Effects of marine noise pollution on Mediterranean fishes and invertebrates: A review.

Marine noise pollution (MNP) can cause a multitude of impacts on many organisms, but information is often scattered and general outcomes difficult to assess. We have reviewed the literature on MNP impacts on Mediterranean fish and invertebrates. Both chronic and acute MNP produced by various human activities - e.g. maritime traffic, pile driving, air guns - were found to cause detectable effects on intra-specific communication, vital processes, physiology, behavioral patterns, health status and survival. These effects on individuals can extend to inducing population- and ecosystem-wide alterations, especially when MNP impacts functionally important species, such as keystone predators and habitat forming species. Curbing the threats of MNP in the Mediterranean Sea is a challenging task, but a variety of measures could be adopted to mitigate MNP impacts. Successful measures will require more accurate information on impacts and that effective management of MNP really becomes a priority in the policy makers' agenda.

Daily variations of endolymph composition: relationship with the otolith calcification process in trout.

Ionic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO(2) concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO(2), total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher (1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca(2+) increases during the dark period and was maximal by the end of the night when the total CO(2) concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87, respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO(3) precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO(3) precipitation starts once the solubility of CaCO(3) is exceeded.

Neurohypophysial hormone regulation of Cl- secretion: physiological evidence for V1-type receptors in sea bass gill respiratory cells in culture.

Neurohypophysial hormone receptors were studied in primary cultures of sea bass (Dicentrarchus labrax) gill respiratory-like cells grown on permeable supports. This preparation was previously shown to provide a functional model for investigating the hormonal regulation of Cl- secretion. Under control conditions, the cultured monolayered epithelium had a short-circuit current (ISC) of 3.5+/-1.1 micro A x cm(-2). This current had previously been identified as an active Cl- secretion. The addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the ISC. Maximal increases of 60.9+/-12.1% and 117.7+/-28.0% above the basal ISC value were obtained for 10(-7) M AVT and IT respectively. Half-maximal effects were obtained for 3.1 x 10(-9) M AVT and for 1.4 x 10(-9) M IT. Mucosal application of 1.0 mM diphenylalamine-2-carboxylic acid (a specific blocker of Cl- channels) after serosal addition of 5 x 10(-8) M AVT or IT inhibited not only the basal but also the stimulated current, revealing a correlation with a hormone-dependent Cl- transport. Specific V1 or V2 receptor analogues of vasopressin (mammalian hormone) were used to characterize the type of neurohypophysial hormone receptors pharmacologically. While the V1 agonist [Phe2,Orn8]-oxytocin stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist [Deamino1,Val4,d -Arg8]-vasopressin had no effect. The V1 antagonist [d(CH2)5 1,O-Me-Tyr2,Arg8]-vasopressin used at a concentration of 5 x 10(-7) M totally reversed the 10-8 M AVT-stimulated Cl- secretion, whereas the V2 antagonist [d(CH2)5 1,d -Ile2,Ile4,Arg8,Ala9]-vasopressin used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10(-8) M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with the efficiency of the V1 receptor antagonist being significantly greater than that of the V2. This study provides evidence for neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests that on a physiological basis the hormonal effect is shared by the two peptides present in fish neurohypophysis (AVT and IT), acting by means of two distinct, although pharmacologically similar, V1-type receptors (according to the mammalian classification). These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish.

23Na NMR study of the interaction between hyaluronan and the bications Ca(++), Mg(++) and Cu(++).

The relaxation rate R = pi Delta nu(1/2) of the quadrupolar (23)Na nucleus was measured at pH approximately 7 using a 200 MHz NMR spectrometer with a view to observe the interaction between hyaluronan and its natural counterion Na(+) and the bications Ca(++), Mg(++) and Cu(++). An interpretation of our results, by means of the "entropy of fluctuations" concept of Na(+), is presented. We show that Cu(++) ions are more effective than Ca(++) and Mg(++). A possible model of complexation of Cu(++) in a cage formed by the 1-4 glycosidic bond, the carboxylate side-chain and the acetoamide side-chain is proposed, according to electrostatic potential computations using the ZINDO1 quantum semi empirical method.

