INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IN MAMMALS, the endothelin (ET) family of peptides comprises three structurally related members: ET-1, ET-2, and ET-3 (6). The amino acid sequences of these peptides have been very strongly conserved among mammals with only mouse ET-2, which contains the substitution Ser⁴  Asn compared with human ET-2, representing a species variant (20). More recently, it has been shown that ETs are synthesized in the tissues of nonmammalian vertebrates, and existing data indicate that the primary structures of these peptides have also been strongly conserved. A peptide that is identical in structure to human ET-1 was isolated from an extract of the stomach of the European green frog Rana ridibunda, and a peptide that is identical to human ET-3 except for a single amino acid substitution (Phe⁴  Tyr) was purified from an extract of the liver in the same species (27). In contrast, only a single molecular component, which differs from human ET-1 by four amino acid substitutions at positions 4-7, was isolated from an extract of the kidney of the trout Oncorhynchus mykiss (26) (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   A comparison of the amino acid sequences of the naturally occurring endothelins (ETs) identified in mammalian and nonmammalian vertebrates. The shaded residues indicate differences from the human orthologs.

Preliminary physiological studies indicated that the biological activities of the ET peptides in nonmammalian species have also been well conserved across evolutionary lines. In several species of mammals, ET-1 stimulates steroidogenesis in dispersed cells from the glomerulosa zone of the adrenal cortex (2), and it has been shown that synthetic frog/human ET-1 and frog ET-3 stimulate both corticosterone and aldosterone production by perifused interrenal slices from R. ridibunda (27). ET-1 was first identified on the basis of its powerful vasocontrictor activity on vascular smooth muscle from a range of mammalian species (29), and synthetic trout ET potently constricts isolated rings of vascular tissue from trout efferent branchial artery, celiacomesenteric artery, and anterior cardinal vein (26). Studies in vivo have shown that bolus intra-arterial injections of trout ET into unanesthetized trout produce complex hemodynamic effects involving increased ventral aortic and central venous pressure, increased gill resistance and systemic resistance, and decreased cardiac output, heart rate (HR), and stroke volume (5).

Before our study, the only ET-like peptides from a reptile to be characterized structurally were the sarafotoxins, a family of five isoforms isolated from the venom of the snake Atractaspis engaddensis (25). The sarafotoxins, like the ETs, comprise 21-amino acid residues, possess the same pattern of disulfide linkages, and exhibit strong vasoconstrictor activity (8), but the evolutionary relationships between the two families are unclear (9). The present study extends our understanding of the evolution of both structure and function of the ET family of peptides by describing the purification, structural characterization, and cardiovascular activity in the species of origin of ET-1 and ET-3 from the American alligator A. mississipiensis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Alligator ET-3, identical in structure to frog ET-3, was synthesized by solid-phase methodology as previously described (27). Human ET-1, identical in structure to alligator ET-1, was supplied by Peninsula Laboratories (Belmont, CA).

Radioimmunoassay. ET-like immunoreactivity (ET-LI) was measured using an antiserum raised against human ET-1 that shows 60% cross-reactivity with human ET-2, 70% cross-reactivity with human ET-3, but only 0.1% reactivity with big human ET-1-(1-38) (24). ¹²⁵I-labeled human ET-1 (Amersham Pharmacia, Piscataway, NJ; sp act 74 TBq/mmol) was used as tracer. The minimum detectable concentration using human ET-1 as standard was ~1 fmol/tube.

