Comparison of vascular and respiratory effects of endothelin-1 in the pig

The haemodynamic and respiratory responses caused by i.v. administration of endothelin-1 (ET-1) (20-100 pmol/kg) were studied in anaesthetized spontaneously breathing pigs. Intravenous bolus administration of synthetic ET-1 (40-100 pmol/kg) caused a transient decrease followed by a long-lasting increase in mean pulmonary arterial pressure and dose dependent vasoconstriction both in the systemic and pulmonary circulations. The effect on pulmonary arterial pressure was biphasic, with an initial transient fall followed by a long-lasting dose dependent increase. A biphasic response of the systemic mean arterial pressure was demonstrated only at a high dose of ET-1 (100 pmol/kg). ET-1 administration did not significantly change breathing pattern or phasic vagal input, but caused a significant decrease in passive compliance. Passive resistances or active compliance and resistances of the respiratory system were not modified. These results suggest that in the pig ET-1 is a more potent constrictor of vascular than of bronchial smooth muscle. The vasoconstrictor activity was greater in the pulmonary than the systemic circulations.     

Long-lasting vasoconstriction and efficient regional extraction of endothelin-1 in human splanchnic and renal tissues

Six healthy subjects were given endothelin-1, intravenously in a dose of 4 pmol.kg-1.min-1 for 20 min. Blood samples were drawn from arterial, hepatic and renal vein catheters for determinations of splanchnic and renal blood flows and the extraction of endothelin-1 in these vascular beds. Intravenous infusion of endothelin-1 increased the mean arterial blood pressure by 6.8 +/- 2.0 mm Hg (p less than 0.05) and reduced splanchnic and renal blood flows by 34% (p less than 0.005) and 26% (p less than 0.001) respectively. Return to basal flow values occurred after about 1 hr for the splanchnic and 3 hrs for the renal blood flow. The fractional extractions of endothelin-1-like immunoreactivity corresponded to 75 +/- 2% and 60 +/- 2% in the splanchnic and renal vascular beds, respectively. The disappearance curve in plasma and two half-lives of 1.4 +/- 0.1 min and 35 +/- 2.8 min respectively.     

The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias).

The aim of the present study was to evaluate the effects of endothelin-l-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg(-1)) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O2. The effects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO2 partial pressure was increased transiently by approximately 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (approximately 0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml(-1)) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by approximately 10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.     

Effect of synthetic physalaemin on splanchnic circulation in dogs

Physalaemin has been reported as one of the most potent vasodilator and hypotensive peptides (1-4). In spite of these studies, however, the effect of the peptide on splanchnic circulation is not known precisely. In the present study, the effect of synthetic physalaemin on superior mesenteric arterial blood flow, portal venous blood flow and pancreatic capillary blood flow was investigated in dogs. Dose dependent increases of superior mesenteric arterial blood flow and portal venous blood flow were induced in response to physalaemin (0.1-10.0 ng/kg). Superior mesenteric arterial blood flow and portal venous blood flow attained maximal increases of 77 +/- 8.9% and 70 +/- 8.6%, respectively, at a dose of 5 ng/kg. Physalaemin caused a dose-related decrease in systemic arterial blood pressure. Pancreatic capillary blood flow did not show significant change with the administration of physalaemin. These data suggest that physalaemin may play some physiological roles in the regulation of splanchnic circulation.     

Comparative responses to endothelin-2 and sarafotoxin 6b in the pulmonary vascular bed of the cat

Pulmonary vascular responses to endothelin-2 and sarafotoxin 6b were investigated in the feline pulmonary vascular bed under natural flow and constant flow conditions. Injections of endothelin-2 and sarafotoxin 6b in a dose of 0.3 nmol/kg iv increased pulmonary arterial and left atrial pressures and cardiac output, and caused a biphasic change in calculated pulmonary vascular resistance. Endothelin-2 caused a biphasic change in systemic arterial pressure, while sarafotoxin 6b only decreased arterial pressure. Under constant flow conditions in the intact-chest cat, injections of endothelin-2 and sarafotoxin 6b in doses of 0.1-1 nmol into the perfused lobar artery increased lobar arterial pressure in a dose-related manner but were less potent than the thromboxane A2 mimic, U46619. An ET analog with only the Cys1-Cys15 disulfide bond and an amidated carboxy terminus had no significant activity in the pulmonary vascular bed. The present data show that endothelin-2 and sarafotoxin 6b have significant vasoconstrictor activity in the pulmonary vascular bed of the cat.     

