INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE KALLIKREIN-KININ SYSTEM in mammals involves the sequential action of a series of well-characterized proteolytic enzymes. Activation of factor XII (Hageman factor) in blood at the site of tissue injury or in vitro by contact with a charged surface results in activation of plasma prekallikrein and generation of bradykinin (BK) by the cleavage of high-molecular mass kininogen. BK is rapidly degraded, primarily in the pulmonary circulation, by the action of carboxypeptidase N (kininase I), angiotensin-converting enzyme (kininase II), and endopeptidase 24.11 (3). The blood of some reptiles appears to contain all the components of the kallikrein-kinin system present in mammals (7). For example, treatment of plasma from the alligator, Alligator mississipiensis (6), and from the red-eared turtle, Pseudemys scripta (10), with a charged surface (glass beads) in the presence of a kinase inhibitor generated a kinin that differs from mammalian BK by one amino acid substitution (Ser⁶  Thr).

The existence of a kallikrein-kinin system in snakes has not been demonstrated unequivocally. When treated with trypsin, plasma from the viper, Bothrops jararaca, generated a peptide that had hypotensive effects in this snake and contracted the snake uterus but did not elicit biological effects in mammals (i.e., no contraction of the rat uterus or guinea pig ileum and no vasodilatation of the perfused dog hindleg) (1). It was proposed therefore that snakes may have developed their own kallikrein-kinin system that generates a structurally different kinin with different bioactivity (1). Consistent with this hypothesis, incubation of heat-denatured plasma of a relatively primitive snake of the Boidea family, the reticulated python, Python reticulatus, with trypsin generated a BK-related peptide that differed from mammalian BK by two substitutions (Arg¹  Ala and Ser⁶  Thr) (9). Similar treatment of plasma from the more derived colubrid snakes, the bullsnake (Pituophis melanoleucus sayi) and the coachwhip (Masticophis flagellum) produced [Val¹,Thr⁶]-BK (19). However, treatment of plasma from these snakes with glass beads, under conditions that generated [Thr⁶]-BK in turtle and alligator, did not produce a kinin, and so it is probable that a component analogous to mammalian factor XII is not present in snake blood (9, 19).

The tachykinins are a family of myotropic peptides that have been identified by immunohistochemical and radioimmunoassay techniques in nervous or neuroendocrine tissues from all classes of vertebrate including reptiles (reviewed in Ref. 16). Tachykinins share a common amino acid sequence at the COOH-terminus, represented by -Phe-X_aa-Gly-Leu-Met-NH₂, where X_aa is an aromatic or branched-chain aliphatic residue (21), and this region interacts with receptors in smooth muscle (24, 25). The mammalian tachykinins comprise substance P (SP), neurokinin A, neurokinin B, and the NH₂-terminally extended forms of neurokinin A, neuropeptide K, and neuropeptide- (5). The primary structures of the tachykinins have been relatively poorly conserved during vertebrate evolution, and only the amino acid residues important for receptor binding are identical across species (reviewed in Ref. 33). In the case of reptiles, the identical SP from the Burmese python, Python molurus (13), and the desert tortoise, Gopherus agassizii (31), shows two amino acid substitutions (Lys³  Arg and Phe⁸  Tyr) compared with mammals, whereas SP from the alligator, Alligator mississipiensis, contains only the substitution (Lys³  Arg) (32).

The biological actions of native kinins in snakes are unknown. Elucidation of the primary structure of python BK and SP has permitted an investigation of the biological activities of these peptides in a snake with a view to gaining insight into their physiological roles. The objective of the present study therefore was to study the cardiovascular effects of synthetic replicates of python BK and SP in the anesthetized ball python, Python regius.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental animals. All experimental procedures were approved by University of Göteborg Ethical Committee (permit number 101/99). Ball pythons (Python regius) of both sexes, weighing between 650 and 900 g, were obtained from a local animal supplier and housed collectively in a vivarium (150 × 60 × 60 cm) with free access to water and under a 12:12-h light-dark photoperiod. The vivarium was equipped with a heating lamp, thus providing a temperature gradient between 25 and 35°C that allowed for behavioral thermoregulation. All animals appeared healthy and fasted for more than 6 wk before experiments.

