Comparative vascular responses in elasmobranchs to different structures of neuropeptide Y and peptide YY.

The vascular responses to neuropeptide Y (NPY) and peptide YY (PYY) were tested in several species of elasmobranchs to assess whether changes in sequence in these neuropeptides from elasmobranchs to mammals are associated with different physiological responses. NPY-like immunoreactivity was detected in the gut and in nerve fibres surrounding some, but not all, blood vessels of six species. Intravenous injection of dogfish, frog and human NPY in anaesthetised fish caused similar vasopressor effects in the three species tested, except human NPY which lowered blood pressure in one of the three. Dogfish NPY and PYY were equipotent pressor agents in two species, but PYY was significantly more potent than NPY in one species. NPY and PYY both contracted isolated gut arteries from three species, but had no effect on isolated efferent arteries tested. In conclusion, differential vascular responses in elasmobranchs are not associated with changes in NPY sequence across vertebrates, but may be with changes in PYY in some species.

The effect of galanin and galanin fragments on blood pressure in the Cane toad, Bufo marinus.

Galanin has previously been shown to have a slight vasodepressor or no effect on blood pressure in placental mammals, but causes potent increases in blood pressure in several other vertebrate species. In this paper, the part of the galanin molecule responsible for the pressor activity was investigated in the Cane road, Bufo marinus by administration of fragments of galanin into anaesthetised toads and isolated arterial segments in an organ bath. In anaesthetised toads, the order of efficacy was human galanin > porcine galanin > Gal 1-16 > Gal 1-15 > Gal 3-15 > Gal 21-29 = 0. In isolated vessels, the first three peptides were equally effective. When four fragments of human galanin were tested in anaesthetised toads, the order of efficacy was human galanin > Gal 1-15 > Gal 5-20 > Gal 10-25 > Gal 15-30. The substitution of alanine for tryptophan at position 2 or for tyrosine at position 9 abolished the pressor response to the human galanin fragment 1-18 in anaesthetised toads. These results suggest that vascular activity in the toad is retained within the N-terminus and that positions 2 (tryptophan) and 9 (tyrosine) are key amino acids in retention of the vascular activity of galanin.

Effect of three galanin antagonists on the pressor response to galanin in the Cane toad, Bufo marinus.

Galanin is a neuropeptide that causes a marked pressor response in several non-mammalian vertebrate species, and some marsupials. In this study, the effect of three galanin antagonists were tested on the pressor response to an intravenous dose (6.3 nmol/kg) of porcine galanin in anaesthetised Cane toads, Bufo marinus. Antagonists were injected at either 20 or 50 times the molar dose (x MD) of galanin. The antagonist, C7 (Galanin 1-13-spantide) reduced the pressor effect of galanin by 32.2 +/- 6.0% when delivered at 20 x MD (n = 4) and by 42.9 +/- 15.7% when delivered at 50 x MD (n = 4) of galanin, the response recovering within 30 min. A second antagonist, M32a (Galanin 1-13-NPY 24-36) had no effect on the pressor response to galanin at 20 x MD (n = 4), but significantly reduced the pressor effect by 54.8 +/- 6.4% at 50 x MD (n = 5), which also recovered within 30 min. Administration of a third antagonist, galantide or M15 (Galanin 1-13-Substance P5-11), resulted in a profound drop in blood pressure, and did not affect the response to galanin at either dose. In conclusion, C7 and M32a are effective, short-term antagonists of the blood pressure effects of galanin in the toad.

Vasoconstrictor effects of galanin and distribution of galanin containing fibres in three species of elasmobranch fish.