The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias)

The aim of the present study was to evaluate the effects of endothelin-1-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg^-1) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O₂. The effects of endothelin-1 on arterial blood partial pressure of CO₂ were not synchronised with the changes in O₂ partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO₂ partial pressure was increased transiently by ~1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO₂ partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (~0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml^-1) eliminated the increase in CO₂ partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO₂ partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by ~10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.     

The effect of calcitonin gene-related peptide on pancreatic blood flow and secretion in conscious dogs

The effects of human alpha-calcitonin gene-related peptide (alpha-CGRP) and beta-CGRP on pancreatic arterial (PA), superior mesenteric (SMA) and left gastric arterial (LGA) blood flows were studied by ultrasound transit-time blood flow meters in five conscious dogs. Intravenous injections of alpha-CGRP and beta-CGRP (5-200 pmol/kg) induced a dose-related increase in PA flow and a dose-related decrease in its resistance. At lower doses, alpha-CGRP was more potent than beta-CGRP, but their maximal responses were similar. The blood flow responses to alpha-CGRP (200 pmol/kg) were 153% of the basal flow in LGA, 313% in PA, and 534% in SMA, while those to VIP (100 pmol/kg) were 467% in LGA, 953% in PA and 163% in SMA. Somatostatin reduced blood flow in all arteries. alpha-CGRP, but not beta-CGRP, at higher doses induced gastric contractions and pancreatic protein-rich secretion, which were blocked by atropine. These results suggest that CGRP in perivascular nerves in the pancreas may regulate pancreatic blood flow in dogs but its physiological function remains to be studied.     

Effect of synthetic neuromedin U-8 and U-25, novel peptides identified in porcine spinal cord, on splanchnic circulation in dogs

Two novel peptides which exert a potent stimulant effect on rat uterus smooth muscle have recently been identified in porcine spinal cord. These peptides designated neuromedin U-8 and U-25 have been reported to exert a hypertensive effect in rats. But further biological activities are not known. In the present study, the effect of these peptides on blood flow in portal vein, superior mesenteric artery and pancreatic tissue and on blood pressure were examined in dogs, utilizing recently developed ultrasonic transit time volume flow meter and laser Doppler flow meter. Neuromedin Us potently reduced blood flow in superior mesenteric artery. The minimum reductions could be observed even at very small doses of neuromedin U-25 (32 fmol/kg) and U-8 (90 fmol/kg), while the maximal reductions of 48.4 and 51.0% were attained at the doses of 320 pmol/kg (U-25) and 900 pmol/kg (U-8), respectively. These peptides also reduced portal vein blood flow, and the maximal reductions of 42.1 and 37.2% were attained at the doses of 32 pmol/kg (U-25) and 90 pmol/kg (U-8), respectively. On the other hand, blood flow in pancreatic tissue increased slightly with the maximal increases of 13.8% at 3.2 pmol/kg (U-25) and 11.8% at 9 pmol/kg (U-8), respectively. The maximal increases of blood pressure were 5.2% at 320 pmol/kg (U-25) and 4.3% at 90 pmol/kg (U-8). Furthermore, neither neuromedin U-25 nor U-8 influenced the axillary artery blood flow, suggesting their selective effect on splanchnic blood flow. Because of the potent and probably selective activity on splanchnic circulation, neuromedin U-25 and U-8 may well be recognized as physiologically significant novel neuropeptides or hormones.     