Anesthesia, surgery, and hemodynamic measurements. The snakes were anesthetized by an intraperitoneal injection of 15-20 mg/kg pentobarbital sodium. This dose was generally sufficient to keep the snakes fully anesthetized for the duration of the experiment, but in one snake it was necessary to administer an additional dose of 10 mg/kg. When the snakes did not exhibit ciliary responses they were placed in a prone position and the trachea was exposed ~5-10 cm from the snout. The trachea was cannulated with tubing of suitable diameter for artificial ventilation at a rate similar to ventilation in resting and undisturbed python (14 ml/breath; 1.3 breaths/min) (22), using a Braun Melsungen type 874052 (Melsungen, Germany) ventilator.

Data recording. The pressure transducer was connected to Grass polygraph (model 7G) recorder for appropriate amplification and filtering. The blood flow probes were connected to a transonic dual-channel blood flowmeter (T206) for measurements of mean blood flows; the blood flowmeter was in turn connected to the Grass polygraph. Heart rate (HR) was derived from the pulsatile flow in the right or left aortic arches using a Grass tachograph (model 7P44D). All the signals were fed into a PC computer for on-line data acquisition using Labview 5.1 (National Instrument, Austin, TX).

Calculation of systemic blood flow and systemic vascular resistance. Systemic blood flow (Q_sys) was calculated as the sum of the recorded blood flows in the left and right aortic arches, respectively. Because we placed the blood flow probe after the carotid artery branched from the right aortic, our measurements of blood flow in the right aortic arch and hence the calculated Q_sys, represents a slight underestimation. Systemic resistance (R_sys) was calculated as the ratio of mean blood pressure to Q_sys (R_sys = P/Q_sys) under the assumption that both atrial pressures are zero. Instantaneous HR was recorded on the basis of systemic blood pressure, and systemic stroke volume was calculated as Q_sys/HR.

Peptide synthesis. Python BK (Ala-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) and python SP (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) were synthesized by solid-phase methodology on a 0.025-mmol scale using an Applied Biosystems model 432 synthesizer. Fluorenylmethoxycarbonyl-labeled amino acids were coupled as their hydroxybenzotriazole-active esters following the manufacturer's standard protocols. After cleavage from the resin, the peptides were purified to near homogeneity by reversed-phase high-performance liquid chromatography (HPLC). Their identities were confirmed by automated Edman degradation and electrospray mass spectrometry (python BK, observed mass 988.5 Da, calculated mass 988.5 Da; python SP, observed mass 1,390.8 Da, calculated mass 1,390.7 Da). Turtle and/or alligator BK (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was synthesized and characterized as previously described (10).

Experimental protocol. All experiments were conducted at 25°C. When the snakes were instrumented for measurements, blood flows and pressures were allowed to stabilize for ~60 min. Python BK and SP, dissolved in 0.9% saline containing 0.1% bovine serum albumin, were injected through the arterial catheter as a 0.1 ml/kg bolus followed by an injection of 0.2 ml/kg physiological saline to rinse the catheters. Hemodynamic variables were allowed to return to baseline levels between each injection, and there were no hemodynamic effects after injection of vehicle only (n = 9). The peptide injections invariably commenced with the lowest concentrations, and the series of python BK and SP injections were performed in random order. The following doses were administered: python SP 0.01, 0.1, 0.3, 1.0, 3.0, 10, and 30 pmol/kg; python BK 0.03, 0.1, 0.3, 1.0, and 3.0 nmol/kg. In six snakes, two doses of turtle and/or alligator BK (0.1 and 1.0 nmol/kg) were injected immediately before or after the same dose of python BK.