Galanin is found in perivascular sympathetic neurons in a wide range of vertebrate species. In placental mammals, galanin has either no effect on blood pressure, or weak depressor effects, but in other vertebrates it has been shown to be a potent pressor agent. To investigate how extensive the vasoconstrictor effects of galanin may be in the vertebrates, the vascular effects of galanin were tested in two species of shark, Heterodontus portusjacksoni, and Hemiscyllium ocellatum, and a ray, Rhinobatos typus. Nerve fibres showing immunoreactivity to galanin were located surrounding gut blood vessels, but were absent from branchial efferent arteries in all three species. Intravenous injection of galanin caused a significant rise in caudal arterial blood pressure in H. portusjacksoni and H. ocellatum, but no change in R. typus. Contraction of segments of pancreatico-mesenteric artery were measured in an organ bath also. Galanin (10(-6) M) caused 21-38% of the maximum K+ induced contraction in all species, but no response in efferent branchial arteries from R. typus. In conclusion, in three elasmobranchs, a galanin-like peptide is present in perivascular nerve fibres, and galanin causes differential vasoconstriction in vascular beds. These data extend the number of vertebrate groups in which galanin has been shown to be a vasoconstrictor peptide.

Physiological correlates of vagal nerve innervation in lower vertebrates.

In lower vertebrates, cardiac vagal innervation shows less anatomic complexity and specialization than in mammals. To assess the physiological development of vagal specialization in the vertebrates, we investigated cardiac chronotropic effects of electrical stimulation of left and right vagus nerves separately and the interactions between both nerves in anesthetized animals from three vertebrate groups, toad (Bufo marinus), shark (Heterodontus portusjacksoni), and lizard (Physignathus lesueurii). Atropine-sensitive slowing was effected equally by left or right vagi in all species, and chronotropic effects of simultaneous stimulation were the same as the sum of left and right responses. In sharks and lizards, no slowing after atropine was detected (10 Hz stimulation). In toads, after atropine, cardiac slowing was elicited equally by left or right vagal stimulation > 2 Hz. Simultaneous stimulation of both vagi after atropine caused significantly greater slowing than the sum of left and right responses. The results suggest even distribution of left and right vagal nerve endings to pacemaker cells, and limited competition for cardiac receptor sites in lower vertebrates.

Selective regional vasoconstriction underlying pressor effects of galanin in anaesthetized possums compared with cats.

1. Intravenous administration of porcine galanin (5 nmol kg-1) caused a rise in mean blood pressure in the brush-tailed possum, Trichosurus vulpecula, from 58 +/- 1.6 to 106 +/- 1.6 mmHg. This effect is in contrast to the cat, in which no significant change in blood pressure was recorded in response to galanin (88 +/- 2.3 vs. 86 +/- 2.4 mmHg). 2. Cardiac output and regional blood flow distribution were assessed by distribution of radioactive microspheres in four anaesthetized possums and four cats, before and after administration of galanin. 3. Cardiac output was 289.8 +/- 14.0 ml min-1 in the cat and 189.9 +/- 25.5 ml min-1 in the possum. Galanin administration did not significantly change cardiac output in either species. 4. In the possum, galanin administration caused large increases in resistance to flow in the spleen, gut, adrenal glands, kidney, skin and carcass. The largest increase was in the kidneys, where renal blood flow fell to 6% of control levels. 5. In the cat, changes in resistance were small. Small increases in resistance to flow in muscle and carcass were offset by small decreases in resistance in the lungs and kidneys. 6. The results suggest that the pressor effect of galanin in the possum is the result of direct vasoconstrictor action in several vascular beds, in contrast to the cat, in which such effects are few and weak.

Effect of frequency and impulse pattern on the non-cholinergic cardiac response to vagal stimulation in the toad, Bufo marinus.