Alteration of the Systemic and Microcirculation by a Single Oral Dose of Flavan-3-Ols

Several systematic reviews have reported that flow mediated dilatation (FMD) was significantly increased in subjects after ingestion of chocolate that contains flavan-3-ols; however, the mechanisms responsible for this effect are not clear. In this study, we evaluated the effects of a single oral dose of flavan-3-ols on the systemic circulation and microcirculation in the cremaster muscle using intravital video microscopy in vivo. The cremaster muscle in rats was spread over a plastic chamber and a gastric tube was placed into the stomach. Blood flow in the cremasteric artery was determined using a laser Doppler flowmeter, while blood pressure and heart rate were measured by the tail-cuff method. Red blood cell velocity in arterioles and blood flow in the artery were significantly increased 5 min after the administration of 10 mg/kg flavan-3-ols compared with distilled water treatment. The number of capillaries recruited in the cremaster muscle was also significantly increased 15 min after treatment. Microscopic observation confirmed that increased shear stress on endothelial cells was maintained during the measurement period. The mean arterial blood pressure and heart rate were also significantly elevated soon after administration and returned to baseline before the end of the observation period. Plasma nitrate and nitrite levels, and NO phosphorylation of aortic tissue were significantly increased at 60 min after administration of flavan-3-ols. According to these results, a single oral dose of flavan-3-ols elevates blood pressure and flow transiently, and these effects induce NO production through increased shear stress on endothelial cells.     

Effect of endothelin-1 on pulmonary resistance in rats

We examined the effect of endothelin-1 (ET-1), a novel 21-residue vasoconstrictor peptide, on pulmonary resistance (RL) in Wistar rats. The lung volume, tracheal flow, and transpulmonary pressure of tracheotomized and paralyzed rats were measured with a fluid-filled esophageal catheter and a pressure-sensitive body plethysmograph. RL was calculated by the method of von Neergaard. The femoral artery was cannulated to measure the mean arterial blood pressure. Intravenous bolus administration of synthetic ET-1 provoked a dose-dependent increase in RL in rats. The bronchoconstricting effect reached maximum at 500 pmol/kg. This bronchoconstriction was observed in less than 5 min, increased up to 15 min, and was sustained for 60 min. ET-1 increased the mean arterial blood pressure in a dose-dependent manner. We conclude that ET-1 is a hitherto unknown potent bronchoconstrictor that has a sustained effect in vivo. The potential physiological and pathophysiological role of this new peptide in the development of respiratory disease warrants further investigation.     

Comparative effects of vasodilator drugs on flow distribution and venous return

Systemic vascular effects of hydralazine, prazosin, captopril, and nifedipine were studied in 115 anesthetized dogs. Blood flow (Q) and right atrial pressure (Pra) were independently controlled by a right heart bypass. Transient changes in central blood volume after an acute reduction in Pra at a constant Q showed that blood was draining from two vascular compartments with different time constants, one fast and the other slow. At three dose levels producing comparable reductions in systemic arterial pressure (30-40% at the highest dose), these drugs had different effects on flow distribution and venous return. Hydralazine and prazosin had parallel and balanced effects on arterial resistance of the two vascular compartments, and flow distribution was unaltered. Captopril preferentially reduced arterial resistance of the compartment with a slow time constant for venous return (-26 +/- 6%, -30 +/- 6%, -50 +/- 5% at 0.02, 0.10, and 0.50 mg X kg-1 X h-1, respectively; means +/- SEM) without altering arterial resistance of the fast time-constant compartment. Blood flow to the slow time-constant compartment was increased 43 +/- 14% at the highest dose, and central blood volume was reduced 108 +/- 15 mL. In contrast, nifedipine had a balanced effect on arterial resistance with the lowest dose (0.025 mg/kg) but caused a preferential reduction in arterial resistance of the fast time-constant compartment at higher doses (-38 +/- 4% and -55 +/- 2% at 0.05 and 0.10 mg/kg, respectively). Blood flow to the slow time-constant compartment was reduced 36 +/- 5% at the highest dose of nifedipine, and central blood volume was increased 66 +/- 12 mL. Total systemic venous compliance was unaltered or slightly reduced by each of the four drugs. These results add further evidence to the hypothesis that peripheral blood flow distribution is a major determinant of venous return to the heart.     