Tissue extraction. Alligator tissues were obtained at the Rockefeller Wildlife Refuge (Grand Chenier, LA) from mature specimens (n = 8), hatched in 1972 or 1973, that had been euthanized and necropsied to investigate reproductive failure. Lung (1.5 kg) and kidney (0.6 kg) were frozen on dry ice and shipped to Creighton University, where they were stored at 80°C for 2 wk before extraction. The frozen tissues were separately homogenized with 10 vol of ice-cold 3:1 ethanol-0.7 M HCl (vol/vol) using a Waring blender. The homogenates were stirred for 3 h at 0°C and centrifuged (1,600 g, 30 min, 4°C), and ethanol was removed from the supernatants under reduced pressure. Peptide material was isolated from the extracts using Sep-Pak C₁₈ cartridges (Waters Associates, Milford, MA) as previously described (26, 27). Bound material was eluted with acetonitrile-water-trifluoroacetic acid (70.0:29.9:0.1 vol/vol/vol) and freeze-dried.

Purification of ET-3 and ET-1 from lung. The lung extract, after partial purification on Sep-Pak cartridges, was redissolved in 1 M acetic acid (8 ml) and chromatographed on a 100 × 2.5-cm column of Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 1 M acetic acid. The column was eluted at a flow rate of 48 ml/h, and fractions (8 ml) were collected. Absorbance was measured at 280 nm. The concentration of ET-LI in the fractions was determined by radioimmunoassay at a dilution of 1:30. Fractions containing ET-LI were pooled and pumped onto a 25 × 1-cm Vydac 218TP510 (C₁₈) reverse-phase HPLC column (Separations Group, Hesperia, CA) equilibrated with 0.1% (vol/vol) trifluoroacetic acid-water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% over 10 min and to 49% over 60 min using linear gradients. Absorbance was monitored at 214 and 280 nm, and fractions (1 min) were collected. The two fractions containing ET-LI were separately rechromatographed on a 25 × 1-cm Vydac 214TP510 (C₄) column equilibrated with acetonitrile-water-trifluoroacetic acid (21.0:78.9:0.1 vol/vol/vol) at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 42% over 40 min using a linear gradient. Alligator pulmonary ET-3 and ET-1 were purified to near homogeneity by successive chromatographies on 250 × 4.6-mm Vydac 214TP54 (C₄), Vydac 219TP54 (phenyl), and Vydac 218TP54 (C₁₈) columns at a flow rate of 1.5 ml/min. The concentration of acetonitrile in the eluting solvent was raised 21 to 42% over 40 min using a linear gradient.

Purification of ET-3 from kidney. The kidney extract, after partial purification on Sep-Pak cartridges, was chromatographed on a Sephadex G-25 column under the same condition used for the renal extract. Pooled fractions containing ET-LI were chromatographed on Vydac C₁₈, C₄, and phenyl columns under the same conditions used for the purification of alligator pulmonary ET-3.

Structural analysis. The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems Procise 491A sequenator (Foster City, CA). Mass spectrometry of the peptide was performed on a Voyager RP MALDI-TOF instrument (Perspective Biosystems, Framingham, MA) equipped with a nitrogen laser (337 nm). The instrument was operated in linear mode with delayed extraction, and the accelerating voltage in the ion source was 25 kV. Approximately 10 pmol of sample was used, and the accuracy of the mass determinations was at least 0.05%.

Surgery for cardiovascular studies. Alligators of both sexes (0.9-12.0 kg) were obtained from the Rockefeller Wildlife Refuge and were kept at 30°C on a 12:12-h dark-light cycle. The animals were not fed for 7 days before surgery. On the day of experimentation, the animals were anesthetized by intramuscular injection of 50 mg/kg pentobarbital sodium (Veterinary Laboratories, Lenexa, KS) ~2 h before surgery. Animals were placed in a supine position, tracheostomized, and ventilated with a gas mixture of composition 67% nitrogen-30% oxygen-3% carbon dioxide (3 breaths/min) at a tidal volume of 30-40 ml/kg (SAR-830 Ventilator, CWE, Ardmore, PA). Occlusive PE-50 polyethylene catheters were inserted into the right femoral artery and vein. The arterial catheter was connected to a Statham P23 pressure transducer, which was used for measurements of systemic arterial blood pressure. The venous catheter was used for injections of test substances. To access the central blood vessels, a 5- to 10-cm ventral incision was made through the skin, and the posterior one-third of the sternum was cut and separated with retractors. The vessels were carefully cleared from connective tissues. For measurements of blood flow, transit-time ultrasonic blood flow probes were placed on the left aorta (Q_LAo), on one branch of the right aorta (Q_RAo), and on the left pulmonary artery (Q_LPA). The right pulmonary artery was nonocclusively cannulated using the Seldinger technique (28) for measurements of pulmonary arterial blood pressure. Body temperature was continuously monitored using a thermistor inserted 3-4 cm into the cloaca.

