Regulation of microvascular prostacyclin and thromboxane with inhibitors of arachidonic acid metabolism

The levels of the stable degradation products of prostacyclin (PGI2) and thromboxane A2 (TXA2): 6-oxo-prostaglandin E1 alpha (6-oxo-PGE1 alpha) and thromboxane B2 (TXB2) respectively were determined in the effluent of the rabbit epigastric skin flap after infusion of exogenous arachidonic acid. The blood to the flap passes through the microcirculation and thus the changes in eicosanoid biosynthesis in this part of the vasculature were recorded. The aim was to use inhibitors of arachidonic acid metabolism to increase the PGI2/TXA2 ratio. This may be potentially beneficial to ischaemic skin flaps by reducing platelet aggregation associated with damaged microvascular endothelium, overcoming vasospasm and increasing microvascular blood flow. Increased PGI2/TXA2 ratios (up to 5-fold) were best achieved using TXA2 synthetase inhibitors such as dazoxiben hydrochloride. These were significantly more potent than the phosphodiesterase inhibitor dipyridamole, and the lipoxygenase inhibitor Bay g6575. No increase in blood flow was achieved. The cyclooxygenase inhibitor indomethacin did slow the blood flow at high concentrations (above 10(-5) M), and inhibited both PGI2 and TXA2 synthesis. Approximately 2-fold higher concentrations of dazoxiben hydrochloride and dipyridamole were required to produce the same TXA2 synthetase inhibition in the flap microvasculature in vivo compared with platelets in vitro.     

Regulation of microvascular prostacyclin and thromboxane with inhibitors or arachidonic acid metabolism

The levels of the stable degradation products of prostacyclin (PGI₂) and thromboxane A₂ (TXA₂): 6-oxo-prostaglandin F_1α(6-oxo-PGE_1α) and thromboxane B₂ (TXB₂) respectively were determined in the effluent of the rabbit epigastric skin flap after infusion of exogenous arachidonic acid. The blood to the flap passes through the microcirculation and thus the changes in eicosanoid biosynthesis in this part of the vasculature were recorded. The aim was to use inhibitors of arachidonic acid metabolism to increase the PGI₂/TXA₂ ratio. This may be potentially beneficial to ischaemic skin flaps by reducing platelet aggregation associated with damaged microvascular endothelium, overcoming vasospasm and increasing microvascular blood flow. Increased PGI₂/TXA₂ ratios (up to 5-fold) were best achieved using TXA₂ synthetase inhibitors such as dazoxiben hydrochloride. These were significantly more potent than the phosphodiesterase inhibitor dipyridamole, and the lipoxygenase inhibitor Bay g6575. No increase in blood flow was achieved. The cyclooxygenase inhibitor indomethacin did slow the blood flow at high concentrations (above 10⁻⁵ M), and inhibited both PGI₂ and TXA₂ synthesis. Approximately 2-fold higher concentrations of dazoxiben hydrochloride and dipyridamole were required to produce the same TXA₂ synthetase inhibition in the flap microvasculature compared with platelets .     

Comparison in anesthetized dogs of the anti-aggregatory and hemodynamic effects of prostacyclin and a chemically stable prostacyclin analog, 6a-carba-PGI2 (carbacyclin)

The intravascular anti-aggregatory and systemic and hemodynamic effects of prostacyclin and carbacyclin were compared by intravenous infusion in pentabarbital anesthetized dogs. Ten times as much carbacyclin was needed to produce comparable inhibition of platelet aggregation in the lumen of partially obstructed circumflex coronary arteries. These doses of carbacyclin caused similar decreases in total peripheral resistance as equi-effective anti-aggregatory doses of prostacyclin. There was a trend for the decrease in blood pressure with carbacyclin to be less than that produced by equi-effective anti-aggregatory doses of prostacyclin because carbacyclin caused somewhat greater increases in cardiac output. Changes in heart rate were similar with both substances. During carbacyclin and prostacyclin infusion resistance in normal (unobstructed) coronary arteries decreased. Both substances had comparable effects on pulmonary vascular resistance, right atrial pressure and left ventricular dp/dt at equivalent anti-aggregatory doses both before and after atropine (1 mg/kg) and hexamethonium (5 mg/kg). During 5 to 6 hour infusions of carbacyclin there was no evidence of desensitization of dog platelets to the anti-aggregatory activity. These results show that carbacyclin has a similar spectrum of activity as prostacyclin and is about one-tenth as potent.     

