Antidiuretic action of angiotensin II in the river lamprey Lampetra fluviatilis: evidence for endocrine control of kidney function in cyclostomes.

Intravenous infusion of angiotensin II ([Asn¹ Val5]-Ang II) at 10⁻9 mol min⁻¹ kg⁻¹ body mass produced a significant antidiuresis in river lamprey Lampetra fluviatilis, captured during upstream migration and maintained in fresh water. Although the renin-angiotensin hormonal system (RAS) is now recognized in jawless fishes, until this study, the role of homologous Ang II in L. fluviatilis kidney function had not been examined. This study provides the first evidence for an antidiuretic action of Ang II in cyclostomes and, in evolutionary terms, suggests a renal function for the RAS in early vertebrates.

Circulating angiotensins in the river lamprey, Lampetra fluviatilis, acclimated to freshwater and seawater: possible involvement in the regulation of drinking.

Plasma angiotensin levels were measured for the first time in a cyclostome, the river lamprey. With the demonstration that angiotensins are present in the circulation, the possibility of a physiological role in the regulation of drinking was re-examined. Angiotensin II and III concentrations and plasma osmolalities were significantly higher in lampreys acclimated to 28 ppt seawater than in those acclimated to freshwater. No changes were found in angiotensin II and III levels 4 h after transfer from freshwater to 50% seawater, although plasma osmolality had started to rise by this time. There was a suggestion that plasma angiotensin II levels might be related to osmolality in the transfer experiment. Injection of Asp(1)Val(5)- or Asn(1)Val(5)-angiotensin II (40-169 microg/kg body wt.) did not stimulate drinking in freshwater-acclimated lampreys, even when they were still capable of drinking. The angiotensin-converting enzyme inhibitor captopril and the smooth muscle relaxant papaverine both reduced drinking rate in 50% seawater-acclimated lampreys. The data do not provide direct evidence for the involvement of the renin-angiotensin system in the control of drinking behaviour in the lamprey. Indirect evidence from the captopril effect is suggestive, but could have other explanations.

Angiotensin II-induced calcium signalling in isolated glomeruli from fish kidney (Oncorhynchus mykiss) and effects of losartan.

Glomeruli were isolated from the kidney of freshwater-adapted rainbow trout, Oncorhynchus mykiss, to qualitatively evaluate changes in cellular calcium associated with angiotensin II ([Asn1Val5]-Ang II) receptor stimulation and antagonism by the Ang II receptor antagonist losartan. Microspectrofluorometry using the fluorescent calcium indicator dye Calcium Green recorded fluorescence changes in isolated single glomeruli. Isolated glomeruli containing ester-loaded Calcium Green showed an Ang-II-induced transient rise in fluorescence. This transient rise showed an increased peak amplitude with increased Ang II concentration (10(-9) to 10(-6) M), but only a very small response was detectable in glomeruli exposed to 10(-9) M Ang II. The biphenylimidazole compound losartan (=DuP 753), an antagonist of the mammalian AT1 subtype Ang II receptor, initiated a transient agonistic rise in glomerular fluorescence at high concentration (10(-5), 10(-4), and 10(-3) M). However, the responses to 10(-6) 10(-7) M losartan were small or very low in each case. Losartan (10(-4) or 10(-7) M) antagonised the Ang-II-induced signalling in isolated glomeruli exposed to 10(-7) or 10(-6) M Ang II, respectively. This is the first evidence for functional AT1-like Ang II receptors coupled to cellular calcium signalling in the glomeruli of rainbow trout.

Acquired immunity to amyloodiniosis is associated with an antibody response.

The dinoflagellate Amyloodinium ocellatum, which causes amyloodiniosis or 'marine velvet disease', is one of the most serious ectoparasitic diseases plaguing warmwater marine fish culture worldwide. We report that tomato clownfish Amphiprion frenatus develop strong immunity to Amyloodinium ocellatum infection following repeated nonlethal challenges and that specific antibodies are associated with this response. Reaction of immune fish antisera against dinospore and trophont-derived antigens in Western blots indicated both shared and stage-specific antibody-antigen reactions. A mannan-binding-protein affinity column was used to isolate IgM-like antibody from A. frenatus serum. The reduced Ig consisted of one 70 kD heavy chain and one 32 kD light chain with an estimated molecular weight of 816 kD for the native molecule. Immunoglobulin (Ig) isolated from immune but not non-immune fish serum significantly inhibited parasite infectivity in vitro. An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal rabbit antibody produced against affinity-purified A. frenatus Ig. Anti-Amyloodinium serum antibody was not always detectable in immune fish, although serum antibody titers in immune fish increased after repeated exposure to the parasite. These results suggest that there may be a localized antibody response in skin/gill epithelial tissue, although antibody was rarely detected in skin mucus.

