Cloning of a fragment of the osteonectin gene from goldfish, Carassius auratus: its expression and potential regulation by estrogen.

During reproduction, female teleost fish display increased plasma estrogen and greatly increased total plasma calcium concentrations; the main source of this calcium seems to be the scale. Osteonectin, a collagen-binding glycoprotein, is a major noncollagenous constituent of mammalian bone and is a product mainly of the osteoblasts. RT-PCT has been applied to clone and sequence part of the osteonectin gene from the goldfish, Carassius auratus. The use of a goldfish scale cell line (GFS) and a specific probe to goldfish osteonectin mRNA has allowed the study of the potential effects of estrogen and other calcitropic hormones on the cells derived from the scales. Osteonectin mRNA was detected in teleost bone, scale, and GFS cells by Northern blot analysis, hybridising to a transcript of approximately 1.6 kb. Expression of osteonectin mRNA was markedly down-regulated by 17beta-estradiol (10(-8) to 10(-11) M) in a dose-dependent fashion but was unaffected by calcitriol, TGFbeta, IL-1beta, calcitonin, and PTHrP. Down-regulation of osteonectin by estrogen is further evidence that estrogen participates in calcium homeostasis during vitellogenesis, acting directly on the cells responsible for matrix and mineral fluxes in scales.

Estrogen receptor mRNA in mineralized tissues of rainbow trout: calcium mobilization by estrogen.

RT-PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)-specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P-labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.

Glyceryl trinitrate enhances nitric oxide mediated perfusion within the equine hoof.

Laminitis, a microvascular disease of the equine hoof leads to severe lameness. Exogenous iv 1-arginine and transdermal nitric oxide donors, such as GTN, applied to the pasterns improve lameness during acute laminitis. Near Infrared spectroscopy in an earlier study showed haemostasis and ischaemia in the hoof during acute laminitis, both were alleviated by 1-arginine. Quantitative NIRS in the present study shows that transdermal GTN increases blood flow in the equine hoof. It is concluded that glyceryl trinitrate enhances nitric oxide mediated perfusion within the equine hoof in normal and chronically laminitic horses and ponies.

Clearance receptors for atrial natriuretic petide in Milan hypertensive and normotensive rats.

OBJECTIVE: Renal responses of anaesthetized Milan hypertensive (MHS) and Milan normotensive (MNS) rats to des-[Glu18-Ser19-Gly20-Leu21-Gly22]r atrial natriuretic peptide-4-23 (cANP4-23) a specific ligand for atrial natriuretic petide (ANP) clearance receptors were examined. METHODS: The peptide was administered intravenously as an initial bolus injection (10 micrograms/kg body weight) followed by constant infusion (1 microgram/min per kg body weight) for 30 min. Glomerular filtration rate, urine flow, sodium excretion and mean blood pressures were measured. Using the same protocol, plasma ANP levels were determined. RESULTS: Plasma ANP levels were significantly increased in both Milan strains (from 9.5 +/- 1.8 to 23.7 +/- 3.2 fmol/ml in MHS rats and from 9.8 +/- 1.2 to 15.9 +/- 1 fmol/ml in MNS rats). This increase was significantly greater in the MHS than in the MNS rats. The cANP4-23 infusions were diuretic and natriuretic in both strains of rats but despite a greater rise in plasma ANP level, the renal response was attenuated in the MHS compared with that in the MNS rats. Furthermore, the time course differed in that the hypertensive rats had a diuresis of slower onset. During cANP4-23 infusion, the mean blood pressure decrease was greater in the MHS rats, consistent with the fact that phenylephrine-precontracted isolated MHS rat aortae were threefold more sensitive to ANP-induced relaxation than were MNS rat aortae. Displacements of [125l]-rANP by rANP and cANP4-23 in isolated renal glomeruli indicated that MHS rats have similar amounts of cANP receptors but with a higher affinity for cANP than have MNS rats. CONCLUSION: cANP4-23 infusion increased plasma ANP more in MHS than in MNS rats. Renal responses were attenuated in the MHS rat compared with those in MNS rats. These differential actions cannot be explained in terms of glomerular ANP receptor densities, although aortic ring sensitivities differ between the two strains of rat.

