Binding and inactivation of prostacyclin (PGI2) by human erythrocytes

Summary . Prostacyclin (PGI₂), the most potent inhibitor of platelet aggregation known, is rapidly hydrolysed in aqueous solution at neutral pH to its inactive derivative 6-keto-PGF_1α. In previous studies (Willems et al, 1979), we found that PGI₂ is stabilized by plasma. Yet, PGI₂ is rapidly inactivated in vivo. These findings prompted us to study the fate of PGI₂ when incubated in whole human blood. After 20 min of incubation (at 37°C, pH 7·8), 29 ± 10% of the amount of PGI₂, added to whole blood, remained in the supernatant plasma whereas, under the same conditions, 80 ± 3% and 86 ± 2% were recovered when PGI₂ was added to platelet-rich plasma or cell-free plasma, respectively. When PGI₂ was incubated with washed erythrocytes resuspended in plasma, 20 ± 15% of the added PGI₂ remained in the supernatant after 10 min of incubation. These findings indicate that PGI₂, when incubated with whole blood, is preferentially bound to erythrocytes. To study the kinetics of binding in more detail, [³H]PGI₂ was incubated with washed erythrocytes resuspended in plasma. The binding was time- and concentration-dependent. The observed binding of label represented binding of [³H]PGI₂, because (1) acid-treated label did not bind to erythrocytes; (2) no substantial binding of [³H]6-keto-PGF_1α occurred, and (3) changes in the specific activity of the PGI₂ preparation did not alter the binding percentage of labelled PGI₂. The binding of [³H]PGI₂ was not influenced by PGE₁ or 6-keto-PGF_1α. Repeated incubations of erythrocytes with PGI₂ revealed that PGI₂ was rapidly degraded into a biologically inactive non-binding substance, presumably 6-keto-PGF_1α. Binding and metabolism of PGI₂ by erythrocytes may explain the apparent instability of PGI₂ in whole blood and provides an explanation for the rapid loss of the biological effects of PGI₂ on termination of the infusion.     

Effect of proteoglycans (Pg) and glycosaminoglycans (GAG) on prostacyclin (PGI2) and PGI2-formation of rat arteries

Heparin had no effect on PGI2-activity if heparin and PGI2 have been incubated-together in an ice bath for 3 minutes. But heparin was able - unlike the other Pg or GAG - to abolish PGI2-activity if it had been incubated with PGI2 in an ice bath for 15 minutes. Pg and GAG, which had been isolated from bovine aortas according to the method of Hascall, and commerically available GAG (hyaluronic acid, chondroitin-40-sulfate, chondroitin-6-sulfate and heparin) had no effect on PGI2-formation of rat aortas in short time incubation (3 min). After long time incubation (15 min) or rat aortas in heparin less PGI2 was detectable compared to a buffer incubation. These data suggest that Pg and GAG do not influence PGI2-formation of arteries. The diminished PGI2-activity after long time incubation should be due to the PGI2-degrading effect of heparin.     

Effect of moderate exercise on salt and water transport in the human jejunum.

The effect of moderate exercise on jejunal absorption was examined in seven healthy subjects using a triple lumen perfusion technique. Moderate exercise on a bicycle ergometer significantly reduced net absorption of water from 32.0 (4.0) to 16.2 (6.1) ml/30 cm/50 min (p less than 0.02), sodium from 2.4 (0.4) to 0.5 (0.9) mmol/30 cm/50 cm (p less than 0.05), chloride from 2.0 (0.4) to 0.3 (0.7) mmol/30 cm/50 min (p less than 0.05), and potassium from 0.20 (0.02) to 0.01 (0.04) mmol/30 cm/50 min (p less than 0.01). After exercise, water, sodium, and chloride absorption returned towards basal values, but potassium absorption remained significantly decreased. These results suggest that moderate exercise can influence jejunal absorption of salt and water in man. They support the possibility that the autonomic nervous system has a physiological role in the control of intestinal transport, although other mechanisms may be involved.     

Cardiovascular responses to PGI2 (prostacyclin) in the dog.

