Digestion of large meals in pythons produces substantial increases in heart rate and cardiac output, as well as a dilation of the mesenteric vascular bed leading to intestinal hyperemia, but the mediators of these effects are unknown. Bolus intra-arterial injections of python neurotensin ([His3, Val4, Ala7]NT) (1 − 1,000 pmol/kg) into the anesthetized ball python Python regius (n = 7) produced a dose-dependent vasodilation that was associated with a decrease in systemic pressure (Psys) and increase in systemic blood flow (Qsys). There was no effect on pulmonary pressure and conductance. A significant (P < 0.05) increase in heart rate (fH) and total cardiac output (Qtot) was seen only at high doses (>30 pmol/kg). The systemic vasodilation and increase in Qtot persisted after β-adrenergic blockade with propranolol, but the rise in fH was abolished. Also, the systemic vasodilation persisted after histamine H2-receptor blockade. In unanesthetized pythons (n = 4), bolus injection of python NT in a dose as low as 1 pmol/kg produced a significant increase in blood flow to the mesenteric artery (177% ± 54%; mean ± SE) and mesenteric conductance (219% ± 74%) without any increase in Qsys, systemic conductance, Psys, and fH. The data provide evidence that NT is an important hormonal mediator of postprandial intestinal hyperemia in the python, but its involvement in mediating the cardiac responses to digestion may be relatively minor.
- blood flow
- mesenteric artery
- heart rate
pythons can eat large meals, and digestion is associated with a marked rise in metabolic rate that is sustained for several days (33, 41). The metabolic response to feeding imposes great demands on the cardiorespiratory system and requires that perfusion of the gastrointestinal tract increases to absorb nutrients from the food. The cardiovascular responses to digestion include a doubling of heart rate and a fourfold increase in cardiac output, as well as a dilation of the mesenteric vascular bed leading to intestinal hyperemia (32, 34, 38, 41). Little is known, however, about the regulation of the postprandial rise in heart rate and dilation of the mesenteric arteries. In addition to the autonomic and enteric nervous systems, the postprandial cardiovascular response is likely to be governed by the direct action of endocrine regulatory peptides released from the gastrointestinal organs (1, 42). In pythons, digestion increases circulating levels of many regulatory peptides, including neurotensin (NT) (35).
NT was first isolated from bovine hypothalami (5) and is widely distributed throughout the central and peripheral nervous system, as well as the gastrointestinal tract in mammals and reptiles (6, 26, 35). In rats and dogs, systemic administration of NT causes hypotension, increases vascular permeability, and increases intestinal blood flow (5, 19, 29). NT also leads to tachycardia and increases cardiac contractility (15, 20). These findings, together with the elevated levels of circulating levels of NT during digestion (30, 35), suggest a role for NT in regulating the postprandial cardiovascular changes.
NT-like immunoreactivity is found in all vertebrate classes, and the primary structure of the peptide has been strongly conserved among reptiles. Python NT is identical to chicken and alligator NT, although this amino acid sequence differs from human NT at three sites (Tyr3 → His, Glu4 → Val, Pro7 → Ala) (9). Only few studies have investigated the biological activity of NT in other vertebrates than mammals. The present investigation extends our understanding of the evolution of the function of regulatory peptides by describing the cardiovascular activity of a synthetic replicate of python NT on the pulmonary and systemic circulations, including the mesenteric vascular bed, in both anesthetized and recovered ball pythons, Python regius. We hypothesized that NT would induce hemodynamic changes resembling the cardiovascular changes in the postprandial python.
MATERIALS AND METHODS
Python NT (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu) was supplied in crude form by GL Biochem (Shanghai) and purified to near homogeneity by reversed-phase HPLC on a (2.2 × 25-cm) Vydac 218TP1022 (C-18) column (Separations Group, Hesperia, CA). The purity of the peptide was >98% and its identities were confirmed by electrospray mass spectrometry. The peptide was dissolved in 0.01% vol/vol acetic acid, and aliquots were stored at −20°C. For injections, the peptide was diluted to the desired concentration with 0.9% (wt/vol) saline containing 0.1% (wt/vol) BSA immediately before use.
Ball pythons, P. regius, weighing between 0.24 and 1.12 kg (0.69 ± 0.09 kg; means ± SE; n = 16) were obtained from a local animal supplier and transported to the University of Aarhus, where they were kept in vivaria at 25–30°C. All snakes appeared healthy and had free access to water, but food was withheld for at least 1 wk before the experiments. All experiments were carried out by authorized investigators according to Danish Federal Regulations.