Neurohypophysial hormone receptors and second messengers in trout hepatocytes.

Neurohypophysial hormone receptors and second messengers were studied in trout (Oncorhynchus mykiss) hepatocytes. Arginine vasotocin (AVT) and isotocin (IT) elicited a concentration-dependent inhibition of cAMP accumulation in the presence of 5x10(-8) M glucagon (maximal effect for 4.5x10(-7) M and 1.4x10(-7) M, half-maximal effect for 2.1x10(-8) M and 0.7x10(-8) M, AVT and IT respectively). The effect of glucagon was inhibited up to 90% by AVT and 80% by IT. While AVT inhibited (up to 50%) the basal cAMP production, IT had no such action. Specific V(1) or V(2) analogues (with reference to vasopressin in mammals) were used for pharmacological characterization of the type of neurohypophysial hormone receptor involved in this inhibition. The V(1) agonist [Phe(2), Orn(8)]-oxytocin inhibited the glucagon-stimulated cAMP production with a maximal effect for 6x10(-7) M and a half-maximal effect for 0.9x10(-8) M concentrations of the analogue. While the V(1) agonist reduced the glucagon-stimulated cAMP level by 70%, it showed only a tendency to reduce the basal level. The V(2) agonist [deamino(1), Val(4),d -Arg(8)]-vasopressin had no effect either on basal or on glucagon-stimulated cAMP production. The V(1) antagonist [d(CH(2))(5)(1), O-Me-Tyr(2), Arg(8)]-vasopressin totally reversed the 10(-8) M AVT-induced inhibition of 5x10(-8) M glucagon-stimulated cAMP production, whereas the V(2) antagonist [d(CH(2))(5)(1),d -Ile(2), Ile(4), Arg(8), Ala(9)]-vasopressin had no such effect. In this particular case, maximal and half-maximal effects of the V(1) antagonist were obtained for 2.3x10(-6) M and 1. 2x10(-6 )M respectively. Changes in intracellular calcium content were measured using the fluorescent probe FURA-2/AM. AVT and IT elicited a concentration-dependent increase in Ca(2+) accumulation. The comparison of the effect of 10(-8) M agonists versus AVT showed the following order of potency: AVT=IT>V(1) agonist>V(2) agonist. The V(1) antagonist reversed the AVT-induced Ca(2+) accumulation whereas the V(2) antagonist had no such effect. These results are taken as evidence for the presence in trout hepatocytes of neurohypophysial hormone receptors functionally close to the V(1a)-type linked to cAMP production and Ca(2+) mobilization.

A V1-type receptor for mediating the neurohypophysial hormone-induced ACTH release in trout pituitary.

We analysed the effects of specific neurohypophysial analogues for pharmacological characterization of the type of vasotocin receptor involved in the control of the adrenocorticotrophin hormone (ACTH) release from the perifused pituitary in the rainbow trout, Oncorhynchus mykiss. Mammalian corticotrophin releasing factor (CRF) and teleostean neurohypophysial peptides (arginine vasotocin (AVT) and isotocin (IT)) stimulated ACTH release. Analysis of concentrations giving half-maximal effects (D50) showed that these peptides affected ACTH release in the following order of potency: CRF (8 x 10(-13) M) > AVT (2 x 10(-10) M) > IT (10(-7) M). Maximal responses (Dmax) were obtained for hormonal concentrations of 10(-10) M, 10(-8) M and 10(-6) M respectively. This suggests that AVT and IT have different roles in the control of ACTH release. The values obtained for AVT and IT were in agreement with the circulating levels we previously found for these peptides. Specific V1 or V2 agonists or antagonists (with reference to vasopressin in mammals) were used to define the specificity of the neurohypophysial peptide receptor involved in this stimulation. The V1 agonist, [Phe2, Orn8]-oxytocin, stimulated ACTH release while the V2 agonist, [deamino1, Val4, D-Arg8]-vasopressin, had no such effect. Maximal and half-maximal responses were obtained in the presence of the V1 agonist with 10(-7) M and 7 x 10(-9) M respectively, and were in the range of values obtained with natural peptides. The V1 antagonist, [d(CH2)5(1), O-Me-Tyr2, Arg8]-vasopressin, and the V2 antagonist, [d(CH2)5(1), D-Ile2, Ile4, Arg8, Ala9]-vasopressin, maximally reversed the 10(-9) M AVT-stimulated ACTH release by 60% and 25% respectively, for a 5 x 10(-10) M concentration of the analogues and a D50 approximately 2 x 10(-11) M. These results demonstrated the presence of only one V1-type receptor in fish pituitary, with some of the structural and functional peculiarities typically displayed by the mammalian V1a-type receptor, but distinct from it. In this sense, the fish pituitary vasotocin receptor may represent a novel type of neurohypophysial hormone receptor, more closely related to the mammalian V1b-type.