Data analysis and statistics. All recordings of blood flows and pressures were analyzed using AcqKnowledge data analysis software (version 3.23; Biopac, Goleta, CA). The effects of python BK and SP injections on hemodynamic variables were assessed by a one-way ANOVA for repeated measures. The effects of python BK and turtle BK were compared by a two-way ANOVA for repeated measures. In both cases, mean values that were significantly different from control values were identified by a subsequent Student-Newman-Keuls test. The effects of python BK injection on plasma catecholamine levels were assessed by a paired t-test. A limit for significance of P < 0.05 was applied, and data are presented as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cardiovascular effects of python BK. Figure 1 shows the effects following a bolus injection of 0.3 nmol/kg python BK as a function of time (Fig. 1, A-E) and the dose-response relationships of the maximum hemodynamic changes at various concentrations (Fig. 1, F-J). Python BK caused an immediate and pronounced tachycardia. At the higher concentrations (0.3-3.0 nmol/kg), HR doubled relative to preinjection levels and remained elevated for up to 15 min. Stroke volume decreased initially but returned to preinjection values within 90 s following injection. Consequently, the massive tachycardia was associated with a large increase in Q_sys and, because R_sys decreased only slightly, the peptide caused a large increase in arterial blood pressure. The hemodynamic changes at the higher python BK concentrations were biphasic, such that Q_sys initially decreased slightly within the first 60 s after python BK injection with a simultaneous increase in R_sys. However, at all concentrations studied, R_sys significantly decreased at 120 s in a dose-dependent manner (Fig. 1I). The effects of 3 nmol/kg BK on any cardiovascular parameter were not significantly greater than the effects of 1 nmol/kg, indicating that the lower dose produced a near maximum response.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Effects of a bolus intra-arterial injection of python bradykinin (BK, 3 nmol/kg) on hemodynamic parameters (HR, heart rate; Qsys, systemic blood flow, BP, mean arterial blood pressure, Rsys, systemic vascular resistance; Vs, stroke volume) in the anesthetized python as a function of time (A-E) and the effects of the peptide as a function of the amount injected (F-J). Data points show means ± SE for 9 independent experiments. * Significantly different (P < 0.05) from the corresponding values following injection of vehicle only. In A-E, the peptide was administered at time +30 s.

Effects of adrenergic blockers and levels of circulating catecholamines. The effects of adrenergic blockade on the hemodynamic responses to python BK injections are presented in Figs. 2 and 3. In untreated snakes, the bolus injections of epinephrine (1 nmol/kg) significantly elevated HR, systemic blood pressure, and Q_sys, whereas R_sys did not change. Injection of the -adrenergic receptor antagonist sotalol did not affect blood pressure but caused a significant increase in R_sys and a small reduction in Q_sys from 9.6 ± 1.4 to 8.0 ± 2.5 ml · min¹ · kg¹ (not significant). Sotalol almost completely abolished the tachycardia following both python BK (3 nmol/kg) and epinephrine (1 nmol/kg) injections (Fig. 2A). Thus in untreated animals, python BK injection resulted in an increase in HR from 24.8 ± 3.3 to 42.2 ± 3.6 beats/min, whereas in sotalol-treated animals, HR did not increase significantly (from 19.9 ± 2.2 to 21.4 ± 2.3 beats/min). After sotalol, python BK did not reduce R_sys, whereas epinephrine injection led to a small increase in blood pressure and R_sys with no changes in Q_sys.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Effect of bolus intra-arterial injections of python BK (3 nmol/kg) and epinephrine (1 nmol/kg) on hemodynanic parameters in the anesthetized python (n = 5) before and after treatment with sotalol. Control, effects following injection of vehicle only. * Significantly different (P < 0.05) from control values

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effect of bolus intra-arterial injections of python BK (3 nmol/kg) and epinephrine (1 nmol/kg) on hemodynanic parameters in the anesthetized python (n = 5) before and after treatment with prazosin. * Significantly different (P < 0.05) from control values following injection of vehicle only.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of a bolus intra-arterial injection of python BK (3 nmol/kg) on the concentrations of plasma catecholamines in the anesthetized python. Blood samples were taken at a time when the increase in arterial blood pressure was at a maximum. * P < 0.05 compared with preinjection concentrations.