Carbacyclin — A potent stable prost acyclin analogue for the inhibition of platelet aggregation

Carbacyclin is a chemically stable analogue of prostacyclin. As an inhibitor of platelet aggregation induced by ADP or collagen in vitro, carbacyclin is 0.03 times as active as prostacyclin in human, dog or rabbit plasma. Carbacyclin, like prostacyclin, reduces systemic arterial blood pressure (BP) in dogs, rabbits and rats and is not inactivated during passage through the pulmonary circulation. Further actions were investigated using a new ex vivo technique which allows rapid preparation of platelet-rich plasma and determination of platelet aggregation. In the dog, intravenous infusion of carbacyclin or prostacyclin inhibits platelet aggregation ex vivo with minimal effects on BP or heart rate. In the anaesthetised or conscious rabbit, carbacyclin and prostacyclin produces similar cardiovascular changes in doses producing an equivalent degree of platelet inhibition. In both rabbit and dog, carbacyclin is 0.1 times as active as prostacyclin in inhibiting ex vivo platelet aggregation. Platelet inhibition is maintained throughout the period of infusion of either compound (up to 3 h) yet is no longer apparent 10 min after terminating the infusion. Carbacyclin is thus a chemically-stable but metabolically-unstable analogue with a biological profile closely similar to prostacyclin.     

The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes

The effect of carbacyclin, a chemically stable analogue of prostacyclin, on the activity of adenylate cyclase in platelet membranes was measured, and compared with the effect of PGE1. When GTP was added in concentrations up to 10 microM the activation of adenylate cyclase by carbacyclin was increased, whereas higher concentrations of GTP were inhibitory. The addition of a non-hydrolysable analogue of GDP, guanosine 5'-[beta-thio]diphosphate (GDP[beta S] ) resulted in a dose-dependent inhibition of adenylate cyclase activation by carbacyclin; this inhibition was relieved by adding increased amounts of GTP.     

Prostacyclin and steroidogenesis in goat ovarian cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 micrograms/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P4) and estradiol-17 beta (E2) production by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10(-6)-10(-3)M) and indomethacin (100 ng-1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 micrograms/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 micrograms- 1 mg/ml) and dipyridamole (10(-6)-10(-3)M), when added alone or along with AA, did not affect steroid production. Up to 100 micrograms/ml of U-51605 (9,11-azoprosta-5,13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGI2) and its stable analog 6 beta PGI1 (0.01-10 micrograms/ml) produced a dose-dependent increase in P4 and E2 production in all the three cell types. Increase at 1 and 10 micrograms/ml was significant in all cases. 6-keto-PGE1 (an active metabolite of PGI2 in certain systems) produced an increase in steroid production which was significant in theca at greater than or equal to 1 microgram/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGF1 alpha (stable metabolite of PGI2) was without effect in the present system. The lack of effect of PGI2 at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolone. The present results indicate that AA-stimulated steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGI2 though PGI2 itself can positively modulate the steroid production.     

Are the renal functional changes of human pregnancy caused by prostacyclin?

