasopressin production in isolated rat inner medullary segments induced cAMP collecting duct.

Previous studies showed that acute chloroquine administration increases plasma arginine vasopressin (AVP) concentration in the rat without influencing urine flow rate. The present study was designed to investigate whether chloroquine inhibits the AVP-induced cAMP production that mediates the antidiuretic effects of vasopressin. Single inner medullary collecting duct (IMCD) segments were pre-incubated at 35 degrees C for 10 min followed by 4 min at 37 degrees C with combinations of AVP and/or chloroquine with 1 mM 3-isobutyl-I-methylxanthine (IBMX) and cAMP concentrations were measured by radioimmunoassay. To establish the possible site of interference in cAMP production IMCD segments were incubated in the presence of chloroquine and forskolin. Chloroquine at concentrations ranging from 10(-9) M to 10(-6) M did not affect cAMP production by comparison with control. However, AVP (10(-8) M) and forskolin (10(-6) M) significantly (p < 0.01) increased cAMP accumulation. Chloroquine at all concentrations significantly suppressed the AVP stimulated cAMP production (e.g., chloroquine (10(-8) M) + AVP (10(-8) M) 41 +/- 12 fmol/4 mm (n = 9 tubules) vs. AVP (10(-8) M) alone 82 +/- 9 fmol/4 min/mm (n = 37 tubules). Chloroquine at all concentrations tested did not have any effect an forskolin-induced cAMP production. The data suggest that chloroquine inhibits the AVP induced cAMP production at the level of hormone/receptor complex. This possibly explains the previously reported lack of the normal antidiuretic responses of AVP in rats following chloroquine administration.

Altered calbindin mRNA expression and calcium regulating hormones in rat diabetic pregnancy.

Offspring of rats with diabetes mellitus are at risk of reduced calcium and bone mineral content. Altered expression of the maternal calcium binding proteins, calbindin-D(9K) and calbindin-D(28K), which are involved in renal and placental calcium transport, may underlie these problems.We have investigated the effect of diabetes on circulating concentrations of regulatory hormones with respect to calbindin-D mRNA concentrations. Three rat groups were studied; control (CP), streptozotocin-induced diabetic (DP), and insulin-treated diabetic (DPI) pregnant rats. Calbindin-D(9K) and calbindin-D(28K) mRNA abundance in placenta and maternal kidney were measured at days 7, 15, 18 and 21 of gestation, together with serum or plasma concentrations of 1,25 dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)), parathyroid hormone (PTH), PTH-related protein (PTHrP), calcitonin, oestradiol and IGF-I. An increase in placental calbindin-D(9K) mRNA abundance between days 18 and 21 in CP and DPI rats was severely blunted in the DP rats. In contrast, renal calbindin-D(28K) mRNA abundance was greater at days 7, 15 and 18 in DP compared with CP rats, as was calbindin-D(9K) at day 18. Calcitonin concentrations showed no differences between the groups, and both PTH and IGF-I were reduced over the first half of gestation, unlike the calbindins. In contrast, the concentrations of PTHrP and 1,25(OH)(2)D(3) were reduced at term in the DP group compared with the other two groups. Plasma oestradiol concentrations were lower in DP than in CP rats at days 7, 15 and 18, and most striking was the absence in DP rats of the peak of oestradiol seen at day 18 in CP rats. Despite the similarity between changes in placental calbindin mRNA and 1,25(OH)(2)D(3), previous work has shown placental calbindin-D(9K) regulation to be vitamin-D-independent. These studies produce suggestive evidence, therefore, that PTHrP and oestradiol may be involved in the altered calbindin-D expression by kidney and placenta in rat diabetic pregnancy.

Renal function in chronically catheterized conscious diabetic rats using constant and servo-controlled infusion.

1. Infusion experiments were performed on chronically catheterized conscious rats to assess kidney function before and after the induction of diabetes mellitus with streptozotocin. 2. Two infusion regimens were used, a conventional constant-infusion protocol and a novel computer-driven, servo-controlled fluid replacement technique. The latter enables body fluid status to be maintained throughout a study occasion by servo-controlled replacement of spontaneous urinary fluid losses. 3. The chronically catheterized conscious rat infused using a servo-controlled system appears to be the optimum model for a study of diabetic renal function. The conscious preparation circumvents problems associated with anaesthesia and acute surgery. The servo-controlled infusion protocol maintains the altered fluid status of the diabetic condition. Both hyperfiltration and polyuria, characteristics of human diabetes often absent in anaesthetized and/or constantly infused diabetic rats, were seen in all conscious servo-controlled diabetic animals. 4. The new regimen enables a more accurate assessment of renal function in experimental diabetes than with previous protocols. It should prove useful in future studies, particularly those assessing the role of anti-diabetic drugs on the kidney.