Hemodynamic measurements. All measurements were made on anesthetized animals at an ambient temperature of 30 ± 2°C. Signals from blood pressure transducers and blood flowmeters were continuously collected onto a computer with a commercial data-acquisition system (AcqKnowledge MP 100, Goleta, CA) sampling at 10 Hz. HR was calculated from the pulsatile systemic pressure signal. Experiments were begun 30 min after surgery. Baseline values of blood flows, blood pressures, and HR were recorded for 10-15 min. Increasing doses of alligator ET-3 (10, 30, 100, and 300 pmol/kg) were injected at intervals of between 15 and 60 min, depending on the time necessary to allow the cardiovascular parameters to stabilize between injections. Approximately 2 h after the last injection of alligator ET-3, ET-1 (10, 30, 100, and 300 pmol/kg) was injected following the same procedures used for ET-3. After the experimental protocol was complete, all animals were euthanized by intracardiac injections of KCl.

Data analysis and statistics. All recordings of blood flows and blood pressures were analyzed with AcqKnowledge data- analysis software (version 3.2.3; Biopac, Goleta, CA). For all cardiovascular parameters measured, mean values were determined for a period of 1 min before injections, and for 1 min during the peak response caused by injections. Total systemic blood flow (Q_sys) was estimated as 2.5 × (Q_RAo + Q_LAo), and total pulmonary flow was calculated as 2 × Q_LPA. The estimations are based on central flow patterns previously reported in the American alligator (21). Systemic vascular resistance (R_sys) and pulmonary vascular resistance (R_pul) were calculated as the respective pressure divided by respective total flow. Mean values that were significantly different from preinjection condition were identified by a Wilcoxon signed-ranks test. Significance for all analyses was at the P  0.05 level. All values are presented as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Purification of ET-3 and ET-1 from alligator lung. The extract of alligator lung, after partial purification on Sep-Pak cartridges, was subjected to gel-permeation chromatography on a Sephadex G-25 column. ET-LI was eluted as a broad peak with partition coefficient K_AV between 0.55 and 0.75. These fractions were pooled and chromatographed on a semipreparative Vydac C₁₈ column, and the elution profile is shown in Fig. 2A. ET-LI was associated with two well-separated fractions denoted by the bars. Alligator pulmonary ET-3 was purified to near homogeneity, as assessed by peak symmetry, by successive chromatographies on a semipreparative Vydac C₄ column (Fig. 3A), an analytic Vydac C₄ column (Fig. 3B), an analytic Vydac phenyl column (Fig. 3C), and an analytic Vydac C₁₈ column (Fig. 3D). Alligator ET-1 was purified under the same conditions of chromatography. The final yields of pure peptides were ~30 pmol (ET-1) and 70 pmol (ET-3).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Reverse-phase HPLC on a semipreparative Vydac 218TP510 (C18) column of an extract of alligator lung (A) and kidney (B), after partial purification on Sep-Pak cartridges and by gel-permeation chromatography. The fractions (1 min) containing ET-like immunoreactivity, denoted by the bar, were pooled for further purification. Dashed line shows the concentration of acetonitrile in the eluting solvent. ABS280, absorbance at 280 nm.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Purification by reverse-phase HPLC of pulmonary alligator ET-3 on a semipreparative Vydac C4 column (A), analytic Vydac C4 column (B), analytic Vydac phenyl column (C), and analytic Vydac C18 column (D). Individual peaks were collected by hand, and arrows indicate where peak collection began and ended. Alligator pulmonary ET-1 and renal ET-3 were purified under similar conditions of chromatography. ABS214, absorbance at 214 nm.