Nitric oxide donors as treatment for grass induced acute laminitis in ponies.

The potential for participation of the arginine-nitric oxide system in the aetiology of acute equine laminitis has been assessed. Nitric oxide (NO), produced by the action of NO synthase (NOS) on its substrate l-arginine, relaxes vascular smooth muscle to cause vasodilation. An attenuated normal vasodilatory tone may characterise the pathogenesis of acute equine laminitis. An intravenous infusion of 10% l-arginine in 0.9% saline caused vasodilatation in the hoof of a normal pony and immediate reperfusion of laminal tissues in an acutely laminitic pony, detected noninvasively by near infrared spectroscopy (NIRS), but the amino acid had little effect on systemic blood pressure. Treatment of acute laminitis with glyceryl trinitrate applied topically to the pasterns reduced the typical 'bounding pulses' in treated limbs, reduced lameness and lowered systemic blood pressure. Nitric oxide is likely to participate in the multifactorial pathogenesis of equine laminitis.

Near infrared spectroscopy of pedal haemodynamics and oxygenation in normal and laminitic horses.

The present study applies near infrared spectroscopy (NIRS) to the haemodynamics of the pedal circulation in normal and laminitic horses. NIRS is a noninvasive technique which uses changes in light absorption at 4 wavelengths to provide information on the changes in cytochrome aa3 (cyt aa3) reduction-oxidation (redox) status, and changes in the tissue concentration of oxyhaemoglobin (O2Hb), deoxyhaemoglobin (HHb) and therefore total haemoglobin (tHb). Other studies have shown NIRS to be sensitive to changes in tissue oxygenation and perfusion in human cerebral and limb circulation. In this study, the NIRS sensor was applied to the dorsal surface of horses' hooves. Normal and laminitic animals (acute and chronic) were subjected to manoeuvres (cuff tourniquet; digital vessel occlusion at the palmar surface of the pastern; lifting of contralateral limb) predicted to change pedal haemodynamics. The procedures produced changes in pedal haemodynamics and oxygenation, which were similar to those observed in the ischaemic/reperfused human forearm. Laminitic differed from normal horses: return of HHb to baseline was slower and the change in cyt aa3 more rapid than normal in cases of chronic laminitis, taken to indicate reduced O2 stores as a result of compromised basal perfusion. In acute laminitis, baseline values did not fluctuate following any of the manoeuvres, suggesting haemostasis in the diseased hoof. NIRS is a useful noninvasive method to assess pedal vascular function in normal and laminitic horses.

Angiotensin(1-7) is an antagonist at the type 1 angiotensin II receptor.

OBJECTIVE: The present study investigated the antagonism of the amino-terminal heptapeptide fragment of angiotensin II ([des-Phe8]-angiotensin II; Ang(1-7)) to angiotensin II (Ang II) both in vitro in rabbit aortae and in vivo in rats. METHODS AND RESULTS: In rabbit isolated endothelium intact aortic rings Ang(1-7) caused a concentration-related rightward displacement of the Ang II curve and depressed the maximum response to Ang II. By applying the data to a Schild plot an apparent pA2 of 5.5 was calculated. This depression of maximum response could be reversed by co-incubation of Ang(1-7) with the competitive angiotensin antagonist losartan. Ang(1-7) had no effect on the contractile responses of several other agonists. Intravenous infusion of 10 or 100 micrograms/kg per min Ang(1-7) had no effect on the resting blood pressure in the anaesthetized rat but inhibited Ang II-induced pressor responses. CONCLUSION: The present results show that Ang(1-7) is a specific non-competitive antagonist of Ang II at type 1 angiotensin II receptors.

Interactions of atrial natriuretic peptide with A7r5 cells.

The interactions of Atrial Natriuretic Peptide with its receptors were investigated with the vascular smooth muscle cells from the A7r5 cell line. Displacement experiments of [125I]rat-Atrial Natriuretic Peptide by rat-Atrial Natriuretic Peptide revealed a single class of receptors with a Kd = 0.21 +/- 0.08 nM and a Bmax = of 35 +/- 16 fmol/mg of protein. Furthermore, the complex [125I]r-Atrial Natriuretic Peptide was internalised by a heatsensitive process. Finally, Atrial Natriuretic Peptide increased cyclic Guanosyl 3',5' Mono Phosphate in a time dependent and dose dependent way, a concentration of 0.1 microM increasing cyclic Guanosyl 3',5' Mono Phosphate level by a factor of 8.3 times when compared to basal level.