Arachidonic acid (AA) produces characteristic hemodynamic changes in the canine circulation. These responses are blocked by prostaglandin (PG) synthetase inhibitors, indicating that AA and its nonprostanoic metabolites are not vasoactive. The hemodynamic effects of the cyclic endoperoxides, thromboxanes, and PGD2, PG2, and PGF2a differ from those produced by AA. PGI2, a newly identified product of AA, is reported to relax arterial strips. However, its cardiac and systemic effects are unknown. In 12 open-chest, anesthetized dogs, PGI2 (0.25-5.0 microgram/kg) produced a dose-related decrease in systemic arterial pressure (BP) and myocardial contractile force (MCF). In five left ventricular bypass preparations, PGI2 produced only a slight decrease in MCF at all doses, whereas the BP decreases were parallel to those in the intact preparation. AA, PGD2, PGE2, and PGI2 were administered in random order by bolus intravenous injections in approximately equidepressor doses to intact dogs. BP fell with each agent (AA, 300 microgram/kg, -25 percent; PGD2, 5 microgram/kg, -26 percent; PGE2, 5 microgram/kg, -26 percent PGI2, 0.5 microgram/kg, -26 percent). The vasodepressor action of PGI2 was approximately 10 times greater than that of PGD2 and PGE2. Pulmonary arterial pressure (PAP) rose significantly with PGD2 and PGE2 (AA, -1 percent; PGD2 +66 percent; PGE2, +20 percent; PGI2, -1 percent). Only PGE2 had a significant effect on MCF (AA, +7 percent; PGD2, +5 percent, PGE2, +20 percent; PGI2, -0.3). At this dose, PGI2 resembles AA in that it has little effect on either PAP or MCF. Of all known AA metabolites the response to PGI2 most closely resembles that of exogenous AA in the dog.     

Relaxation of human isolated pulmonary arteries by prostacyclin (PGI2)

The effects of indomethacin (IND) and PGI₂ on the tone of human isolated pulmonary arteries were studied. Baseline tone of the vessels was increased by IND, electric stimulation (ES), norepinephrine (NE), prostaglandin F_2α (PDF_2α) or K⁺-excess. This high tone was decreased by PGI₂ in a concentration dependent manner. IC₅₀ values (molar concentrations producing 50% relaxation) for PGI₂ were in the same concentration range (10 to 58.8 nmoles/l). The potency of the relaxant effect of PGI₂ was inversely related to the magnitude of tone induced prior to the addition of PGI₂ and independent of the type of tone inducer. It is suggested that the relaxant effect of prostacyclin on human pulmonary artery may be of clinical importance in the treatment of conditions associated with a rise in pulmonary vascular resistance.     

Pulmonary vasodilator activity of prostacyclin (PGI2) in the cat.

We studied the pulmonary vascular effects of prostacylin, PGI2, in the cat with intact chest under conditions of controlled blood flow. Intralobar injections of PGI2, 0.03--1 microgram, decreased arterial pressure in the perfused lobe in a dose-dependent manner. Inasmuch as lobar blood flow was held constant and left artrial pressure was unchanged, the fall in lobar arterial pressure reflects a decrease in lobar vascular resistance. Prostaglandin E1 (PGE1) and nitroglycerin also decreased lobar arterial pressure; however, PGI2 had greater vasodilator activity than did these substances. Vasodilator responses to PGI2, PGE1, and nitroglycerin in absolute terms were dependent on the baseline level of tone in the pulmonary vascular bed. Prostacyclin reversed the hypertensive and platelet aggregating effects of ADP in the lobar vascular bed. These data indicate that PGI2 has significant vasodilator activity in the feline pulmonary lobar vascular bed.     

Effect of peptide histidine isoleucine on water and electrolyte transport in the human jejunum.