Enzyme linked immunosorbent assay for the neurohypophyseal hormones arginine vasotocin and isotocin.

A competitive enzyme linked immunosorbent assay (ELISA) using new polyclonal antibodies was developed for the first time for each of two neurohypophyseal hormones: arginine vasotocin (AVT, ubiquitous in vertebrates) and isotocin (IT, restricted to teleost fish). The antibodies obtained were highly specific, showed no cross-reaction between the two peptides and were able to suppress the activity of the peptides in in vitro bioassays. An original feature of the assays was the covalent binding of the peptidic antigen to Covalink microplates. Competition was thereafter made between this bound antigen and the antigen in samples, for a fixed amount of the relevant antibody. Revelation used peroxidase-labeled antibodies. These ELISAs were sensitive enough to detect 1 ng/ml and 0.1 ng/ml for AVT and IT respectively. Moreover, for the first time, the two hormones were measured separately, each in the presence of the other, and shown to exist as circulating hormones in fish.

ELISA measurements of vasotocin and isotocin in plasma and pituitary of the rainbow trout: effect of salinity.

The fish neurohypophysial hormones arginine vasotocin (AVT) and isotocin (IT) were measured for the first time by ELISAs (in comparison with other techniques) in plasma and hypophysis of rainbow trout adapted stepwise from freshwater (FW) to seawater (SW). AVT concentrations were higher than IT in plasma and, conversely, lower in hypophysis. No difference appeared between FW and SW conditions, but plasma hormone concentrations fell when FW fish were moved to 1/3 SW and increased progressively when fish were moved from 1/3 SW to SW. Peptide values obtained in 1/3 SW may correspond to the lowest osmoregulatory constraints occurring in an isosmotic medium in comparison to FW or full SW. The data suggest that storage and/or release of AVT and IT differ, but vary in a similar way with external salinity, and that these peptides should play a role in teleost fish osmoregulation.

Gi protein mediates adenylate cyclase inhibition by neurohypophyseal hormones in fish gill.

Adenylate cyclase activity was measured on membrane fractions from the gill epithelium of rainbow trout Salmo gairdneri. Basal and glucagon-stimulated activities responded negatively to homologous neurohypophyseal peptides (arginine-vasotocin and isotocin). This inhibitory effect was totally abolished in the presence of pertussis toxin (IAP). The guanine nucleotide dependence of the enzyme was further explored by using GTP, GDP, and their stable analogs Gpp(NH)p, GTP gamma S, and GDP beta S. The results suggest that neurohypophyseal peptides at low concentrations inhibit the adenylate cyclase system directly by way of a Gi-protein, thus implying the intervention of a new type of membrane receptor for these hormones in fish gills.

Evidence for presence of a new type of neurohypophysial hormone receptor in fish gill epithelium.