Role of tachykinins in the control of pancreatic secretion and circulation.

Tachykinins (TK) are family of peptides including substance P (SP), substance K (SK) and neuromedin K (NK) that have been found in the nerves of the gastrointestinal tract and proposed to act as neurotransmitters to affect the motor, secretory and circulatory functions of the gut, but little is known about their action on the pancreas. In this study three series of tests were carried out to determine the action of SP, SK and NK on pancreatic secretion in conscious dogs and amylase release from the dispersed rat pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects of TK in anesthetized dogs. SP, SK and NK infused i.v. in graded doses (0.12-1.0 microgram/kg per h) in conscious dogs stimulated pancreatic protein outputs reaching, respectively, 38% and 23% of the maximal response to CCK (40 pmol/kg per h). HCO3- outputs were also significantly increased but the highest response did not exceed about 5% of secretin (328 pmol/kg per h) maximum. Cholinergic blockade by atropine abolished the pancreatic responses to tachykinins. When added at various concentrations (10(-11)-10(-7) M) to the incubation medium of rat dispersed pancreatic acini, SK, SP and NK increased in concentration-dependent manner the release of amylase from the resting pancreatic acini and augmented the enzyme release induced by CCK-8 and by urecholine. In anesthetized dogs infused with a background dose of secretin (82 pmol/kg per h), addition of SP, SK and NK caused an immediate and dose-dependent increase in the pancreatic blood flow, oxygen consumption and pancreatic secretion accompanied by a dose-dependent decrease in arterial blood pressure. This study shows that TK are potent pancreatic circulatory stimulants and moderate secretagogues both in vivo and in vitro, acting, at least in part, via cholinergic pathway.     

Effects of pancreatic polypeptide on the pancreatic exocrine secretion stimulated by secretin and cholecystokinin in the conscious pig

Fourteen castrated male Large White pigs, weighing 42.5 +/- 1.0 kg, were fitted with pancreatic and duodenal fistulae for pancreatic secretion studies. Moreover, catheters were placed in a carotid artery for blood sampling and in a jugular vein for peptide infusion. Pancreatic juice was automatically restituted to the animals and continuously sampled for analysis on experimental days. Following an 8-day recovery period, perfusion studies were performed after an overnight fast. After a 30-min basal period, sustained pancreatic flow and protein output were obtained and maintained throughout the assay with secretin (36 pmol/kg/h) and CCK-8 (600 pmol/kg/h) infusion. Then, 200, 400, 600, 800 or 1200 pmol/kg/h of porcine pancreatic polypeptide (PP) were infused for 60 min. Secretin + CCK infusion was continued for 1 h after PP infusion was stopped. Each dose of PP was given on a separate day. Neither pancreatic flow nor bicarbonate output were affected whatever the dose of infused PP. On the contrary, protein concentration and output decreased with the lowest dose of PP (200 pmol/kg/h) and the diminution was more pronounced with the other doses. With 600 pmol/kg/h as well as with 800 and 1200 pmol/kg/h of PP, pancreatic protein output fell to about 20% of values obtained with secretin + CCK. Plasma levels of PP were below or similar to postprandial values for 200, 400 and 600 pmol/kg/h and they were significantly larger with 800 and 1200 pmol/kg/h. Protein concentration and output returned to values obtained with secretin + CCK infusion after cessation of PP infusion. In conclusion, porcine PP given in physiological doses to the pig decreases pancreatic protein output whereas pancreatic flow remains unaffected.     