Separate receptors mediate oxytocin and vasopressin stimulation of cAMP in rat inner medullary collecting duct cells.

The two neurohypophysial hormones arginine vasopressin (AVP) and oxytocin have actions in the inner medullary collecting duct (IMCD) where both peptides induce an increase in cAMP accumulation. The present study has employed a novel IMCD cell line to determine whether these two hormones induce cAMP accumulation via common or separate receptors, and to characterize the potential receptors responsible. Equal volumes of vehicle (150 mM NaCl) or hormone/antagonist solutions were added to aliquots of 10(4) IMCD cells in the presence of 10(-3) M 3-isobutylmethylxanthine (IBMX) and incubated at 37 degrees C for 4 min. cAMP levels were determined by radioimmunoassay and protein concentration by Bradford assay. Both AVP and oxytocin elicited dose-dependent increases in cAMP generation, though oxytocin was less potent than AVP (EC50 = 1.6 x 10(-8) M vs. 7.4 x 10(-10) M). AVP at 10(-8) M and oxytocin at 10(-8) M, concentrations sufficient to elicit near-maximal cAMP accumulation, resulted in cAMP levels of 73.4 +/- 1.7 and 69.0 +/- 3.3 pmol (mg protein)-1 (4 min)-1, respectively (n = 10), compared with the vehicle-treated basal value of 37.7 +/- 2.2 pmol (mg protein)-1 (4 min)-1 (P < 0.001, n = 10). Combined AVP (10(-8) M) and oxytocin 10(-6) M) resulted in cAMP accumulation of 63.8 +/- 3.1 pmol (mg protein)-1 (4 min)-1 (n = 10), which was not significantly different from the effect of oxytocin alone, but slightly less than that for AVP alone (P < 0.05). A submaximal concentration of AVP (10(-10) M) induced cAMP accumulation of 48.6 +/- 2.5 pmol (mg protein)-1 (4 min)-1 (P < 0.01 compared with basal level of 34.9 +/- 2.4 pmol (mg protein)-1 (4 min)-1, n = 10), which was blocked in the presence of a vasopressin V2 receptor antagonist (10(-7) M OPC-31260) but not by the oxytocin receptor antagonist (10(-6) M [Pen1,pMePhe2, Thr4,Orn8]oxytocin) (36.3 +/- 6.1 and 45.1 +/- 1.3 pmol (mg protein)-1 (4 min)-1 respectively, P < 0.05, n = 10). A submaximal concentration of oxytocin (10(-7) M) induced a cAMP accumulation of 45.8 +/- 1.8 pmol (mg protein)-1 (4 min)-1 (n = 10), which was reduced by addition of 10(-6) M oxytocin antagonist (36.3 +/- 2.1 pmol (mg protein)-1 (4 min)-1, P < 0.05, n = 10), whereas co-incubation with 10(-6) M of the V2 receptor antagonist had no effect (43.2 +/- 1.3 pmol (mg protein)-1 (4 min)-1, n = 10). These results indicate that AVP and oxytocin induce cAMP accumulation from a common ATP pool in IMCD cells, and that separate vasopressin V2 and oxytocin receptor systems are involved, perhaps coupled to a common adenylate cyclase system.

Adrenergic receptor activation hyperpolarizes the caudal neurosecretory cells of the flounder, Platichthys flesus.

The physiological factors that govern activity of the caudal neurosecretory system in teleost fish are poorly understood. Immunocytochemical evidence indicates that the neurosecretory Dahlgren cells are innervated by descending monoaminergic fibres. Using intracellular recording techniques in an isolated preparation of the posterior spinal cord of the flounder (Platichthys flesus) we have demonstrated that superfusion of adrenaline or noradrenaline (10(-7) - 10(-3) M) causes hyperpolarization of Dahlgren cells (up to -30 mV). This hyperpolarization is likely to reflect an inhibitory effect of noradrenergic nerves on the neurosecretory system in vivo, reducing the rate of hormone release. Fluctuations in the input resistance and membrane time constant suggest involvement of a multiplicity of cellular mechanisms, including the opening and closing of populations of ion-selective channels. Superfusion with dopamine (10(-7) - 10(-3) M) had no effect. Superfusion with the beta-adrenoreceptor agonist, isoprenaline, caused hyperpolarization but to a markedly lesser extent than the maximum effect of adrenaline or noradrenaline, suggesting that their effects are mediated, only in part, by a beta-adrenoreceptor subtype. Superfusion of the preparation with a membrane permeable, non-hydrolysable cyclic AMP analogue (8-[4-chlorophenylthio]-cAMP) resulted in a slight hyperpolarization which was accompanied by a small, but significant, increase in input resistance. These data are consistent with at least part of the beta-adrenoreceptor mediated effect involving closure of cAMP-sensitive ion channels. Superfusion with the alpha 1-adrenoreceptor agonist, phenylephrine, had no effect on any electrophysiological parameter studied. However, the alpha 2-adrenoreceptor agonist, clonidine, caused hyperpolarization which again failed to reach the maximum level produced by adrenaline or noradrenaline. Together, these data suggest that the adrenergic inhibition of Dahlgren cell activity is mediated by both alpha 2- and beta-adrenoreceptor subtypes.