Peptide histidine isoleucine, a 27 amino acid peptide with close amino acid sequence homology to vasoactive intestinal peptide and secretin, is distributed throughout the mammalian intestinal tract, where it has been localised to intramural neurones. An intestinal perfusion technique has been used to study the effect of intravenous peptide histidine isoleucine (44.5 pmol/kg/min) on water and electrolyte transport from a plasma like electrolyte solution in human jejunum in vivo. Peptide histidine isoleucine infusion produced peak plasma peptide histidine isoleucine concentrations in the range 2000-3000 pmol/l, flushing, tachycardia and a reduction in diastolic blood pressure. Peptide histidine isoleucine caused a significant inhibition of net absorption of water, sodium, potassium and bicarbonate and induced a net secretion of chloride, these changes being completely reversed during the post-peptide histidine isoleucine period. These findings suggest that endogenous peptide histidine isoleucine may participate in the neurohumoral regulation of water and electrolyte transport in the human jejunum.     

Haemodynamic effects of prostacyclin (PGI2) in pulmonary hypertension.

An infusion of prostacyclin (PGI2) in four patients with pulmonary hypertension resulted in a dose-related decrease in the systemic and pulmonary vascular resistance and an increase in cardiac output. No selectivity for the pulmonary vasculature was observed and the drug was not therapeutically beneficial.     

Effect of psychological stress on salt and water transport in the human jejunum

The possibility that the central and autonomic nervous system might be involved in the control of intestinal absorption was assessed by measuring the effect of psychological stress, induced by dichotomous listening, on jejunal transport in 15 healthy subjects. Using a triple-lumen perfusion technique to determine jejunal absorption, dichotomous listening significantly reduced mean net water absorption from 31.2 to 8.7 ml X 30 cm-1 X h-1 (p less than 0.005) and changed mean net sodium and chloride absorption to secretion (sodium +1.14 to -1.2 mEq X 30 cm-1 X h-1, p less than 0.01; and chloride +1.5 to -0.9 mEq X 30 cm-1 X h-1, p less than 0.02). During the hour following dichotomous listening these changes were maintained. Jejunal water, sodium, and chloride absorption measured in 9 subjects during three consecutive stress-free periods did not change significantly. Mean transit time over the 30-cm test segment, measured by a dye dilution technique, in 7 subjects was not significantly different during control and dichotomous listening periods, being 2.8 and 4.0 min, respectively. During the hour following dichotomous listening there was a significant increase in mean transit time to 6.4 min (p less than 0.02). In 7 subjects who received a constant intravenous infusion of atropine throughout the control, dichotomous listening, and recovery periods, jejunal sodium, chloride, and water absorption did not change significantly. These results suggest that psychological stress can influence jejunal absorption of salt and water and that this effect may be mediated by a cholinergic parasympathetic nervous mechanism.     

Effect of cold-induced pain on salt and water transport in the human jejunum

The possibility that the central and autonomic nervous systems might be involved in the control of intestinal absorption was assessed by measuring the effect of physical stress (cold-induced pain) on jejunal transport in 13 healthy subjects. Using a triple-lumen perfusion technique to determine jejunal absorption, cold pain significantly reduced net water absorption from 34.9 to 15.7 ml/30 cm.50 min (p less than 0.005), net sodium absorption from 2.6 to 0.2 mEq/30 cm.50 min (p less than 0.005), and net chloride absorption from 2.2 to 0.6 mEq/30 cm.50 min (p less than 0.02). These changes were associated with an elevation of blood pressure and plasma norepinephrine during cold pain. During the period following cold pain, systolic blood pressure remained significantly elevated and net jejunal water absorption significantly reduced. These results suggest that physical stress can influence jejunal absorption of salt and water in humans and support the possibility that the central and autonomic nervous systems have a physiologic role in the control of intestinal function.     

Effect of experimentally induced diabetes on swine vascular prostacyclin (PGI2) synthesis

Repeated administration of streptozotocin to G?ttingen miniature swine led to the development of a mild form of diabetes. The amount of PGI2 generated by the vessels (abdominal aorta, thoracic aorta, pulmonary artery), as estimated by reference to inhibition of ADP-induced platelet aggregation, was lower in the treated animals than in the controls. The decrease in the synthesis of PGI2 may contribute to the prethrombotic state in diabetes mellitus.     

Microcirculatory effects of prostacyclin (PGI2) in the hamster cheek pouch.