Surgery and Instrumentation
Studies on anesthetized snakes.
Twelve pythons were anesthetized by an intramuscular injection of pentobarbital sodium (mebumal, 25 mg/kg). All reflexes disappeared within 30 min, and the animals were then tracheostemized for artificial ventilation at 4 breaths/min and a tidal volume of 50 ml/kg using a Harvard Apparatus mechanical ventilator. A 5-cm lateroventral incision was made cranial to the heart, and a polyethylene (PE)-50 catheter, filled with heparinized saline, was advanced into the vertebral artery for measurements of systemic blood pressure (Psys). The left pulmonary artery, which perfuses the smaller left lung and carries less than a quarter of the total pulmonary blood flow (unpublished observations), was occlusively cannulated with PE-50 catheter for measurements of pulmonary blood pressure (Ppul). For measurements of blood flows 1.5R transit-time ultrasonic blood flow probes (Transonic Systems, NY) were placed around the left aortic arch (LAo) and the right pulmonary artery.
Studies on recovered snakes.
Four pythons were anesthetized by ventilation with 1–2% isofluran (Isofluran, Baxter, Denmark) during surgery. For measurements of systemic blood pressure, a PE-50 catheter was advanced into the vertebral artery. To record left aortic blood flow (QLAo) and mesenteric blood flow (Qmesen) probes (1–1.5 R) were placed around the left aortic arch and mesenteric artery branching of the aorta caudally to the gall bladder. The catheter was externalized, the incisions closed, and the snake was allowed to recover from surgery in a box at 25°C until the following day.
All catheters were connected to Baxter Edward (model PX600, Irvine, CA) disposable pressure transducers, and the signals were amplified using an in-house built preamplifier. The pressure transducers were positioned at the level of the heart of the animal and were calibrated daily against a static water column. Acoustical gel was infused around the blood flow probes to enhance the signal. Flow probes were connected to a Transonic dual-channel blood flow meter (T206).
Signals from the pressure transducer and the blood flow meter were recorded with a Biopac MP100 data acquisition system (Biopac Systems, Goleta, CA) at 100 Hz.
Studies on anesthetized pythons.
After instrumentation, all basal hemodynamic variables were recorded for up to 45 min. To assess whether the vehicle for injections had hemodynamic effects, a 1 ml/kg injection of 0.9% (wt/vol) saline containing 0.1% (wt/vol) BSA (vehicle only) was given. A series of bolus injections of increasing doses of NT was given (1, 3, 10, 30, 100, 300, and 1,000 pmol/kg). The lower range of the dosages is within the physiologically relevant range for postprandial pythons, whereas the higher dosages are primarily of pharmacological relevance. Hemodynamic variables were allowed to return to baselines between each injection. All drugs were given in 1.0 ml/kg aliquots and injected through the systemic catheter.
To investigate whether the effects of NT were mediated through the release of adrenaline, bolus injections of NT (100 pmol/kg) and adrenaline (2 μg/kg) were given before and after β-adrenergic blockade with propranolol (4 mg/kg). To investigate whether the effect of NT was mediated through the release of histamine, a different set of animals received bolus injections of NT (100 pmol/kg) before and after the histamine H2-receptor antagonist ranitidine (40 mg/kg). This dose of ranitidine abolishes the effect of histamine (10 nmol/kg) in pythons (N. Skovgaard and T. Wang, unpublished observation). The antagonists were allowed 20 min to take affect before subsequent injections.
Studies on recovered pythons.
NT failed to stimulate changes in mesenteric blood flow in the anesthetized snakes and we, therefore, included experiments on recovered snakes. Basal hemodynamic variables were recorded for up to 1 h to allow for the animals to return to a quiescent state after handling. The animals curled up under a bowl and remained inactive during the protocol. Two bolus injections of NT (1 and 100 pmol/kg) were given, and hemodynamic variables were allowed to return to baseline between injections. All experiments were carried out at 25°C. After completion of protocols, both anesthetized and recovered snakes were killed with a lethal dose of Mebumal.