Atrial natriuretic peptide in the Milan hypertensive rat and the Milan normotensive rat: plasma concentration and binding to renal glomeruli in young, adult and aged rats.

METHODS: Blood pressures were determined in Milan hypertensive (MHS) and Milan normotensive (MNS) rats at different ages. Mean blood pressure, plasma atrial natriuretic peptide (ANP) concentration and renal glomerular receptors numbers and affinities were determined in young (25-day-old), adult (60- to 80-day-old) and aged (300-day-old) rats. RESULTS: Mean blood pressures, always higher in the MHS than in the MNS rats, increased with age in both strains. Plasma ANP concentrations were similar in the young and aged rats of both strains, but were higher in the adult MHS than in the adult MNS rats. There were no quantitative differences in the ANP receptors between young and old rats of the two strains, but an increase in the maximal binding capacity was observed, in both strains, when adult rats were compared with young rats. Moreover, saturation experiments with [125I]-rat ANP revealed a downregulation of the ANP receptors in the renal glomeruli isolated from the adult MHS rats. In isolated glomeruli the cyclic GMP stimulation by ANP was similar in adult rats of both strains. CONCLUSION: Downregulation in glomeruli of MHS rats, probably involving the clearance receptors for ANP, is concluded to occur.

Evidence of abnormalities in corticosteroid secretion leading to volume-dependent hypertension in Milan rats.

We examined corticosteroid secretory patterns and their relation to altered salt and water metabolism in Milan hypertensive and normotensive rats. Hypertensive rats had significantly higher blood pressures, exchangeable sodium (hypertensive, 41.2 +/- 0.3 mmol.kg-1; normotensive, 38.4 +/- 0.03 mmol.kg-1, P < .001), plasma volume (hypertensive, 5.39 +/- 0.12 mL.100 g-1; normotensive, 4.84 +/- 0.10 mL.100 g-1, P < .001), and plasma concentrations of atrial natriuretic peptide (hypertensive, 38.8 +/- 4.0 pg.mL-1, normotensive, 22.4 +/- 3.1 pg.mL-1, P < .02). These features coincide with those of mineralocorticoid-induced hypertension. Adrenal venous secretory rates (picomoles per minute) of corticosterone (hypertensive, 1696 +/- 202; normotensive, 873 +/- 139), 18-hydroxycorticosterone (hypertensive, 49.7 +/- 8.3; normotensive, 25.7 +/- 3.3), and aldosterone (hypertensive, 1.16 +/- 0.17; normotensive, 0.52 +/- 0.08) were higher in the hypertensive than the normotensive strain, but that of 11-deoxycorticosterone (DOC) (hypertensive, 94.4 +/- 14.9; normotensive, 114.3 +/- 33.9) was similar in the two strains. The corticosterone-DOC, 18-hydroxycorticosterone-DOC, and aldosterone-DOC ratios were higher in the hypertensive than the normotensive strain (P < .02), but the 18-hydroxycorticosterone-corticosterone and aldosterone-18-hydroxycorticosterone ratios were not. These results indicate increased activity of the "late" aldosterone biosynthetic pathway in the hypertensive compared with the normotensive strain caused by an increased conversion rate of DOC to corticosterone. The comparison of corticosterone secretion between the two strains indicates that 11 beta-hydroxylase rather than aldosterone synthase activity is more active in the hypertensive than the normotensive rats.

Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells.

The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in fresh-water (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.

Renin and angiotensin converting enzyme in elasmobranchs.