Purification of ET-3 from alligator kidney. The extract of alligator kidney, after partial purification on Sep-Pak cartridges, was subjected to gel-permeation chromatography on a Sephadex G-25 column. ET-LI was eluted as a broad peak in the same fractions as the pulmonary ET. These fractions were pooled and chromatographed on a semipreparative Vydac C₁₈ column, and the elution profile is shown in Fig. 2B. ET-LI was associated with the single fraction denoted by the bar. Alligator renal ET-3 was purified to near homogeneity, as assessed by peak symmetry, by successive chromatographies on semipreparative Vydac C₄, analytic Vydac C₄, analytic Vydac phenyl, and analytic Vydac C₁₈ columns under the same conditions used for the purification of ET-3 from lung (chromatograms not shown). The final yield of pure peptide was ~40 pmol.

Structural characterization. The primary structures of the alligator ET peptides were determined by Edman degradation using an automated microsequence analyzer. The amino acid sequence of alligator ET-1 was established as Xaa-Ser-Xaa-Ser-Ser-Leu-Met-Asp-Lys-Glu-Xaa-Val-Tyr-Phe-Xaa-His-Leu-Asp-Ile-Ile-Trp. No phenylthiohydantoin-coupled amino acid derivatives were detected during cycles 1, 3, 11, and 15, which is consistent with the presence of cystine residues at these positions. The structure of alligator ET-1, including the presence of two disulfide bridges, was confirmed by mass spectrometry. The observed molecular mass of the peptide was 2,493 ± 1 Da compared with a calculated average molecular mass of 2,492 Da for the proposed structure.

Hemodynamic effects of alligator ET-3. Intravenous injection of synthetic alligator ET-3 in the dose range 10-100 pmol/kg produced an immediate monophasic and concentration-dependent hypotensive response of short duration. The time course of the responses of P_sys to increasing doses of the peptide in a single animal is shown in Fig. 4. The fall in P_sys was accompanied by a decrease in R_sys, but there was no significant change in Q_sys, R_pul, pulmonary pressure (P_pul), or pulmonary blood flow (Q_pul; Fig. 5). At the highest dose tested (300 pmol/kg), the response was biphasic, with the initial fall in P_sys being followed by significant rise in R_sys, leading to a small but nonsignificant increase in blood pressure. Q_sys, Q_pul, and P_pul decreased, but because of the variability of the response, there was no significant change in R_pul. There was no change in HR at any dose of ET-3 tested.

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Effect on systemic arterial blood pressure (Psys; in kPa) of bolus injections of increasing doses of alligator ET-3 (10, 30, 100, and 300 pmol/kg) in a single animal.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Mean values of the absolute changes from controls of hemodynamic parameters after injection of increasing doses of alligator ET-3: systemic vascular resistance (Rsys; A), Psys (B), systemic blood flow (Qsys; C), pulmonary vascular resistance (Rpul; D), pulmonary blood pressure (Ppul; E), and pulmonary blood flow (Qpul; F). For A-C and E, n = 6; for D and F, n = 5. Error bars indicate SEs. * Statistically significant difference from control (P  0.05).