Calculations of Blood Flows, Stroke Volume, and Vascular Conductances
Because the left pulmonary artery was occlusively cannulated, blood flow measurements in the right pulmonary artery represent total pulmonary blood flow (Qpul). In anesthetized pythons, total systemic blood flow (Qsys) can be estimated as 2.5 times QLAo (36). Total cardiac output (Qtot) was calculated as Qsys + Qpul. Heart rate (fH) was calculated from the instantaneous blood flow trace from the left aortic arch and total stroke volume (VStot; pulmonary + systemic) was calculated as Qtot/fH. Pulmonary, systemic and mesenteric conductance (Gpul, Gsys, and Gmesen, respectively) was calculated from mean blood flow and mean blood pressure (Gpul = Qpul/Ppul, Gsys = Qsys/Psys and Gmesen = Qmesen/Psys), assuming that central venous blood pressures are negligible.
Data Analysis and Statistics
All recordings of blood flows and pressures were analyzed using AcqKnowledge data analysis software (version 3.7.1., Biopac, Goleta, CA). Hemodynamic effects of NT were assessed using a one-way ANOVA for repeated measurements followed by a Dunnett's post hoc test to identify values that were significantly different from control values. Effects on hemodynamic variables of NT and adrenaline before and after antagonists were evaluated using a paired t-test. Differences were considered statistically significant at a 95% level of confidence (P < 0.05). All data are presented as means ± SE.
The effects of increasing amounts of NT on maximum changes are presented in Figs. 1 and 2. Injection of NT produced a dose-dependent systemic vasodilation that was associated with a decrease in Psys and a rise in Qsys, causing Gsys to increase (Fig. 1, A–C). There were no effects on Ppul and Gpul, but Qpul increased together with a rise in fH and Qtot (Figs. 1, D–F and 2). However, a significant increase in fH was seen only at doses ≥100 pmol/kg and in Qtot in doses ≥30 pmol/kg.
The systemic vasodilation produced by NT (100 pmol/kg) persisted after β-adrenergic blockade with propranolol and after histamine H2-receptor blockade with ranitidine (Table 1). The effect of NT on fH was completely blocked after propranolol, but the increase in Qtot persisted due to the rise in VStot (Table 1). Bolus injections of adrenaline significantly increased Psys from 4.1 ± 0.4 to 6.0 ± 0.5 kPa and fH from 31.2 ± 5.6 to 38.8 ± 4.6 min−1. After administration of propranolol, adrenaline increased Psys from 4.2 ± 0.5 to 7.6 ± 0.8 kPa, but the heart rate response was abolished (from 22.9 ± 1.8 to 22.5 ± 2.0 min−1), showing that the blockade of the β-adrenergic receptors was successful.
At the low dose of NT, there was a vasodilation of the mesenteric artery followed by a significant increase in Qmesen from 0.78 ± 0.13 ml·min−1·kg−1 to 1.97 ± 0.13 ml·min−1·kg−1 (Figs. 3 and 4). There were only small, nonsignificant changes in fH, Psys, and in overall systemic blood flow and conductance (Qsys and Gsys). At the high dose of NT, there was an increase in Gmesen as well as Gsys concomitant with an increase in Qsys, Qmesen, and reduction in Psys. There was no significant change in fH.
Neurotensin elicited tachycardia, systemic vasodilation, and a rise in mesenteric blood flow in pythons. These effects resemble the hemodynamic changes during digestion, which has been proposed to be governed by regulatory peptides (35, 41, 42). Plasma NT levels increase markedly during digestion in pythons (35), and although the mechanism of release remains to be characterized in reptiles, NT is likely to be released in response to the presence of intraluminal fat in the intestine, as described for mammals (30). Apart from increasing gastrointestinal blood flow, the postprandial rise in circulating levels of NT has been implicated in the stimulation of intestinal motility and pancreatic and biliary secretions (10, 44). Also, it increases intestinal capillary permeability and is likely to be involved in fatty acid translocation in the intestine (12, 44). Oppositely, NT inhibits gastric motility, gastric mucosal blood flow, and gastric acid secretion in mammals (11, 31). In crocodiles, NT also decreases celiac blood flow but increases heart rate (14).
NT-like immunoreactivity is widely distributed in the nervous system and the gastrointestinal tract of all classes of jawed vertebrates (3, 6, 13, 21, 25). In mammals, NT is primarily found in the ileum and jejunum (6), whereas NT is distributed throughout both the small and large intestine of reptiles (4, 25). In both groups, NT is stored in endocrine cells (N cells) in the mucosa (4, 6).