Comparison of the cardiovascular actions of python and turtle and/or alligator BK. The hemodynamic effects of bolus injections of turtle and/or alligator BK (0.1 and 1.0 nmol/kg) are compared with the corresponding effects of the injections of the same dose of python BK in Table 1. The effects of 0.1 nmol/kg turtle and/or alligator BK on HR and arterial blood pressure were significantly less that the corresponding effects of 0.1 nmol/kg of python BK but the effects of 1.0 nmol/kg injections of the peptides were not significantly different. There was no difference in the effects of python BK and turtle and/or alligator BK on cardiac output and R_sys at either dose tested.

Cardiovascular actions of python SP. The mean hemodynamic effects of a bolus intra-arterial injection of 3.0 pmol/kg python SP as a function of time are shown in Fig. 5, A-E, and the maximum hemodynamic changes following injections of the various concentrations of python SP are depicted in Fig. 5, F-J. Injection of python SP elicited a large reduction in systemic peripheral resistance (Fig. 5I) with an attendant decrease in systemic blood pressure (Fig. 5H), whereas Q_sys increased (Fig. 5G). There were no significant effects of python SP on HR, except for a slight increase after an injection of 10 pmol/kg (Fig. 5F), but the peptide produced an increase in stroke volume (Fig. 5J). All hemodynamic effects, except HR, were augmented as the dose of python SP increased reaching a maximum response at a dose of 10 pmol/kg.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effects of a bolus intra-arterial injection of python substance P (3 pmol/kg) on hemodynamic parameters in the anesthetized python as a function of time (A-E) and the effects of the peptide as a function of the amount injected (F-J). Data points show means ± SE for 9 independent experiments. * Significantly different (P < 0.05) from the corresponding values following injection of vehicle only. In A-E, the peptide was administered at time +30 s.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The cardiovascular actions of BK-related peptides differ appreciably among tetrapods (7). In mammals, vascular injection of BK rapidly reduces blood pressure by an arteriolar vasodilatation that arises from activation of prostaglandins and nitric oxide synthesis (15). At high doses and in unanesthetized animals, the hypotensive effect is followed by an increased blood pressure, HR, and renal vascular resistance caused by activation of nociceptive pathways and stimulation of afferent sympathetic fibers (29). In the chicken, bolus injections of ornithokinin ([Thr⁶,Leu⁸]-BK) produced a strong hypotensive response of short duration (<1 min) (18) and a similar response was observed following bolus injections of [Thr⁶]-BK in anesthetized alligators (6). In contrast, bolus injections of [Thr⁶]-BK produced no change in systemic blood pressure in the anesthetized turtles but an observed increase in blood flow in the left aortic arch indicated that this peptide reduced peripheral vascular resistance, as observed in alligators and mammals (10).

The present study was designed to investigate the direct effects of python BK and SP on cardiovascular parameters in pythons. We performed the experiments on phenobarbitone-anesthetized animals because barbiturates produce a general depression of central nervous system activity, including a reduction in respiratory drive and a dampening of cardiorespiratory reflexes. The use of relatively deep anesthesia, such that we did not observe behavioral or cardiovascular responses to stimuli such as tail pinching, means that effects arising from the activation of nociceptive pathways are probably not important. Furthermore, a study in anesthetized animals allows a direct comparison with data obtained with native BK peptides in the anesthetized turtle (10) and alligator (6). The mean values for arterial blood pressure and for concentrations of circulating catecholamines measured in the unstimulated state in the anesthetized python are comparable to those measured in the unrestrained resting snake, the black racer, Coluber constrictor (average daytime blood pressure 43 cmH₂O, HR 30-40 beats/min, norepinephrine 0.3 ± 0.1 ng/ml, epinephrine 1.1 ± 1.4 ng/ml) (28). However, it was shown that handling the snakes resulted in an increase in HR to near 100 beats/min and 51- and 26-fold increases in plasma epinephrine and norepinephrine, respectively (28). It is hoped that a future study will investigate the cardiovascular action of kinins in the unanesthetized python but clearly, in the light of such a marked stress response, this kind of study presents severe technical problems.