Hemodynamic effects of human/alligator ET-1. In contrast to the effects of ET-3, the response to synthetic ET-1 was biphasic at all doses tested. The time course of the response of P_sys to increasing doses of ET-3 in a single animal is shown in Fig. 6. As shown in Fig. 7, intra-arterial injection of ET-1 produced an immediate fall in P_sys that was significant even at a dose of 10 pmol/kg, but this was followed by a prolonged hypertensive response. The hypertensive phase lasted for approximately 10-15 min at the 10-pmol/kg dose and 45-60 min at the 300-pmol/kg dose, before the pressure returned to preinjection levels. During the initial hypotensive phase, R_sys decreased, and a significant increase in Q_sys was observed at the 10- and 30-pmol/kg doses (Fig. 7). Q_pul showed a dose-dependent decrease and mean P_pul did not change, indicating that R_pul increased. During the second hypertensive phase, R_sys increased and Q_sys progressively decreased (Fig. 7). The only change in hemodynamic parameters was a small increase in P_pul at the highest dose (300 pmol/kg). As in the case of alligator ET-3, there was no change in HR after injection of ET-1 at any dose tested.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effect on Psys (in kPa) of bolus injections of increasing doses of human/alligator ET-1 (10, 30, 100, and 300 pmol/kg) in a single animal.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Mean values of the absolute changes from controls of hemodynamic parameters after injection of increasing doses of human/alligator ET-1: Rsys (A), Psys (B), Qsys (C), Rpul (D), Ppul (E), and Qpul (F). For A-C and E, n = 8; for D and F, n = 6. Error bars indicate SEs. * Statistically significant difference from control (P  0.05).

Effects of L-NAME. As shown in Fig. 8, separate intravenous injections of ET-1 (300 pmol/kg) and L-arginine (150 mg/kg) produced a rapid and significant hypotensive response in animals equipped only with arterial and venous catheters. After administration of L-NAME, the mean arterial blood pressure was significantly elevated, but there was no difference in the hypotensive response to ET-1 before or after L-NAME injection. In contrast, the response to L-arginine was significantly attenuated after administration of L-NAME.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Effect on Psys of successive bolus intra-arterial injections of human/alligator ET-1 (A), L-arginine (L-Arg; B), N-nitro-L-arginine methyl ester (L-NAME; C), human/alligator ET-1 after L-NAME treatment (D), and L-Arg after L-NAME treatment (E). Data show means ± SE; n = 5.  Significant change in Psys after injection; * significant difference compared with injection of same drug before L-NAME treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In most mammalian tissues, ET is secreted by the constitutive pathway (19), resulting in steady-state concentrations of the peptide that are very low. This poses a challenge to the peptide chemist wishing to obtain sufficient pure material to permit structural characterization, but recent advances in the instrumentation of microsequence analysis allow amino acid sequence determination of very low picomole amounts of a specific peptide. Studies in mammals have shown the lung (15) and kidney (1) not only synthesize ET but are sites of uptake of circulating ET that is internalized through receptor-mediated endocytosis. The alligator lung and kidney were chosen as the tissues from which to extract the peptides, but it is unclear to what extent the ET isoforms isolated in this study represent peptide that is synthesized in the tissues rather than material that has been taken up by the organ from the circulation. This article describes the purification of two components corresponding to ET-1 and ET-3 from lung, together with a single molecular form corresponding to ET-3 from kidney. The primary structures of these peptides are compared with those of previously characterized ET isoforms in Fig. 1.

Our study has demonstrated that the primary structures of the ET isoforms have been very strongly conserved throughout the evolution of tetrapods. ET-1 is structurally identical in an alligator, human, and frog. Alligator ET-3 is the same as in a frog and differs from human ET-3 by a single conservative amino acid substitution (Phe⁴  Tyr). It has been proposed that the ET family of peptides arose from a series of gene duplication events (9). Duplication of the gene encoding an ancestral ET gave rise to a gene encoding ET-3 and a second gene encoding the ancestor of ET-1 and ET-2. A subsequent duplication of this latter gene gave rise to separate genes encoding ET-1 and ET-2. As all mammalian species yet studied synthesize the three ET isoforms, both gene duplications are presumed to have occurred before the appearance of mammals. Our failure to isolate ET-2 from the alligator tissues examined suggests, but of course does not prove, the hypothesis that the second gene duplication took place after the appearance of the reptiles, possibly within the mammalian lineage.