NT elicited a strong dose-dependent systemic vasodilation and hypotension in anesthetized pythons, while the pulmonary circulation was not affected. At higher doses, NT increased heart rate and cardiac output. The effects of NT appeared to be more pronounced in recovered compared with anesthetized snakes. Thus, upon injection of 100 pmol/kg, the rise in systemic flow was considerably larger in the anesthetized snakes, reflecting a larger increase in Gsys. This may indicate that pentobarbital reduces the sensitive of the systemic vasculature. The vasodilation could not be blocked by the β-adrenergic antagonist propranolol, showing that it is not mediated through elevated sympathetic activity. However, upon β-adrenergic blockade, NT caused VStot to increase in the anesthetized snakes, which may reflect a direct inotropic effect of NT on the cardiomyocytes. In recovered snakes, the low dose of NT that is likely to result in plasma levels comparable to those measured previously in postprandial pythons (35) dilated the mesenteric beds and increased intestinal blood flow. This dose only elicited small nonsignificant changes in systemic pressure and overall systemic conductance, indicating that NT specifically regulates intestinal blood flow. It is likely, therefore, that increased circulating NT concentration contributes to the increased perfusion of the gastrointestinal organs during digestion in pythons (32, 38). Only a few other studies have investigated the biological activity of NT in nonmammalian vertebrates. In the Australian lungfish, NT increases dorsal aortic pressure and decreases heart rate and mesenteric blood flow (13).
In mammals, NT causes pronounced effects on blood pressure, but the responses differ among species. In rats, low doses of NT cause hypotension, whereas larger doses elicit a biphasic response (8, 40). In guinea pigs, NT causes a dose-dependent pressor effect (15). In mammals, NT vasodilates the mesenteric vascular bed and, at concentrations below those eliciting hypotension, NT increases intestinal blood flow without significant changes in flows to other organs (12, 19, 29). The effects on the intestinal vasculature occur at considerably higher plasma NT concentrations than those measured during digestion (22), and the physiological role is accordingly uncertain. Given that NT is released from the intestinal mucosa, it is likely that local concentrations exceed that of plasma and exert a paracrine rather than an endocrine action in the intestine.
In anesthetized pythons, NT caused a marked tachycardia that was blocked by the β-adrenergic receptor antagonist. Because propranolol did not block the systemic vasodilation, it is likely that tachycardia is not mediated through increased levels of circulating catecholamines but rather by a NT-mediated presynaptic release of catecholamines from sympathetic nerves innervating the heart. This is consistent with the presence of NT immunoreactive nerve fibers in the sinoatrial and atrioventricular nodal cells, as well as the coronary vasculature and cardiomyocytes in mammals (26, 43). Thus, consistent with our observations in python, several studies on mammals have suggested that the positive chronotropic effect of NT may be mediated through increased activity of the sympathetic nervous system and an NT-mediated presynaptic release of catecholamines. Indeed, the hypertensive effects and tachycardia produced by high doses of NT in guinea pigs and rats could be abolished with a ganglion blocker and with α- and β-blockers (2, 15), but other studies on mammals indicate a direct positive chronotropic and ionotropic effect of NT (15, 23, 39). Alternatively, an activation of capsaicin-sensitive sympathetic visceral afferent nerve fibers may produce a secondary reflex activation of preganglionic sympathetic nerve fibers (2, 8, 27, 28). The NT-induced tachycardia occurred only at dosages producing much higher plasma concentrations than those measured during digestion in the python (35) and so may represent a secondary pharmacological response with minor physiological significance.
In mammals, NT causes histamine release from mast cells (7, 16, 17, 18), and several of the effects produced by NT, including increased vascular permeability and hypotension, are mimicked by histamine and can be blocked by histamine antagonists, mast cell depletion, or mast cell stabilization (8, 24). Mast cells have been identified in snakes (37), and histamine dilates the systemic vasculature of the python P. regius that can be blocked by the H2-receptor antagonist, ranitidine (N. Skovgaard and T. Wang, unpublished observation). However, the systemic vasodilation produced by NT in pythons could not be blocked by H2 receptor blockade, indicating that the vascular effects of NT are unlikely to be mediated through the release of histamine. Therefore, it is possible that, in the python, the selective vasodilator action of NT on the vasculature is mediated through a direct action of the peptide on receptors on vascular smooth muscle and/or through generation of other second messenger molecules such as nitric oxide and prostacyclin.
In conclusion, NT exerts potent cardiovascular responses in pythons that resemble the postprandial cardiovascular changes. Thus, it is likely that the increased circulatory levels of NT upon feeding contribute significantly to cardiovascular responses associated with increased GI performance and metabolism during digestion.
This study was supported by the Danish Research Council, the NOVO Foundation and the Weis-Fogh Fund.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society