Our study has shown that bolus arterial injections of python BK ([Ala¹,Thr⁶]-BK) into the anesthetized python increase arterial blood pressure, HR, and cardiac output in a concentration-dependent manner. These effects are clearly in marked contrast to those reported for the effects of [Thr⁶]-BK in the anesthetized turtle (10) and alligator (6). However, as in these reptiles, BK reduces systemic vascular resistance in the python, and therefore the major difference between snakes and other reptiles is the pronounced tachycardia observed in the present study. Because sotalol treatment almost completely abolished the tachycardia following python BK injection, the effect appears to be mediated primarily by activation of cardiac -adrenergic receptors. Similarly, the attenuation of the hypertensive response by prazosin indicates that this effect of python BK is mediated at least in part by activation of -adrenergic receptors in the peripheral circulation. The involvement of catecholamines was confirmed by the significant rise in the concentration of circulating norepinephrine following administration of the peptide. Because the circulating levels of epinephrine did not change after BK administration, it appears plausible that the peptide primarily activates sympathetic neurons, as in mammals (29), rather than stimulating catecholamine release from the adrenal gland. Morphological studies, particularly in the colubrid snake, Elaphe obseleta (rat snake), have demonstrated an extensive adrenergic innervation of the arteries and veins with a particularly dense innervation of the arteries posterior to the heart (12, 14). Lillywhite and Donald (20) comment that this density is much greater than in any other class of reptile examined and postulate that this is indicative of the importance of adrenergic regulation of hemodynamic parameters in snakes. In rats, microinjections of mammalian BK into the anterior hypothalamic medial preoptic nuclei produce tachycardia that arises from activation of cardiac sympathetic nerves (13). It is not known whether BK is able to cross the blood-brain barrier in the python so that central effects of the peptide on stimulation of HR cannot be excluded.

Structure activity studies with mammalian BK have shown that the amino acid substitution (Ser⁶  Thr) in the peptide has little or no effect on its cardiovascular actions in the rat (23) and turtle (10). However, [Ala¹]-BK is only one-tenth as potent as BK in lowering blood pressure in the rat after intra-arterial injection of the peptides (23). Our observation that [Thr⁶]-BK (1 nmol/kg) is equally effective as the same dose of [Ala¹,Thr⁶]-BK in the python suggests that the anomalous cardiovascular actions of python BK in the python are not a consequence of the substitution (Arg¹  Ala) in the peptide. It follows therefore that the ligand-binding properties of the BK receptor(s) in python tissues are appreciably different from the mammalian BK receptors.

The ability of SP to produce hypotension in mammals was one of the properties leading to its identification (30). In addition to the systemic vasodilatation that is mediated at least in part through release of nitric oxide (24), administration of SP to mammals and crocodilians increases cardiac output and in particular elevates blood flow to the intestine (17). In the chicken (27) and the toad, Bufo marinus (11), SP also causes a transient hypotension. In contrast to the anomalous cardiovascular responses to python BK, bolus intra-arterial injections of python SP in the python had similar effects to those of mammalian SP in mammals that is a concentration-dependent decrease in arterial blood pressure and R_sys and an increase in cardiac output. In our experiments, there was only a minute increase in HR during the hypotensive phase, which is consistent with the absence of baroresponses in the phenobarbitone-anesthetized animals. Python SP contains conservative amino acid substitutions at positions 3 (Lys  Arg) and 8 (Phe  Tyr) compared with mammalian SP. However, a structure-activity study, in which each residue in the peptide was replaced by alanine, demonstrated that amino acids at these positions are not involved in the interaction with the SP receptor in vascular tissue of the rabbit (26). Unfortunately, sufficient animals were not available to study the mechanism of action of python SP in the python.

Perspectives

Immunohistochemical studies have shown the sinus venosus, atria, and ventricle of E. obsoleta (14) are innervated by SP-containing neurons, and there is also an extensive and dense innervation by SP of both anterior and posterior arteries and veins where it is often colocalized with vasoactive intestinal peptide (12). We speculate therefore that release of SP from these neurons may have a functionally important role in maintaining adequate blood flow to vital anterior organs, particular when the python is adopting an upright posture during climbing (20).