Electrophysiology and Innervation of the Photosensitive Epistellar Body in the Lesser Octopus Eledone cirrhosa.

The innervation and responses to light of the cephalopod epistellar body were investigated in preparations isolated from the stellate ganglia of the lesser or northern octopus, Eledone cirrhosa. Extracellular generator potentials in response to flashes of light were recorded from these photosensitive vesicles, with the amplitude of the response being found to be dependent upon the intensity of the flash and the level of ambient illumination. Intracellular recordings from photoreceptor cells of the epistellar body showed that they had resting potentials of about -49 +/- 7 mV (mean +/- SD, n = 43) and were depolarized by flashes of white, but not red (>650 nm) light. The evoked depolarization consisted of a transient component, followed by a steady plateau in which the amplitude of the depolarization was well correlated with the log of the stimulus intensity. The evoked depolarizations induced action potentials in the photoreceptor cells, with the frequency of firing being well correlated with the stimulus intensity. The morphologies of individual photoreceptor cells were visualized by intracellular injections of the fluorescent dye Lucifer yellow, and the path of the epistellar nerve across the stellate ganglion, into the pallial nerve, toward the brain was traced using the lipophilic dye Di-I. This pathway was confirmed physiologically by recording light-evoked responses from the cut end of the pallial nerve.

Characterisation of angiotensin II binding to glomeruli from rainbow trout Oncorhynchus mykiss adapted to fresh water and seawater.

Specific binding of 125I-[Asn1, Val5]-angiotensin II to isolated glomeruli of freshwater-adapted and seawater-adapted rainbow trout, Oncorhynchus mykiss, reached a plateau after 20 min at 10 degrees and was displaced by nonradiolabelled [Asn1, Val5]-angiotensin II. Seawater adaptation increased the rate of dissociation of 125I-[Asn1, Val5]-angiotensin II from putative receptors. Competitive binding experiments revealed a single population of high-affinity receptors with a KD 0.3-0.4 x 10(-10) M, which was unchanged by seawater adaptation. Seawater adaptation did, however, reduce the density (Bmax) of the binding sites, which may reflect down-regulation by the higher plasma concentration of angiotensin II in seawater-adapted trout.

Characterization of putative glomerular receptors for angiotensin II in the rainbow trout Oncorhynchus mykiss using the antagonists Losartan, PD 123177, and saralasin.

Isolated glomeruli from the rainbow trout Oncorhynchus mykiss have been examined for the presence of receptors specific for angiotensin II (AII). Specific binding of 125I-Asp1Val5-AII was saturable, plateaued after 20 min, and increased with glomerular protein. Saralasin (Sar1Val5Ala8-AII), a nonselective peptide AII receptor antagonist of AII binding to mammalian tissues, was a poor inhibitor of 125I-Asn1Val5-AII binding to trout glomeruli. However, the nonpeptide antagonist, Losartan (= DuP 753), which is specific for AII subtype AT1 receptors in mammalian tissues, was an effective inhibitor of binding of both 125I-Asp1Val5-AII and 125I-Asn1Val5-AII to trout glomeruli. The IC50 for Losartan inhibition of binding of Asp1Val5-AII was 1.25 +/- 0.07 x 10(-8) M while that for inhibition of binding of Asn1Val5-AII was 2.73 +/- 0.45 x 10(-8) M. Statistical analysis of logistic curve fits confirmed the easier displacement of Asp1Val5-AII than of Asn1Val5-AII. PD 123177, a nonpeptide antagonist specific for AII subtype AT2 receptors in mammalian systems, was ineffective as an antagonist of AII binding to trout glomeruli. These results are consistent with the presence of a specific AII receptor in the trout glomerulus and imply a novel receptor configuration.

Angiotensin II binding to tissues of the rainbow trout, Oncorhynchus mykiss, studied by autoradiography.

Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to 125I-angiotensin II (1.01.10(-9) M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10(-5) M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.