In the toad, Bufo marinus, stimulation of the vagosympathetic trunk to the heart in the presence of cholinergic and adrenergic blockade results in cardiac slowing. This study investigates the importance of impulse pattern and frequency of neural stimulation in determining this cardiac response. When 360 stimuli were delivered to the heart either continuously at 3 Hz for 2 min, 4 Hz for 1.5 min or 6 Hz for 1 min, or in pulses at 6 Hz for 1 s every 2 s, over a 2 min period, there were no significant differences in the size of the chronotropic responses observed. However, when 360 stimuli were delivered in pulses at 6 Hz for 0.5 s every 1 s over 2 min, the resulting cardiac slowing was significantly greater than in response to the other stimulus regimens. In addition, the cardiac slowing in response to 8 Hz for 0.5 s every 1 s over 2 min was significantly greater than the response to 4 Hz continuously for 2 min. The results provide evidence to support the suggestion that the non-cholinergic, non-adrenergic cardiac response to stimulation of the vagosympathetic trunk is peptidergic in origin, and that the frequency of impulses is important in the gain of the response.

Cardiac vagal effects in the toad are attenuated by repetitive vagal stimulation.

The neuropeptide, somatostatin is co-localised with acetylcholine and galanin in cardiac vagal nerve fibres in the toad, Bufo marinus. Cardiac responses attributed to the release of somatostatin are profound bradycardia, and potentiation of cardiac vagal action by increased acetylcholine release. Cardiac slowing in response to a standard electrical stimulus applied to the vagus (1-2 Hz for 10 s) was potentiated after a 2 min high frequency stimulation (10 Hz). This potentiation of cardiac vagal action was abolished after a 1-hour period of repetitive vagal stimulation. In the presence of atropine, increases in pulse interval recorded in response to vagal stimulation at various frequencies for 2 min each, were significantly reduced after the hour of repetitive stimulation. Potentiation of cardiac vagal action and increases in baseline pulse interval were recorded also in response to intravenous injection of exogenous somatostatin. These responses were not significantly different after the hour of repetitive stimulation. It is concluded that attenuation of the cardiac responses described after the hour of repetitive stimulation is due to depletion of the stores of the neuropeptide somatostatin in the vagal nerve endings.

Desensitisation of the cardiac response to somatostatin in isolated toad atria but not in whole toads.

The cardiac slowing measured in response to repeated application of somatostatin (SOM) was compared in an isolated toad sinus venosus/atrial preparation and in a whole, anaesthetised toad, Bufo marinus. Repeated doses of 0.5-1 x 10(-7) M SOM in the organ bath progressively reduced the cardiac response to 25% or less of the response to the first dose given. When preparations were allowed a recovery period of 50-80 min, the response recovered to 51.0 +/- 3.7% of the control. In contrast, the cardiac chronotropic response in anaesthetised toads to repeated intravenous injections of 2 micrograms or 1.2 nmol SOM over 3 h was not changed. Prolonged exposure to SOM (0.1-0.25 mumol/kg/h infusion), for up to 4 h in anaesthetised toads caused no significant reduction either in the cardiac responses to a bolus dose of SOM (0.6 nmol) or nerve released SOM (3 Hz for 2 min) in the presence of atropine. These opposing results suggest that the half-life of a peptide and the method of exposing receptors to a peptide are important factors in receptor desensitisation.

Cardiovascular effects of amphibian and mammalian tachykinins in the toad, Bufo marinus.

In this study, we investigated the cardiovascular effects of four tachykinins in the anaesthetized toad, Bufo marinus. The potencies were compared of the amphibian peptides ranakinin and physalaemin and the mammalian peptide substance P, all of which interact preferentially with tachykinin NK-1 receptors. Neurokinin B, which is found in both mammals and amphibians, was also tested. All tachykinins produced dose-dependent decreases in arterial blood pressure. Ranakinin caused significantly greater falls in blood pressure than substance P, and the response was of longer duration. Both ranakinin and physalaemin were significantly more potent at decreasing blood pressure than neurokinin B. The NK-3 receptor selective agonist, senktide, caused no change in blood pressure. No tachykinin, at doses up to 10 nmole/kg, produced effects on baseline heart rate or affected the ability of the vagus nerves to slow the heart. A non-peptide NK-1 receptor antagonist, CP96,345 (in the dose range 10(-10)-5 x 10(-7) moles/kg) had no effect on the depressor action of ranakinin or substance P. It is concluded that amphibian tachykinins cause depressor effects via an NK-1-like receptor which differs substantially from its mammalian counterpart.

