Rat Atriopeptin Dilates Vascular Smooth Muscle of the Ventral Aorta from the Shark (Squalus Acanthias) and the Hagfish (Myxine Glutinosa)

Most studies on the presence and function of AP in fishes have dealt with teleosts, but a limited data base indicates that AP probably plays some physiological role(s) in the two other major groups of fishes, Chondrichthyes (sharks, etc.) and Agnatha (hagfishes and lampreys). The plasma of representatives of both groups contains AP_ir (Evans et al. 1989) and it is clear that AP may be a major stimulant of spiny dogfish shark (Squalus acanthias) rectal gland salt secretion via both indirect (Silva et al. 1987) and direct (Karnaky et al. 1990) means. AP has also been found to lower dorsal aortic blood pressure in S. acanthias (Solomon et al. 1985; Benyajati and Yokota, 1990), but either it does not change (Solomon et al. 1985) or it reduces (Benyajati and Yokota, 1990) urinary glomerular filtration rate and sodium excretion in this species. No physiological studies of the effects of AP on Agnatha have been published, but AP receptors have been identified autoradiographically in the ventral aorta and kidney of the Atlantic hagfish (Myxine glutinosa; Kloas et al. 1988), and AP_ir has been described in the plasma of the sea lamprey (Petromyzon marinas;, Freeman and Bernard, 1990). AP_ir levels are significantly higher in seawater-adapted than in 10% seawater-adapted lampreys (Freeman and Bernard, 1990). To explore the potential for AP to affect gill hemodynamics in non-teleostean fishes, we have examined the sensitivity of isolated, aortic vascular smooth muscle of an elasmobranch and agnathan species to mammalian AP.

Spiny dogfish sharks were collected in Frenchman Bay, Maine, and maintained in submerged, seawater tanks at the Mount Desert Island Biological Laboratory (MDIBL). Atlantic hagfish were purchased from the Huntsman Marine Laboratory, St Andrews, NB, Canada, transported to the MDIBL and maintained in running seawater tanks. Endothelium-free, vascular smooth muscle rings from the shark aorta were prepared and mounted as previously described (initial tension=500mg; Evans and Weingarten, 1990). Hagfish were anesthetized in 1% MS222 and the ventral aorta was dissected free proximal to the last branchial arch nearest to the heart. The vascular endothelium was removed with a roughened polyethylene tube under iced hagfish Ringer’s solution (HRS; in mmol l^-1: NaCl, 501; KC1, 8.7; CaCl₂, 5.4; MgCl₂, 12.6; Na₂SO₄, 3.2; NaH₂PO₄, 0.4; NaHCO₃, 11; glucose, 0.7) bubbled with 1 % CO₂ in air. Rings were mounted in 5 ml of HRS in the same system (12°C) as that used for the shark rings (Evans and Weingarten, 1990); initial tension was set at 50 mg and maintained for approximately 30 min until stable tensions were achieved. Preliminary experiments indicated that it was unnecessary to preconstrict shark aortic rings before application of AP, but it was necessary to preconstrict the hagfish rings by the addition of 10⁻⁴moll^-1 carbachol, which produced an initial tension of 114±7.3 mg (N=8). We assume that this was necessary because of the extremely low initial tensions maintained in the hagfish rings. Low initial tensions were chosen because of the extremely low blood pressures normally found in vivo in the hagfish ventral aorta (approx. 0.67kPa; Satchell, 1986; D. H. Evans, unpublished results). Synthetic rat AP (101-126) was dissolved in 0.2 mol l^-1 acetic acid, sampled into polyethylene microfuge tubes, lyophilized in a Speedvac (Savant, Farmingdale, NY) and stored at —70°C until used. Rings of both species were exposed to the cumulative addition of AP producing a concentration range of 10⁻¹¹ to 2x10∼⁷mol l^-1. Endothelium removal was judged complete because both rings responded to carbachol addition with contraction. Maximal actual tension changes were 120±15mg (11) for the shark and 69.7±7.4mg (6) for the hagfish.

Synthetic rat AP (101-126) was purchased from Bachem, Inc. (Torrance, CA). Graphics were performed on a Macintosh II microcomputer using Cricket Graph (1.3; Cricket Software, Inc., Malvern, PA). All data are expressed as means±s.E. Curve fitting and apparent EC50 values of AP effects were estimated using Superpaint (2.0; Silicon Beach Software, San Diego, CA).

