Radioimmunoassay of prostaglandins

The earlier radioimmunoassays were mainly intended for the measurement of prostaglandins of the E-F-A or B type in blood plasma/serum or urine. Many recent studies, however, explain the use of radioimmunoassay to measure the prostaglandin content of tissues, and many other studies are concerned with the prostaglandin production in a single cell type, or in a few cell types, rather than the whole tissue. To date, however, by far the greatest number of quantitative prostaglandin studies have been carried out on blood plasma or serum, while assay for primary prostaglandins are now fairly seldom applied to the peripheral circulation, unless it is to study the prostaglandin production in vivo. It has been proposed that prostglandins of the A type are circulating hormones in contrast to other prostglandins, and a number of laboratories have developed quantitative methods for the measurements of PGA compounds. The sensitivity and specificity of the prostaglandins radioimmunoassays have increased considerably in later years through the use of labelled ligands of better quality; on the other hand, the accuracy of many radioimmunoassays seems to be very low when they are applied to biologic materials.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2 alpha on human umbilical cord vessels

Human umbilical blood vessels have the ability to close spontaneously following delivery at term. It has been suggested that prostaglandins may have a possible physiological role in its closure. This study investigates the effects of 6 naturally occurring prostaglandins (A1, A2, B1, B2, E2, F2a) on the umbilical blood vessels. Umbilical cords were collected from cases of normal spontaneous vaginal deliveries and cesarian section at term. A total of 41 strips of umbilical arteries and 26 strips of umbilical veins from 24 cords were used. A 4-point bioassay method was used to compare the potency of prostaglandins A1, A2, B1 and F2a with PGE2. The effect of Polyphloretin Phosphate (PPP) on prostaglandin-induced contractions was studied on umbilical artery strips from 12 cords. The 6 prostaglandins exerted a stimulant effect on the isolated strips of human umbilical arteries. Prostaglandin B2 was the most potent compound on the umbilical vein, followed by PGA2. PPP in the concentration range of 10 to 40 mcg/ml completely eliminated the responses of PGE2, F2a, A1, A2, and B1. Responses to PGB2 were considerably but not completely abolished. PPP (up to 40 mcg/ml) did not affect contractions induced by 5-hydroxytryptamine, suggesting the presence of discrete receptor sites in the blood vessels for different pharmacologically active compounds. This is the first report of the constrictor effect of PGA and PGB compounds. These naturally occuring prostaglandins with high potencies (compared with other prostaglandins and other vasoactive substances) may play a role in spontaneous closure of umbilical vessels. PGE1, E2, F1 and F2a are found in umbilical blood vessels obtained at term.     

Effect of prostaglandins on 3H-thymidine uptake into human epidermal cells in vitro.

Prostaglandins A2, B1, E1, E2, F1alpha and F2alpha were added to cultures of human epidermal cells (keratinocytes) for 24 hours at 37 degrees C, and the effects on 3H-thymidine uptake into DNA was measured. At 70 mu/ml all prostaglandins tested except PGF2alpha inhibited the uptake of 3-thymidine greater than 50%. However, at 35 mug/ml, PGA2 and PGB1 were the only two prostaglandins to show significant inhibition, 96% and 51% respectively. At 17.5 mug/ml only PGA2 caused substantial inhibition, 68%. In order to determine if the PGA2 action was mediated by membrane receptors propranolol, phentolamine, metiamide and prostynoic acid were added in conjunction with PGA2. None of the above receptor antagonists were able to reduce the PGA2-induced inhibition of 3H-thymidine uptake. These results indicate that the pre-incubation of human keratinocytes with prostaglandins for 24 hours results in a decrease of 3H-thymidine incorporation in DNA. The precise mechanism of action is unknown at this time.     

Uptake and inactivation of a-type prostaglandins by human red cells

Incubation of A type prostaglandins with whole blood or washed red cells at 37°C converted them to more polar products with negligible vasodepressor and smooth muscle-contracting activities. This conversion did not occur in platelet-rich plasma. Uptake of the prostaglandins by red cells was demonstrated at both 4°C and 37°C. The data suggest 1) that if PGA is released from tissues into the blood stream or is administered for therapeutic purposes, its biological activity would be diminished by human red cells, and 2) that development of an assay for PGA in blood should take into account its uptake and metabolism by human red cells.     

Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.     