Inhibition of cardiac vagal action by galanin but not neuropeptide Y in the brush-tailed possum Trichosurus vulpecula.

1. Stimulation of the right cardiac sympathetic nerve for 2 min at 16 Hz in the presence of either beta- or alpha- and beta-adrenoceptor blockade evoked attenuation of cardiac vagal action in eight possums: 31.3 +/- 10.3% maximum inhibition of cardiac vagal action on prolonging pulse interval, with a time to half-recovery of 4.9 +/- 1.1 min. 2. Intravenous injection of galanin (2-3.5 nmol kg-1) evoked similar inhibition of cardiac vagal action: 41.3 +/- 4.1% maximum inhibition of cardiac vagal action on pulse interval, with a time to half-recovery of 13.4 +/- 2.3 min. 3. Intravenous injection of neuropeptide Y (NPY) at greater molar doses (6.5-10 nmol kg-1) caused no inhibition of cardiac vagal action. 4. The galanin injections caused a powerful pressor response: 57.1 +/- 4.9 mmHg increase in systolic blood pressure. NPY caused a smaller pressor response, despite administration of higher molar doses: 36.7 +/- 3.0 mmHg increase in systolic blood pressure. 5. In the possum, galanin but not NPY can mimic the effects of cardiac sympathetic nerve stimulation on vagal action. Galanin also causes large pressor effects.

Vagal stimulation and somatostatin potentiate cardiac vagal action in the toad, Bufo marinus.

Cholinergic postganglionic neurones of the cardiac vagus in the toad, Bufo marinus, have been shown to contain the peptide somatostatin (SOM), which causes direct negative inotropic and chronotropic effects on the heart. In anaesthetised toads, high frequency stimulation (10 Hz) of cardiac vagus nerves results in prolonged cardiac slowing and potentiation of the cardiac slowing measured in response to a train of vagal stimuli at low frequency. Intravenous administration of the tetradecapeptide form of SOM also results in prolonged cardiac slowing and potentiation of cardiac vagal action. Effects on heart rate of small bolus doses of acetylcholine (ACh) were unaltered by administration of SOM, at the same time as cardiac vagal slowing was enhanced. It is suggested that SOM is released from vagal nerve endings by high frequency stimulation and enhances cardiac vagal action by a presynaptic mechanism.

Effect of neuropeptide-Y and galanin on autonomic control of heart rate in the toad, Bufo marinus.

The effects of neuropeptide-Y (NPY) and galanin (GAL) on the autonomic control of heart rate were investigated in the anaesthetised toad, Bufo marinus. Both vagosympathetic trunks were sectioned to prevent reflex changes in heart rate, and the cardiac responses to electrical stimulation of either the vagal or sympathetic fibres to the heart assessed. Intravenous, bolus doses of 10 or 20 micrograms (2 or 4 nmol) NPY and 5 or 10 micrograms (1.5 or 3 nmol) GAL caused pronounced pressor responses but small direct changes in heart rate. Pulse intervals measured after peptide administration were within 5% of control values. All doses of both peptides caused inhibition of action of the cardiac vagus nerves, the maximum inhibition observed in response to 20 micrograms NPY: mean 49.5 +/- 14% (SEM). No significant changes in cardiac sympathetic nerve action were observed. It is concluded that NPY and GAL have similar, important cardiovascular actions in the toad. Similarities between the responses of toads and mammals to NPY suggest a phylogenetic conservation of function for this peptide.

Effect of temperature on cardiac vagal action in the toad Bufo marinus.