The isolated, endothelium-free, aortic vascular smooth muscle from both the spiny dogfish shark and Atlantic hagfish is very sensitive to heterologous mammalian AP (Fig. 1). The apparent EC₅₀ values of the vasodilatory effect of rat AP are 7x10⁻⁹moll^-1 (shark) and 4x10⁻⁹moll⁻¹ (hagfish), nearly identical to that previously described for the aortic vascular smooth muscle of the teleost Opsanus beta (4x10⁻⁹moll^-1; Evans et al. 1989) and in the same range as that described for various mammalian vessels (Genest and Cantin, 1988; Winquist, 1985). Even greater sensitivity might be expected with homologous (at least in terms of vertebrate class) peptide, but we have recently found that the aortic vascular smooth muscle from O. beta is actually slightly less sensitive (Price et al. 1990) to eel (Anguilla japonica) AP (Takei et al. 1989) and a killifish (Fundulus heteroclitus) brain AP (Price et al. 1990) than to mammalian AP. Whether an AP-induced dilation of the ventral aorta in sharks and hagfish in vivo results in alteration of perfusion pressures and patterns in the branchial vasculature remains to be determined, but in O. beta AP does produce a net fall in branchial resistance of the perfused head (Evans et al. 1989). Moreover, several hormones (Oduleye et al. 1982) and neurotransmitters (Nilsson, 1984) and metabolites such as adenosine (Colin et al. 1979; Evans and Walton, 1990) have been shown to control gill hemodynamics. Nevertheless, the present data and our earlier study (Evans et al. 1989) certainly suggest that AP may also play a role in controlling gill hemodynamics in all three major groups of extant fishes.

Therefore, it is instructive to consider the theoretical effects of an AP-induced increase in branchial perfusion. Sharks are slightly hyperosmotic to sea water because of the retention of high concentrations of urea and trimethylamine oxide (TMAO) (approx. 350 and 70 mmol l^-1, respectively) in the plasma. The resulting osmotic influx of water takes place across highly water-permeable gills, but is balanced by production of significant volumes of urine and rectal gland fluid. Since the plasma NaCl content is approximately 50% that of sea water, there is also a net influx of Na⁺ and Cl∼ across the gills (despite a very low ionic permeability), which is balanced by rectal gland, and possibly gill, salt excretion (Evans, 1979). A theoretical increase in gill perfusion produced by the vasodilatory action of atriopeptin, as found in the present work, would presumably increase both net water uptake and net ionic influx, thereby exacerbating rather than reducing the osmoregulatory problems faced by sharks.

The agnathan hagfishes are iso-osmotic to sea water, solely by the maintenance of plasma NaCl concentrations near to those of sea water; urea and TMAO are not retained in the plasma. However, plasma Na⁺ concentrations are slightly above those in sea water (486 mmol l^-1 vs 439 mmol l^-1; Robertson, 1966). Gill water permeabilities are quite high, presumably because no significant net osmotic fluxes of water occur, but gill ionic permeabilities appear to be rather low (Evans, 1979; Evans and Hooks, 1983). In these fishes, a theoretical increase in gill perfusion could potentially increase net loss of Na⁺, but would have little effect on volume regulation since no substantial gradients exist.

Thus, marine chondrichthyan fishes which are presumably slightly volume- and salt-loaded, hagfishes which are neither volume-nor salt-loaded in sea water and teleosts which are volume-depleted and salt-loaded in sea water all have aortic vascular smooth muscle which dilates in response to heterologous AP (present study and Evans et al. 1989), presumably leading to an increase in gill perfusion, and have AP_ir in their plasma (Evans et al. 1989). It is difficult to propose a common osmoregulatory parameter in these distinct fish groups associated with a hormone that has the array of known effects elicited in mammals and lower vertebrates by AP (Evans, 1990). However, they all share the need to maximize gas exchange by maintenance of consistent gill perfusion. One might propose, therefore, that, in the earliest vertebrates, AP played a major role in control of gas exchanger hemodynamics, rather than osmoregulation. This very tentative suggestion can only be supported or rejected by a careful assessment of the presence and role of AP in the primary aquatic vertebrates.

These studies were supported by DCB 8801572 from the National Science Foundation and by NIEHS EHS-1P30 ESO3828-03 to the Center for Membrane Toxicity Studies, Mount Desert Island Biological Laboratory. Excellent technical assistance was provided by Karl E. Weingarten and Julie S. Walton. Abstracts of portions of this work have appeared Bull. Mt Desert Isl. biol. Lab28 (1989) and 29 (1990).