To investigate the hypothesis of others that the many characteristic physiological alterations of pregnancy are due to prostacyclin, the effects of this vasodilator prostaglandin on renal function and related variables were measured. Intravenous infusion of prostacyclin (7 ng/kg/min) for 90 minutes in seven healthy male volunteers resulting in a significant fall in diastolic blood pressure and increase in heart rate. No significant change occurred in renal plasma flow or glomerular filtration rate in response to the prostacyclin, but there were significant falls in uric acid clearance (16 +/- 1.3----10 +/- 1.5 ml/min/ 1.73m2) and in free water clearance (4.0 +/- 1.1-----0.5 +/- 0.4 ml/min) both of which rose rapidly to pre-infusion levels upon cessation of prostacyclin infusion. Plasma renin and aldosterone levels rose sharply during prostacyclin infusion, falling to pre-infusion levels rapidly afterwards. Although the haemodynamic alterations were similar, the renal functional effects of prostacyclin infusion were quite different from (and often opposite in direction to) those of normal pregnancy. This acute study therefore does not add support to the hypothesis suggested by others that these alterations in pregnancy are due to prostacyclin, and that deficiency of prostacyclin will lead to the changes of pregnancy-associated hypertension.     

Effect of the prostacyclin analogue iloprost in sodium-depleted rats pretreated with captopril.

Previous studies have shown that administration of captopril to sodium-depleted rats decreases the glomerular filtration rate (GFR) and blunts the increase in glomerular prostacyclin synthesis normally occurring in response to sodium depletion. To clarify the relationship between these two responses, iloprost, a stable analogue of prostacyclin, was administered to Na-depleted, captopril-treated (LNC) rats. At a dosage not affecting systemic blood pressure (12.5 ng/kg/min), iloprost increased GFR in LNC rats by 25% (from 0.26 +/- 0.03 to 0.35 +/- 0.03 ml/min/100 g body wt, P less than 0.01), without significant effects on renal plasma flow. No effect was observed in control rats. The results suggest that altered prostacyclin synthesis could contribute to the decrease of GFR in this model.     

A comparison of the inhibitory effects of prostacyclin and carbacyclin on platelet adhesion to collagen

The effect of carbacyclin, a chemically stable analogue of prostacyclin (PGI2), on the adhesion of platelets to collagen has been examined. The compound was compared to PGI2 which is unstable and rapidly hydrolysed to the inactive derivative, 6-oxo-PGF 1 alpha. The adhesion of 111Indium-labelled human platelets to collagen in the absence of platelet aggregation and secretion was measured. The cAMP level in the platelets was also monitored. Both PGI2 and carbacyclin inhibited platelet-collagen adhesion and caused a rise in the platelet cAMP level. Carbacyclin was approximately 15-fold less effective than PGI2, however, its effect was longer lasting, remaining constant for at least 30 minutes.     

Effect of prostacyclin (PGI2) on platelet adhesion to rabbit arterial subendothelium.

The effect of prostacyclin on platelet aggregation and adhesion was investigated in everted pieces of rabbit abdominal aorta, from which the endothelium had previously been removed. Citrated human blood, to which different, concentrations of prostacyclin (0.1-100 ng/ml) were added, was perfused through the vessels, after which sections were examined and evaluated by light microscopy. Prostacyclin inhibited thrombus formation at concentrations greater than 0.1 ng/ml, whereas 20 ng/ml were required to reduce the amount of adhesion to the subendothelial surface. Thus prostacyclin prevents thrombus formation at much lower concentrations than are needed to inhibit platelet-vessel wall interaction.     

Effect of prostacyclin (PGI2) on platelet adhesion to rabbit arterial subendothelium

The effect of prostacyclin on platelet aggregation and adhesion was investigated in everted pieces of rabbit abdominal aorta, from which the endothelium had previously been removed. Citrated human blood, to which different concentrations of prostacyclin (0.1–100 ng/ml) were added, was perfused through the vessels, after which sections were examined and evaluated by light microscopy. Prostacyclin inhibited thrombus formation at concentrations greater than 0.1 ng/ml, whereas 20 ng/ml were required to reduce the amount of adhesion to the subendothelial surface. Thus prostacyclin prevents thrombus formation at much lower concentrations than are needed to inhibit platelet-vessel wall interaction.     