Interaction between adrenergic agonists and forskolin on adenylate cyclase activity in the rabbit proximal tubule.

Using a micro-radioimmunoassay, cAMP was measured in single, isolated S2 proximal straight tubules dissected from rabbit kidneys to investigate the effects of adrenergic agonists on adenylate cyclase activity. The baseline activity of adenylate cyclase was low and unaffected by either the alpha 2 agonist clonidine, the beta agonist isoprenaline (in the absence or presence of 1 microM forskolin) or 1 microM forskolin. Adenylate cyclase activity was markedly stimulated by 20 microM forskolin, an effect which was inhibited by 1 microM clonidine. The inhibition by clonidine was not apparent in the presence of the alpha 2 antagonist yohimbine. These results confirm the inability of isoprenaline to stimulate adenylate cyclase in the rabbit proximal tubule and demonstrate the coupling of alpha 2 receptors, in an inhibitory fashion, to adenylate cyclase. The inhibitory action of the alpha 2-receptor agonist was independent of other hormone activity in the renal proximal tubule.

Lack of effect of atrial natriuretic peptide and urodilatin on vasopressin-stimulated cyclic AMP production in microdissected rat inner medullary collecting ducts.

It is unclear whether the diuretic effects of atrial natriuretic peptide (ANP) result, in part, from an inhibition of the renal actions of vasopressin. Moreover, accruing evidence suggests that the kidneys themselves may produce an ANP-like peptide, urodilatin, which shares many of the renal actions of ANP. The mechanism underlying the diuretic action of urodilatin has not yet been examined. Accordingly, we have investigated the potential modulatory actions of both ANP and urodilatin on vasopressin-stimulated cyclic AMP (cAMP) production in microdissected inner medullary collecting duct (IMCD) segments of rat kidney. ANP and urodilatin alone (at 10(-8) or 10(-6) M) had no demonstrable effect on cAMP accumulation in IMCD segments. Moreover, neither ANP nor urodilatin (each at 10(-6) M) significantly altered either the profile or the absolute magnitude of the cAMP response stimulated by vasopressin. These findings indicate that neither ANP nor urodilatin interacts with the vasopressin-sensitive adenylate cyclase site in the rat IMCD to contribute to its diuretic actions.

Peptide hormone-stimulated second messenger production in the teleostean nephron.

Renal actions of arginine vasotocin (AVT) and atrial natriuretic peptide (ANP) in teleosts are poorly defined. Administration of these hormones changes renal function, largely explained by systemic vascular effects. The present experiments examine the potential direct action of these hormones on the teleost nephron. This has been assessed from the hormones' abilities to stimulate their second messenger systems within isolated nephroi of the rainbow trout Onchorhynchus mykiss. Following initial experiments to define the experimental conditions, AVT was found to induce a dose-dependent increase in cAMP content of a suspension of nephroi incubated for 10 min at 10 degrees; 10(-5) M AVT provoking an increase of 846% (SW) and 829% (FW) above basal cAMP accumulation of 88 and 125 pmol/mg protein, respectively. This V2-type receptor response to AVT (or AVP) has previously been considered to be present only in the tetrapod kidney. ANP evoked a dose-dependent stimulation of cGMP accumulation from 1.3 (SW) or 1.9 (FW) pmol/mg protein basal levels to 5806 and 4405% of these levels, respectively, at the highest concentration used of 3.3 x 10(-7) M. Further experiments using isolated glomeruli from the eel Anguilla anguilla localized at least a part of the response to ANP to a glomerular site.

Renal, cardiovascular and endocrine effects of centrally administered galanin in the anaesthetised rat.

Several studies implicate galanin as a central neuromodulator with an ability to influence hypothalamic and pituitary secretion. Central galanin content is also sensitive to the state of body hydration. Cardiovascular, renal and peripheral endocrine changes evoked by intracerebroventricular administration of galanin have been examined in the anaesthetized rat. Central galanin infusion consistently induced a transitory diuresis, the increase in urine flow being associated with a reduction in urine osmolality. There was no demonstrable change in plasma vasopressin concentration at the end of a 40 min galanin infusion. However, plasma aldosterone and corticosterone concentrations were significantly reduced by comparison with time-matched vehicle infused controls. There were no clear changes in renal electrolyte excretion or in heart rate or mean arterial blood pressure during the study period. The findings of this study support a participatory role for galanin in body fluid homeostasis, though the mechanisms responsible for mediating its central action on urine production remain unclear.