The effect of temperature on the action of the vagus nerve on the heart was studied in the toad Bufo marinus. Experiments were performed on two groups of toads, in one the heart was perfused at a constant rate with oxygenated Ringer's solution and in the other the circulation was left intact. In all toads there was a linear relationship between pulse interval (PI) and the frequency of vagal stimulation (fv) at any one temperature. The slope of this relationship changed with temperature, the effectiveness of the vagus (delta PI/delta fv) increasing with decreasing temperature. At low temperatures the vagus nerves of intact toads were more effective than in those with perfused hearts. It is suggested that, in intact toads at low temperatures, cardiac output decreases and the consequent accumulation of acetylcholine leads to increased vagal effectiveness.

Enhancement of cardiac vagal action during ischaemia of the sino-atrial node.

The effect of ischaemia of the sino-atrial node on cardiac vagal action was studied in anaesthetized dogs. The cut, cardiac end of the right vagus nerve was stimulated with a standard supramaximal stimulus every 10 s. The arterial supply to the sinoatrial node was occluded for periods of 1-3 min during this intermittent vagal stimulation. Vagal action on heart rate was potentiated during ischaemia of the sino-atrial node.

Cardiac vagal action during hypoxia in adult and fetal sheep.

The effects of hypoxia on the potential for the vagus to slow the heart, and on resting heart rate, were compared in anesthetized, vagotomized adult and fetal sheep, and in a chronically catheterized fetus in utero. In adults, the action of the cardiac vagus was potentiated at and below an arterial pO2 in the range 13-27 mm Hg. In contrast, in the fetus and the neonate, vagal action was not potentiated as pO2 fell through this range to 10-12 mm Hg. Below 10-12 mm Hg baseline heart rate fell markedly, and the effect of the cardiac vagus on heart rate was diminished, but its effect on pulse interval was not consistently changed. It is concluded that potentiation of vagal action during hypoxia occurs in adult but not fetal sheep and the bradycardia seen in the fetus during severe hypoxia is probably due to direct myocardial depression.

Modification of vagal action on the toad heart by changes in flow.

Hearts of toads (Bufo marinus) were perfused in situ with oxygenated Ringer's solution. The flow of the perfusate was varied during periods of vagal stimulation and in the absence of vagal stimulation. Pulse interval and cardiac contraction were monitored, and results only from those animals that showed no change in pulse interval with changes in flow in the absence of vagal stimulation were used. In these animals, during vagal stimulation, cardiac slowing was greater when flow was low than when it was high. In addition, in cases where vagal stimulation caused atrio-ventricular block, the block could be reversed by an increase in flow of the perfusate. The results are consistent with flow through the heart being an important determinant of the concentration of neurally released ACh at the pacemaker sites and therefore of the effectiveness of the vagus nerve.

Excitation of the cardiac vagus by vasopressin in mammals.

In unanaesthetized sheep, the sensitivity of the baroreceptor-cardio-inhibitory reflex was greater when intravenous vasopressin was used to raise blood pressure, than when intravenous phenylephrine was used to raise blood pressure. This difference was still evident in animals in which beta-adrenergic blockade had been carried out using propranolol. In the presence of combined beta-adrenergic and muscarinic blockade, a direct negative chronotropic effect of intravenous vasopressin could not be demonstrated. It was concluded, therefore, that intravenous vasopressin enhanced cardiac vagal tone. This effect of vasopressin on efferent cardiac vagal tone was confirmed directly in anaesthetized dogs by recording from single cardiac vagal efferent fibres. Furthermore, recordings from single carotid sinus baroreceptor fibres did not demonstrate a direct action of vasopressin on the sensitivity of the baroreceptors. However, the pressor effect of vasopressin is associated with a greater increase in efferent cardiac vagal discharge than that seen when equipressor doses of phenylephrine are given, or when blood pressure is raised by a similar amount by inflation of an intra-aortic balloon. Studies in isolated guinea-pig atrial preparations and in anaesthetized rabbits and dogs, revealed no consistent peripheral action of vasopressin on the action of the vagus at the heart.