Renin-like activity (RLA) and angiotensin I converting enzyme-like activity (ACELA), the two key enzymes of the renin-angiotensin system (RAS), were sought in the elasmobranch Scyliorhinus canicula. Renal extracts were desalted in a G-25 and eluted in a G-100 Sephadex column (calibration 15,000-70,000). The fractions were concentrated in a vacuum device. A 48,000-MW fraction incubated with synthetic and porcine angiotensiongen generated angiotensin I estimated by RIA. This same fraction was vasopressor in rats and dogfish. ACELA was sought in gill, heart, liver, spleen, pancreas, intestine, kidney, gonads, brain, skin, and muscle of dogfish using a spectrophotometric assay. The highest level of ACELA was found in the gills followed by spleen, kidney, and brain (33.79 +/- 2.3, 29.56 +/- 1.0, 14.62 +/- 1.0, and 13.80 +/- 2.3 nmol hippurate/min/mg protein, respectively). Intestine, gonads, skin and muscle contained no measurable amounts of ACELA. Captopril inhibited enzymatic activity from all ACELA containing tissues.

Characterization of a kallidin receptor in the eel intestine.

Edman degradation of an eel bradykinin (BK) -like peptide isolated and detected by gel filtration and HPLC and RIA gave an amino acid sequence of Arg1-Pro-Pro-Gly-X-Ser-Pro-Leu-Arg9. Kallidin but not BK and des-Arg9-BK contracted eel intestine. The contractile effect of kallidin was not decreased by B1 and B2 receptor antagonists (up to 10(-6)M), nor by anticholinergics, antiadrenergics, ganglion blockers and an angiotensin II receptor antagonist but was attenuated by 10(-5)M indomethacin. Kallidin appears to interact with a receptor different from the BK B1 and B2 receptor types and prostaglandins may participate in the response.

Trans-thoracic fluid shifts and endocrine responses to 6 degrees head-down tilt.

A tomographic method of measuring electrical impedance known as Applied Potential Tomography (APT) has been used to image the impedance changes within the thoraxes of 8 healthy volunteers (4 male, 4 female) during 4-h periods of 6 degrees head-down tilt (HDT). A large decrease in impedance, reflecting an increase in thoracic fluid, was apparent within 1 min of tilting, peaked after 45 min, and was maintained throughout, although during the 4 h there was an 8% return towards baseline resistivity. Resistivity changes were most obvious in the region of the lungs. Simultaneous measurements of the key fluid regulating hormones revealed a significant increase in atrial natriuretic peptide (ANP) and a significant decrease in angiotensin II (AII) and aldosterone. There was no significant difference in plasma antidiuretic hormone level. These results illustrate the dynamic nature of fluid shifts during HDT, the spatial distribution of the fluid within the thorax and the associated endocrine responses.

Effects of altered dietary sodium intake on hormonal profiles in salt-sensitive hypertensive rats.

Blood pressure and selected putatively influential hormones were measured in Brattleboro rats which were without diabetes insipidus and which were subjected to various manipulations in dietary sodium intake. Rats fed a control diet from weaning to 16 weeks of age showed a slow increase in blood pressure whereas rats fed a sodium-enriched diet for the same period exhibited sustained hypertension (115 +/- 3 versus 169 +/- 5 (S.E.M.) mmHg). In animals fed a sodium-enriched diet plasma concentrations of antidiuretic hormone (ADH) were significantly increased from 55 +/- 8 to 108 +/- 5 fmol/l. Rats fed the control diet from weaning (group A) and subsequently maintained on that diet or changed to a sodium-enriched diet or sodium-deficient diet showed no differences in their blood pressure. Plasma hormone concentrations were similar in these groups, with the exception of aldosterone suppression in rats switched from control to a sodium-enriched diet (0.26 +/- 0.04 versus 0.08 +/- 0.03 nmol/l; P less than 0.001). Animals fed the sodium-enriched diet from weaning to 16 weeks of age (group b) and either maintained on that diet or changed to a control diet showed little change in their established hypertension. Transfer to the control diet was associated with increased plasma renin concentrations (PRC) (13.8 +/- 2.1 to 122.6 +/- 6.2 nmol/l) and plasma aldosterone concentrations (0.04 +/- 0.01 to 0.08 +/- 0.01 nmol/l; P less than 0.001) but corticosteroids and ADH concentrations were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the renin gene intron A tandem repeat region of Milan and Lyon hypertensive rat strains.