Intestinal blood flow and oxygen consumption

In this study, we examined the effects of both pharmacologically and mechanically induced increases in intestinal blood flow on intestinal oxygen consumption. Intraarterial infusions of prostacyclin (1-20 ng X kg-1 X min-1) significantly increased both blood flow and oxygen consumption under free flow conditions. However, the increase in oxygen consumption appears to be due to the corresponding increase in blood flow rather than a direct effect of prostacyclin on intestinal metabolism. This conclusion is supported by the finding that a mechanically induced increase in intestinal blood flow (60%) can also produce an increase in intestinal oxygen consumption (24%). These findings support the hypothesis that intestinal oxygen consumption is flow-dependent over a wide range of blood flows.     

Pulmonary prostacyclin production with increased flow and sympathetic stimulation

We evaluated the effects of an abrupt increase in flow and of a subsequent sympathetic nerve stimulation on the pulmonary production of prostacyclin (PGI2) and thromboxane A2 (TXA2) in canine isolated left lower lobes perfused in situ with pulsatile flow. When flow was abruptly increased from 50 +/- 3 to 288 +/- 2 ml/min, mean pulmonary arterial pressure (Ppa) increased by 15 +/- 2 Torr and then declined by 2.4 Torr over the next 5 min. This secondary decrease in Ppa was associated with a significant 0.26 +/- 0.11 ng/ml increase in the pulmonary venous concentration of the stable PGI2 hydrolysis product 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) as determined by radioimmunoassay. Stimulation of the left stellate ganglion usually resulted in an increase in Ppa which peaked at 1.1 +/- 0.6 Torr above its prestimulus level and then declined over the next 5 min. Associated with this decline was a 0.24 +/- 0.11 ng/ml increase in 6-keto-PGF1 alpha at 1 min. We suggest that the decline in Ppa is due to the synthesis and release of PGI2 by the endothelial cells in response to an increase in perfusion pressure.     

Are the renal functional changes of human pregnancy caused by prostacyclin?

To investigate the hypothesis of others that the many characteristic physiological alterations of pregnancy are due to prostacyclin, the effects of this vasodilator prostaglandin on renal function and related variables were measured.

Intravenous infusion of prostacyclin (7 ng/kg/min) for 90 minutes in seven healthy male volunteers resulting in a significant fall in diastolic blood pressure and increase in heart rate. No significant change occurred in renal plasma flow or glomerular filtration rate in response to the prostacyclin, but there were significant falls in uric acid clearance (16 ± 1.3 → 10 ± 1.5 ml/min/ 1.73m²) and in free water clearance (4.0 ± 1.1 → −0.5 ± 0.4 ml/min) both of which rose rapidly to pre-infusion levels upon cessation of prostacyclin infusion. Plasma renin and aldosterone levels rose sharply during prostacyclin infusion, falling to pre-infusion levels rapidly afterwards.

Although the haemodynamic alterations were similar, the renal functional effects of prostacyclin infusion were quite different from (and often opposite in direction to) those of normal pregnancy. This acute study therefore does not add support to the hypothesis suggested by others that these alterations in pregnancy are due to prostacyclin, and that deficiency of prostacyclin will lead to the changes of pregnancy-associated hypertension.     

Effects of uterine stimulant drugs on prostacyclin production by the pregnant rat myometrium. I. Oxytocin, bradykinin and PGF2α

The release of prostacyclin from chopped myometrial fractions of 18–20 day pregnant rats was assayed by inhibition of ADP-induced aggregation of citrated rabbit platelet-rich plasma. Preincubation of myometrial tissue with oxytocin 10 mU/ml increased prostacyclin generation from 2.25 ± 0.48 (control) to 3.75 ± 0.73 ng/mg over 15 minutes. Bradykinin 20 μg/ml also caused a significant increase in myometrial prostacyclin output from 2.26 ± 0.19 to 4.26 ± 0.64 ng/mg. PGF_2α 1 μg/ml did not increase prostacyclin release significantly. Pretreatment of myometrial tissue with the phospholipase inhibitor mepacrine significantly reduced the peptide-stimulated release of prostacyclin. The results suggest that prostacyclin production may play an important role in modulating the actions of oxytocin and bradykinin in the pregnant rat myometrium.     