Bosentan antagonizes the effects of endothelin-1 on rat gastric blood flow and mucosal integrity

Bosentan, a new type of orally effective, mixed (ETA+ETs) endothehn receptor antagonist has been recently introduced and tested in a variety of experimental models. We studied the effect of bosentan on the changes in gastric mucosal hemodynamics and mucosal integrity, induced by the exogenous application of endothelin-1, in rats. Bosentan (10 mg/kg iv) pretreated rats were injected with endothelin-1 (500-1000-2000 pmol/kg, iv) and gastric mucosal hemodynamics were monitored. After combined oral (30 mg/kg) and systemic pretreatment with bosentan we studied the effects of submucosal injection of endothelin-1 (50 pmol) on blood flow and gastric mucosa. Bosentan antagonized the vasodilator, vasoconstrictor and ulcerogenic effects of endothelin-1 in the rat gastric mucosa. These results show that bosentan can be a useful probe in the study of endogenous endothelin in the gastrointestinal tract.     

Evidence that neurotensin mediates postprandial intestinal hyperemia in the python, Python regius

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 ([His(3), Val(4), Ala(7)]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 (P(sys)) and increase in systemic blood flow (Q(sys)). There was no effect on pulmonary pressure and conductance. A significant (P < 0.05) increase in heart rate (f(H)) and total cardiac output (Q(tot)) was seen only at high doses (>30 pmol/kg). The systemic vasodilation and increase in Q(tot) persisted after beta-adrenergic blockade with propranolol, but the rise in f(H) was abolished. Also, the systemic vasodilation persisted after histamine H(2)-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 Q(sys), systemic conductance, P(sys), and f(H). 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.     

Glucagon-like peptide-2 acutely increases proximal small intestinal blood flow in TPN-fed neonatal piglets

Glucagon-like peptide-2 (GLP-2) is a gut hormone that is secreted in response to enteral feeding and stimulates small intestinal mucosal growth. We have previously shown that GLP-2 infusion acutely increases portal venous blood flow in TPN-fed piglets. The aim of this study was to localize the vasoactive effect of GLP-2 within the gastrointestinal tissues and other visceral organs in TPN-fed piglets. Tissue blood flow rates were quantified using fluorescent microsphere deposition in anesthetized TPN-fed piglets given intravenous infusion of GLP-2 at either 500 pmol x kg(-1) x h(-1) (low GLP-2, n = 7 pigs) or 2,000 pmol x kg(-1) x h(-1) (high GLP-2, n = 8 pigs) for 2 h. Compared with baseline, the low and the high GLP-2 treatment significantly increased the blood flow rate in the duodenum (+77%) and jejunum (+40% and 80%), respectively, but blood flow to the distal small intestine and colon (-15%) was unchanged or slightly decreased. Baseline mucosal blood flow was five-fold higher than serosal blood flow; however, high GLP-2 treatment increased serosal (+140%) to a larger degree than mucosal blood flow (+73%). The high GLP-2 dose increased pancreatic flow (+34%) but decreased blood flow in the kidneys (-14%) and stomach (-12%), whereas the spleen and brain were unaffected. These findings suggest that the acute GLP-2-mediated stimulation of portal blood flow in TPN-fed piglets occurs principally via increased blood flow through the superior mesenteric artery to the proximal small intestine, a tissue region where the GLP-2R mRNA abundance and trophic GLP-2 effects are greatest.     