The effects of prostacyclin (PGI₂) on small blood vessels in the hamster cheek pouch microcirculation were studied microscopically. Prostacyclin applied systemically or locally caused an increase in the diameter of precapillary arterioles (10–50 μm diameter) with inherent or induced tone. It was more potent prostaglandin PGE₁, PGE₂, or bradykinin. In animals treated with indomethacin (4 mg/kg po), the relative vasodilator potency of prostacyclin to PGE₁ and PGE₂ was increased. The cyclic endoperoxide precursor of prostaglandins, PGH₂, and the stable chemical breakdown product of prostacyclin, 6-oxo-PGF_1α, were also dilators but were less potent than prostacyclin itself. In some experiments, responses of arterioles to PGH₂ were biphasic, a long-lasting dilatation being preceded by a short-lasting vasoconstriction. The postcapillary venules (20–50 μm diameter) in this preparation were inactive and did not respond by dilatation or constriction to PGI₂, PGE₁, PGE₂, PGH₂, 6-oxo-PGF_1α, bradykinin, or noradrenaline.     

Protective action of endogenous prostacyclin (PGI2) and prostaglandin E2 (PGE2) in endoscopic polypectomy-induced human ulcers

To assess how endogenous prostaglandin (PG) in gastric mucosa acts against ulcer formation, we determined the mucosal prostacyclin (PGI2), PGE2, PGF2 alpha, and thromboxane A2(TXA2) concentrations before and after polypectomy in 6 patients in whom gastric ulcers were produced by electric burning resection of gastric polyps. These artificially induced ulcers all healed within short periods (25.7 +/- 7.4 days, mean +/- SE). Of the PGs assayed, the level of PGI2 was highest. The pG levels were increased at 4 and 7 days post-polypectomy; the most remarkable increase took place in the mucosa along the ulcer margin rather than the mucosa far from the ulcer site. We suggest that the observed increase in endogenous PGs represents a physiological response against polypectomy-induced ulcer formation.     

Atrial natriuretic peptide and water and electrolyte transport in the human jejunum.

The effects of atrial natriuretic peptide were investigated on water and electrolyte transport in the human jejunum. Six healthy male volunteers (aged 21-33 years) were studied using a triple lumen perfusion technique. A plasma like electrolyte solution containing polyethylene glycol (5 milligrams) as a non-absorbable marker was perfused into the jejunum at 10 ml/min, and net water and electrolyte transport and transepithelial potential difference were measured. Subjects were studied single blind on two occasions with either intravenous atrial natriuretic peptide (6 pmol/min/kg for 90 minutes) or placebo (saline), both after controlled sodium intake over three days. Plasma atrial natriuretic peptide concentrations rose from (mean (SD)) 10.3 (3.6) pmol/l to a peak of 96.0 (61.8) pmol/l. Jejunal net water and electrolyte fluxes and potential difference were identical in both the atrial natriuretic peptide and the control studies. Compared with placebo atrial natriuretic peptide induced a significantly greater diuresis (peak 10.2 (6.0) v 1.8 (1.0) ml/min, p less than 0.05) and natriuresis (peak 1069 (351) v 376 (208) mumol/min, p less than 0.01) and haemoconcentration (haematocrit 0.405 (0.040) v 0.368 (0.018), p less than 0.01). There was no difference in blood pressure, pulse rate, plasma electrolytes, and plasma osmolality between the two studies. There was no evidence to suggest an effect of atrial natriuretic peptide on jejunal water and electrolyte transport in healthy human subjects.     

Effect of bicarbonate or base precursor on water and solute absorption from a glucose-electrolyte solution in the human jejunum

A modified perfusion technique was used to examine the effect on water and solute absorption in the healthy human jejunum of replacing bicarbonate (18 mmol/l) by equivalent amounts of different base precursors in a glucose-electrolyte solution. Acetate, citrate and lactate were absorbed from the perfusion solutions. Absorption of these base precursors appeared to have no effect on water uptake, but greater sodium (p less than 0.05) absorption occurred from solutions containing either acetate or lactate compared with the bicarbonate-containing solution. These data suggest that oral rehydration solutions with base precursors other than bicarbonate are as effective as bicarbonate-containing solutions in promoting absorption of water and electrolytes.     