Prostacyclin and steroidogenesis in goat ovari cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 μg/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P₄) and estradiol-17β (E₂) productin by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10⁻⁶−10⁻³M) and indomethacin (100 ng−1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 μ/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 μg− 1 mg/ml) and dipyridamole (10⁻⁶−10⁻³M), when added alone or along with AA, did not effect steroid production. Up to 100 μg/ml of U-51605 (9,11-azoprosta-5, 13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGl₂) and its stable analog 6βPGl₁(0.01–10μg/ml) produced a dose-dependent increase in P₄ and E₂ production in all three cell types. Increase at 1 and 10μg/ml was significant in all cases. 6-keto-PGE₁ (an active metabolite of PGl₂ in certain systems) produced an increase in steroid production which was significant in theca at 1μg/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGf₁ alpha (stable metabolite of PGl₂) was without effect inthe present system. The lack of effect of PGl₂ at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolene. The present results indicate that AA- stimualted steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGl₂ though PGl₂ itself can positively modulate the steroid production.     

Effect of prostacyclin on perfusion pressure, electrical activity, rate and force of contraction in isolated rat and rabbit hearts.

Prostacyclin when added to medium perfusing rat and rabbit hearts caused an increase in perfusion pressure at concentrations from 1 pg/ml ? 1 ng/ml (2.8 × 10^?12 ? 2.8 × 10^?9M) and a decrease at higher concentrations. Rhythm disturbances were observed with low prostacyclin concentrations in 6 of 10 rat hearts and 2 of 5 rabbit hearts studied. Increased heart rates were seen in the isolated rat hearts but not in the rabbit hearts. Force of contraction of isolated rat hearts was increased with increasing prostacyclin concentrations up to 100 pg/ml. Higher concentrations decreased contractile force. No inotropic effects were seen with rabbit hearts.     

The effect of prostaglandin synthetase inhibitors on renal function: studies in normal rats during sodium or water diuresis and in rats with chronic renal insufficiency

The effect of acute infusion of the prostaglandin synthetase inhibitors - meclofenamate or indomethacin - was examined in awake rats. Studies were performed in normal rats undergoing either sodium or water diuresis and in salt-replete rats with chronic renal insufficiency. Prostaglandin synthetase inhibitors had no effect on renal plasma flow, glomerular filtration rate or fractional excretion of sodium in any of the groups. Absolute urinary excretion rates for sodium and potassium decreased only in the normal, salt-replete rats. In contrast, prostaglandin synthetase inhibitors consistently decreased urinary flow and osmolar clearance under all experimental conditions studied. In the normal, salt-replete rats the fall in urine flow was preceded by an increase in urinary excretion of cyclic AMP. These results show that inhibitors of prostaglandin synthesis enhance the ability of the kidney to reabsorb water. This effect may be secondary to increased cyclic AMP generation and to increased urea recirculation resulting in higher urea accumulation in the renal medulla.     

Effect of thromboxane A2 synthetase inhibitor and prostacyclin analogue on arterial blood pressure, fibrinolysis and platelet function in patients with hypertension]

An effect of the specific thromboxane A2 synthetase inhibitor and stable prostacyclin analogue on arterial blood hypertension was investigated in 12 patients with spontaneous hypertension of II degree and in 12 healthy subjects. The patients were given a 3-hour intravenous infusion of Iloprost (Schering) in the dose of 2 ng/kg b.w. per minute and OKY-046 (ONO, Japan) in a single oral dose of 400 mg. Iloprost shortened euglobin fibrinolysis time without an effect on tissue plasminogen activator levels or blood pressure. OKY-046 decreased TBX2 to undetectable values, increased 6-keto-PGF1 alpha by 8-fold, and significantly reduced both systolic and diastolic blood pressures in hypertensive patients. Such effects may dependent upon an increase in the endogenous prostacyclin or an inhibition in thromboxane production in the affected arterial walls. If the present observations would be confirmed by double blind trial, they would constitute the base for new pharmacotherapy of hypertension.