Arginine vasotocin (AVT) and isotocin (IT) induced direct inhibition of adenylate cyclase activity in gill plasma membranes of the rainbow trout adapted to freshwater (FW) and seawater (SW). The maximal inhibition was obtained with 10(-11)-10(-10) M (50% inhibitory concentration approximately 10(-13) M), values in agreement with the circulating levels of AVT in trout blood. Specific V1 or V2 agonists or antagonists (with reference to vasopressin) were used to define the specificity of the neurohypophysial peptide receptors involved in this inhibition. The V1 agonist [Phe2,Orn8]vasotocin ([Phe2]OVT) inhibited the basal and glucagon-stimulated adenylate cyclase activity, and this effect in SW (20%) was twice more than in FW (10%). The V2 agonist 1-deamino[Val4,Arg8]-vasopressin (dVDAVP), however, produced a stimulation that was of the same amplitude (10%) in both media. The V1 antagonist [(1-beta-mercapto-beta-beta-cyclopentamethylenepropionic acid), 1-(O-ethyl)Tyr2,Orn8]vasotocin (d(CH2)5[Tyr(Et)2]OVT) totally reversed the AVT- or IT-induced inhibition of basal or glucagon-stimulated cyclase activity, whereas the V1/V2 antagonist [(1-beta-mercapto-beta-beta-cyclopentamethylene propionic acid), 1-(O-ethyl)D-Tyr2,Val4,Arg8]vasopressin (d(CH2)5[D-Tyr(Et)2]-VAVP) (previously used as V2 antagonist in amphibians) had no such effect. When active, all analogues had their maximal activity at 10(-11)-10(-10) M (50% maximal activity approximately 10(-13) M), as observed with the natural peptides. These results, together with our previous observations, point to the gill epithelium as a direct target organ for neurohypophysial peptides and suggest that the hormone receptors involved in fish belong predominantly, if not exclusively, to a new type that we propose to designate as fish neurohypophysial hormone (NHF) receptors while awaiting further characterization.

Inhibitory effect of neurohypophysial peptides on cyclic AMP accumulation by isolated hepatocytes of the trout.

Cyclic AMP levels were measured in freshly isolated hepatocytes of the rainbow trout. Compared with basal values, the average levels were increased up to 60 times in a dose-dependent manner either by mammalian glucagon (concentration range 1 nmol-1 mumol/l; dose giving half maximum response (EC50) 0.18 mumol/l) or by forskolin (concentration range 0.1-100 mumol/l; EC50 about 10 mumol/l). These stimulatory effects were partially inhibited by fish or mammalian neurohypophysial hormones used at relatively high concentrations (1-5 mumol/l). It is suggested that these results are evidence for the presence of V1-type receptors in fish hepatocytes. Together with previous results obtained with gills on the hormonal inhibition of adenylate cyclase activity, they suggest that teleost fish may possess only V1-type receptors (or two V1-related types), while the V2 receptors have evolved (or have become functional) in higher vertebrates.

Arginine vasotocin binding to isolated branchial cells of the eel: effect of salinity.

Binding of 125I-labelled arginine vasotocin (AVT) was studied in isolated intact gill cells obtained from eels (Anguilla anguilla) adapted to fresh water (FW) or to sea water (SW). Experiments carried out at 20 degrees C showed maximum and stable binding beyond 10 min of incubation. Specific binding, determined by using labelled peptide in the presence or absence of an excess of unlabelled hormone, represented 30-50% of total and was reversible, with a half-time of less than 5 min. Scatchard plot analysis revealed the presence of a single population of saturable, high-affinity sites. Maximum binding capacity (Bmax: fmol AVT/10(6) cells) and dissociation constant (Kd: nM) were respectively 5.16 and 3.21 in FW and 24.25 and 1.05 in SW. Analysis of chloride cell number and size in gills and of binding characteristics of AVT revealed parallel changes with external salinity. These results are taken as evidence for the direct intervention of neurohypophysial peptides on the gill epithelium of teleost fishes.

Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout.

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membrane of freshwater-adapted trout by measuring the conversion of [alpha-32P]ATP into [alpha-32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20 degrees. The Km for ATP was 0.5 mM in the presence or absence of the stimulators. The presence of 10(-5) M guanosine-5'-triphosphate and 4 X 10(-3) M MgCl2 (2.41 X 10(-3) M free Mg2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca2+ was inhibitory. IC50 for CaCl2 was 5 X 10(-4) M (10(-7) M free Ca2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400-450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE1 10(-5) M (50% increase over basal activity) or by glucagon 5.7 X 10(-10) M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10(-2) M.

Adenylate cyclase activity in fish gills in relation to salt adaptation.

The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [alpha-32p]-ATP into [alpha-32p]-cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted-mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones (glucagon, VIP) and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE1. These results are interpreted in relation to the general process of osmoregulation.