The biosynthesis of the ET isoforms in mammals is atypical in that posttranslational processing of preproendothelin at the site of dibasic amino acid residues by the well-characterized prohormone convertases produces a big ET, of between 38 and 41 amino acids depending on the species, that has low biological potency (19). Big ET is further processed by a highly selective enzyme, ET-converting enzyme, that exists in several isoforms and cleaves at the Trp²¹-Val²² bond in big ET-1 and big ET-2 and at the Trp²¹-Ile²² bond in big ET-3 (22). The isolation of alligator ET-1 and ET-3 in 21-amino acid residue forms indicates that preproendothelins are processed in alligators, as in a teleost fish (26) and an amphibian (27), by a pathway similar to that in mammals.

The cardiovascular actions of the ET isoforms in mammals are mediated through interaction with two well-characterized receptors: the ET_A receptor that is selective for ET-1 and ET-2 and the ET_B receptor that exhibits similar affinities for all three isopeptides (23). Activation of the ET_A receptor on vascular smooth muscle cells leads to vasoconstriction, whereas activation of the ET_B receptor on endothelial cells elicits vasodilatation by a mechanism that involves the formation of nitric oxide (14). However, studies using ET_B-selective agonists and antagonists have identified ET_B receptors on vascular smooth muscle that also mediate vasoconstriction (13).

Our data demonstrate that the hemodynamic responses of the alligator to ET-1 and ET-3 are qualitatively similar to the responses in mammals, but the magnitude and duration of the hypotensive phase are appreciably greater. For example, intravenous bolus injection of ET-1 (250 pmol/kg) into unanesthetized, normotensive rats produces a fall in mean arterial blood pressure of 3.5 kPa of <1-min duration followed by a sustained rise with a maximum increase of 3.7 kPa occurring ~10 min after administration (12). The effects of alligator ET-1, at all doses tested, are also biphasic with an initial systemic vasodilation and fall in P_sys followed by a prolonged constriction of the systemic vasculature and hypertension. The effects of ET-1 on the systemic circulation were greater than on the pulmonary circulation. The changes in Q_pul are probably reflections of changes in cardiac output rather than direct effects on the pulmonary circulation elicited by ET-1. The lack of effect of ET-1 on HR contrasts with the bradycardia seen in unanesthetized trout (5, 10) and is probably a consequence of the fact that anesthetic used in this study abolishes the baroreceptor-mediated cardioinhibitory reflex in reptiles (3).

Alligator ET-3, at doses up to 100 pmol/kg, elicits dose-dependent decreases in R_sys, leading to a fall in arterial blood pressure that persisted for up to 10 min. At the highest dose tested (300 pmol/kg), the sustained hypotensive phase (up to 20 min) was followed by a significant increase in R_sys, but the effect on arterial blood pressure was not significant. By way of comparison, a bolus injection of ET-3 (1.1 nmol) into pentobarbital sodium-anesthetized cats produced a decrease in P_sys of ~4.3 kPa whose duration was only 30-120 s (11). As found with ET-1, effects of ET-3 in the systemic circulation were more pronounced than in the pulmonary circulation. The only effects of ET-3 on the pulmonary circulation were a decrease in pressure and flow after the maximum dose, similar to the effects seen in the systemic circulation at the same time. The reduction in blood pressure was probably initiated by a decrease in R_sys, leading to a decreased venous return to the heart and a subsequent reduction in stroke volume and cardiac output.

The circulating concentrations of ET-LI in the alligator are below the detection limit of the radioimmunoassay used in this study (unpublished data) so that injection of ET, even in a dose as low as 10 pmol/kg, probably produces plasma levels of the peptide that are supraphysiological. It may be argued, therefore, that the cardiovascular effects reported here may be more pharmacological than physiological in character. On the other hand, the study has shown that ET is synthesized in alligator lung and kidney and may also be made in the endothelial cells of vascular tissue, as in mammals (29). Consequently, the peptide may exercise its regulatory effects on cardiovascular function by a mechanism that involves a paracrine rather than an endocrine action.

Perspectives