The inhibitory effect of PGI2 (prostacyclin) on white platelet arterial thrombus formation.

Prostaglandins play an important role in the platelet-vessel wall interactions. Superfusion with PGI2 of arterial segments prepared in an in vivo model for thrombus induction resulted in a very marked dose-effect-related decrease of white arterial thrombus formation.     

Topical methotrexate alters solute and water transport in the rat jejunum in vivo and rabbit ileum in vitro.

The topical effect of methotrexate (MTX) on small intestinal hexose and ion transport has been studied using an in vivo steady state jejunal perfusion technique in the rat, and short circuited rabbit terminal ileum in Ussing chambers in vitro. In rat jejunum, perfusion with MTX (1 mumol/l) caused significant reductions in water, sodium, and glucose absorption within 110 minutes of exposure. Fructose absorption was, however, unimpaired. The same concentration of MTX, when added to the mucosal side of distal rabbit ileum caused significant increases in transmucosal potential difference, short circuit current and the unidirectional flux of chloride from serosa to mucosa. In the presence of a subphysiological magnesium concentration (0.3 mmol/l), MTX resulted in the abolition of net sodium absorption and the conversion of net chloride absorption to secretion. We conclude that MTX has a topical effect on small intestinal transport which is independent of its effect on crypt cell kinetics.     

Effect of prostacyclin (PGI2) and a prostaglandin analogue BW 245C on galactosamine-induced hepatic necrosis

The reported cytoprotective effects of prostaglandins against noxious stimuli in the liver was the basis for the present investigations of the effects of prostacyclin (PGI2) and a prostaglandin analogue (BW 245C) in an animal model of severe liver failure. Rats were given galactosamine at two dose levels and the prostaglandins were given in repeated doses from 0 to 6 h during the development of the liver damage or in another group from 24 to 30 h at the time of maximal liver injury. For PGI2 significant cytoprotection was found as assessed by a reduction in blood Normotest at 24, 48 and 72 h (P less than 0.05) and the plasma level of aspartate aminotransferase at 24 and 48 h (P less than 0.02) and the lysosomal markers N-acetyl-beta-glucosaminidase at 24, 48 and 72 h (P less than 0.001) and cathepsin D at 48 h (P less than 0.005) as compared to appropriate controls. Early administration of PGI2 reduced the mortality rate from 63% in the control group to 0% (P less than 0.01) in the treated group, but no significant effects were found when either compound was given later in the 24-h to 30-h period.     

Effects of prostacyclin (PGI2) on hypoxic pulmonary vasoconstriction in the conscious adult sheep

The effects of prostacyclin (PGI2) on alveolar hypoxic pulmonary vasoconstriction were investigated in the conscious adult sheep. In our model, hypoxia also produced increases in pulmonary arterial pressure (PPA) and pulmonary vascular resistance (PVR), indicating pulmonary vasoconstriction. PGI2 was injected rapidly as a 0.5 microgram/kg bolus via the right atrium in five sheep during normoxia and hypoxia. During normoxia, PGI2 increased PPA and cardiac output, and decreased systemic arterial pressure (PSA), systemic vascular resistance (SVR) and PVR. Left atrial pressure did not change. During hypoxia following PGI2 administration, PPA decreased, CO increased, and PVR decreased, suggesting dilator action on the pulmonary resistance vessels. As the same time PSA and SVR decreased, suggesting dilator action on the systemic resistance vessels. However, the degree of the decline in PVR caused by PGI2 was much greater during hypoxia than during normoxia. The decreases in PSA and SVR induced by PGI2 were not significant between hypoxia and normoxia. These findings confirm that PGI2 decreases pulmonary and systemic vascular resistances in normoxic and hypoxic sheep. Moreover, during hypoxia, associated with the increased PPA and PVR, the administration of PGI2 appears to be particularly effective in "normalizing" these parameters.