The region of intron A of the rat renin gene containing a unique tandemly repeated sequence was analysed in the Milan and Lyon hypertensive rat strains and their controls, and in several Sprague-Dawley rats, using an oligonucleotide probe complementary to the tandemly repeated sequence and a renin complementary DNA probe. In the Milan rats, the size of the Bgl II DNA fragment encompassing the tandem repeat region was the same in the hypertensive (MHS) and normotensive (MNS) strains. In the Lyon model, a difference of 1.1 kilobase (equivalent to about 28 copies of the 38 basepair tandem repeat sequence) was observed in the size of the Bgl II fragment of the hypertensive (LH) and normotensive (LN) strains. However, the finding that the size of the fragment in the Lyon low-blood-pressure (LL) strain was the same as that in the LH strain rather than the LN strain suggests that the difference between the two latter strains is not by itself a major cause of the blood pressure difference between them in the intron A tandem region. An analysis of Sprague-Dawley rats, from which the Lyon strains are derived, showed that at least three different renin gene alleles, two with Bgl II fragments of the same size as those seen in the Lyon strains, are randomly segregating in this population.

Secretory patterns of 1 alpha-hydroxycorticosterone in the isolated perifused interrenal gland of the dogfish, Scyliorhinus canicula.

An isolated in-vitro perifused interrenal gland preparation from the dogfish Scyliorhinus canicula was used to study production of quantitatively the major corticosteroid 1 alpha-hydroxycorticosterone (1 alpha-OH-B), measured by radioimmunoassay. Basal secretory rates were 877.1 +/- 145 (S.E.M.) fmol/mg per 15 min (n = 14) and the preparation remained viable for up to 22 h, as reflected in a brisk response to 10 microM cyclic AMP (cAMP) after this time. Steroid production responded in a dose-dependent manner to porcine ACTH, with 10 microM producing a maximum stimulation of 225% above the basal secretory rate. cAMP (10 microM) produced an increase of 278% above basal, while 1 microM forskolin increased basal secretory rates by 127%. [Val5]- and [Ile5]-angiotensin II (0.1 microM) increased 1 alpha-OH-B production by 120 and 372% respectively over basal secretory rates. Increasing the concentration of K+ in the perfusate from 8 mM to 12, 18, 28 and 40 mM produced a significant rise only at 28 mM. Alterations in the concentration of Na+ and osmolarity of the perifusion medium had inconsistent effects on steroid production. Increased concentrations of urea (from 360 to 720 mM) increased the basal secretory rate by 121%, whilst reducing the concentration of urea (from 360 to 90 mM) had no effect.

Water and salt disturbances under condition of microgravity.

Under conditions of microgravity severe alterations in body fluid composition and volume take place largely as a result of "cardiothoracic pooling" or headward shift of blood. Inappropriate endocrine, renal and cardiovascular responses result from the "misreading" of homeostatic signals by physiological receptors to produce an as yet incompletely defined syndrome under microgravitational conditions.

Somatostatin-related and glucagon-related peptides with unusual structural features from the European eel (Anguilla anguilla).

Peptides derived from prosomatostatins I and II and from two distinct proglucagons have been isolated from the pancreas of a teleost fish, the European eel (Anguilla anguilla). The product of prosomatostatin I processing, somatostatin-14, is identical to mammalian somatostatin-14. A 25-amino-acid-residue peptide (Ser-Val-Asp-Asn-Gln5-Gln-Gly-Arg-Glu-Arg10-Lys-Ala-Gly-Cys- Lys15-Asn-Phe-Tyr- Trp-Lys20-Gly-Pro-Thr-Ser-Cys25) is derived from prosomatostatin II. Compared with the corresponding peptides from other teleost fish, the eel somatostatin-25 contains the unusual substitution Pro for Phe at position 22. This peptide was also isolated in a form containing a hydroxylsyl residue at position 20. A 29-amino-acid-residue eel glucagon contains four substitutions relative to human glucagon Asn for Ser8, Glu for Asp15, Thr for Ser16, and Ser for Thr29). In common with mammalian and avian glucagons but unlike most other fish glucagons, the eel peptide possesses a glutamine residue at position 3. A peptide derived from a second proglucagon comprises 36 amino acid residues. A 7-residue C-terminal extension to the glucagon sequence shows structural similarity to the corresponding extension in ratfish (Hydrolagus colliei) glucagon and mammalian oxyntomodulin.