Cardiopulmonary effects of recombinant interleukin-2 infusion in sheep

The systemic administration of recombinant interleukin-2 (rIL-2) with or without lymphokine-activated killer (LAK) cells, a new treatment for patients with advanced cancer, is associated with a presumed "third-space" syndrome. To further define the extent and time course of this toxicity, we established a chronic sheep model and monitored changes in systemic and central vascular pressures, cardiac function, and gas exchange during a 72-h continuous intravenous infusion of rIL-2 at a total dose of 5 (group 3) or 9 x 10(5) U/kg (group 4). At 72 h, caudal mediastinal lymph flow, histology, and extravascular lung water-to-dry lung weight ratio (EVLW/DLW) were obtained. During the rIL-2 infusion there was a dose-dependent significant decrease in systemic blood pressure and arterial Po2 and an increase in core temperature. In group 4, pulmonary arterial pressure increased from a base line of 13 +/- 5 to 21 +/- 6 mmHg (P less than 0.05). Lung lymph flow was significantly increased in groups 3 and 4 compared with animals receiving 0.9% NaCl or excipient infusions (groups 1 and 2). EVLW/DLW values were elevated in groups 3 and 4 (P less than 0.01). In animals receiving rIL-2, histological evaluation revealed a dose-dependent infiltration of lung tissue by lymphoblastoid cells that stained esterase negative. We conclude that rIL-2 infusion in doses comparable to those given to humans results in alterations in systemic and central hemodynamics, gas exchange, high-protein lung lymph flow, and infiltration of lymphoblastoid cells into the lung parenchyma.     

An automated high-performance liquid chromatography fluorescence method for the analyses of endothelins in plasma samples.

A high-performance liquid chromatographic method with fluorescence detection was developed to simultaneously analyze endothelins, a class of vasoactive peptides, in plasma samples. Sample preparation for HPLC analysis was carried out by initial stabilization of blood and plasma samples against transformation of big endothelins to mature endothelins and breakdown of mature endothelins by serine proteases, as well as oxidative modifications of endothelins. Deproteinization of plasma samples was achieved with acidified acetone, and the samples were further purified on molecular weight cutoff filters. Endothelins were separated on a reversed phase LC-318 column by gradient elution using a mobile phase containing acetonitrile and water (0.1% trifluoroacetic acid) and were analyzed by fluorescence detection (lambda(Ex), 280 nm; lambda(Em), 340). Limit of detection values were in the range of 0.2-0.5 pmol. Linear (R(2), 0.99) calibration curves were established for analyte amounts in the range of 1 to 100 pmols. Recoveries of endothelins from spiked plasma samples analyzed ranged from 60-95%. Under optimized conditions the HPLC-fluorescence method was determined to be sensitive and specific for the analysis of big endothelin-1, endothelin-1, endothelin-2, and endothelin-3 in plasma. Simultaneous measurement of these endothelins by the HPLC method should permit a better understanding of their specific roles and relationships under various pathological conditions.     

Synthesis of porcine neuropeptide Y(NPY) in solution

The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. beta-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10 micrograms/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.     

Comparison of helodermin, VIP and PHI in pancreatic secretion and blood flow in dogs

Helodermin, VIP and PHI, which share a high degree of homology with secretin, have been identified in the gut but their physiological role is unknown. In this study 3 series of tests were carried out to determine the actions of helodermin, VIP and PHI on pancreatic secretion in 6 conscious dogs and amylase release from the dispersed canine pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects in 24 anesthetized dogs. Helodermin, VIP and PHI infused i.v. in graded doses (12.5-200 pmol/kg.h) resulted in a dose-dependent increase in pancreatic HCO3 secretion reaching, respectively, 100%, 7% and 2% of secretin maximum. When combined with constant dose infusion of CCK-8 (100 pmol/kg.h), helodermin but not VIP or PHI augmented dose-dependently the HCO3 secretion. When added in various concentrations (10(-10)-10(-5)M) to the incubation medium of dispersed pancreatic acini only helodermin but not VIP or PHI increased dose-dependently amylase release reaching about 50% of CCK-8 maximum. In anesthetized dogs, the pancreatic blood flow (PBF) measured by electromagnetic blood flowmetry showed an immediate and dose-dependent increase following the injections of various doses of helodermin, VIP, PHI and secretin, the peak blood flow preceding by about 1 min the peak secretory stimulation. This study shows that helodermin resembles secretin in its potent pancreatic HCO3 stimulation but differs from VIP or PHI which are poor secretagogues but potent vasodilators. We conclude that if tested peptides are released in the gut, helodermin, like secretin, may be involved in the hormonal stimulation of exocrine pancreas, whereas VIP and PHI may serve mainly as vasodilators in the pancreatic circulation.