Single session exercise stimulates formation of pre beta 1-HDL in leg muscle

Physical activity can raise the level of circulating HDL cholesterol. Pre beta 1-HDL is thought to be either the initial acceptor of cellular cholesterol or virtually the first particle in the pathway of the formation of HDL from apolipoprotein A-I and cellular lipids. We have therefore sought to identify pre beta 1-HDL in arterial and venous circulations of exercising legs in healthy individuals and in subjects with stable Type 2 diabetes mellitus. Blood samples were taken simultaneously from the femoral artery and vein before and after 25 min cycling exercise. The major findings were, first, that exercise significantly increased plasma concentration of pre beta 1-HDL (20% increase, P < 0.05) and second, that the pre beta 1-HDL concentration was significantly higher in the venous compared with the arterial blood both before and after exercise in both diabetics and controls. In the combined population, formation of pre beta 1-HDL at rest was 9.9 +/- 5.2 mg/min and exercise enhanced pre beta 1-HDL formation 6.6-fold in both groups.     

Vasoactive properties of dihomo-gamma-linolenic acid and series one prostaglandins in a freshwater teleost, Channa maculata

1. Prostaglandins A1, B1, E1 and F1 alpha (2-120 micrograms/kg), arachidonic acid and dihomo-gamma-linolenic acid (0.1-2 mg/kg) were injected intravenously into Channa maculata and changes in arterial blood pressure were recorded. 2. Injection of PGF1 alpha had no significant effect on arterial blood pressure. Injection of PGA1 and PGE1 was followed by dose-dependent hypotension whereas injection of PGB1 elicited significant dose-dependent increase in arterial blood pressure. 3. Both dihomo-gamma-linolenic acid and arachidonic acid were also depressor agents but dihomo-gamma-linolenic acid was more potent. 4. A single bolus intravenous injection of indomethacin (5 mg/kg) or 4 daily intraperitoneal injections (4 x 10 mg/kg) significantly lowered arterial blood pressure. One hour after pre-treatment of indomethacin, the vascular effects of both prostaglandin precursors were abolished. 5. It appears that the vascular effects of prostaglandins in Channa maculata are qualitatively different from those reported for mammals.     

Prostaglandin A1 metabolism and inhibition of cyclic AMP extrusion by avian erythrocytes

Prostaglandins (PG) inhibit active cyclic AMP export from pigeon red cells, PGA1 and PGA2 most potently (Brunton, L.L., and Mayer, S.E. (1979) J. Biol. Chem. 254, 9714-9720). To probe the mechanism of this action of PGA1, we have studied the interaction of [3H]PGA1 with suspensions of pigeon red cells. The interaction of PGA1 with pigeon red cells is a multistep process of uptake, metabolism, and secretion. [3H] PGA1 rapidly enters red cells and is promptly metabolized (Vmax greater than or equal to 1 nmol/min/10(7) cells) to a compound(s) that remains in the aqueous layer after ethylacetate extraction. The glutathione-depleting agent, diamide, inhibits formation of the PGA1 metabolite. In agreement with the order of potency of other prostaglandins to inhibit cAMP efflux (A much greater than E congruent to B greater than F), PGA2 forms a polar adduct whereas prostaglandins E2, B1, and F2 alpha do not. The red cells secrete the polar metabolite of PGA1 by a saturable mechanism (at 37 degrees C, Km congruent to 0.6 microM, Vmax congruent to 0.5 pmol/min/10(7) cells) that lowered temperatures inhibit (Eact congruent to 21 kcal/mol). Because uptake and metabolism progress with much greater rates than metabolite secretion, red cells transiently concentrate the polar compound intracellularly. Onset and reversal of inhibition of cyclic AMP export by PGA1 coincide with accumulation and secretion of PGA1 metabolite, suggesting that the polar metabolite acts at an intracellular site to inhibit cyclic AMP efflux. In the accompanying Appendix, we present chromatographic and amino acid analyses demonstrating that the polar metabolite is a glutathione adduct of PGA1.     

Lactate concentration differences in plasma, whole blood, capillary finger blood and erythrocytes during submaximal graded exercise in humans

The aim of the study was to investigate the distribution of lactate in plasma, whole blood, erythrocytes, and capillary finger blood, before and during submaximal exercise. Ten healthy male subjects performed submaximal graded cycle ergometer exercise for 20-25 min. Venous blood samples and capillary finger blood samples were taken before exercise and every 5th min during exercise for lactate determination. The plasma lactate concentration was significantly higher (P less than 0.001, approximately 50%) than in the erythrocytes. This difference was not altered by the venous blood lactate concentration or exercise intensity. A significant difference (P less than 0.01) in lactate concentration was also found between capillary whole blood and venous whole blood. It was concluded that direct comparisons between lactate in capillary finger blood, venous whole blood and plasma could not be made.     

Differential effects of prostaglandins A1 and A2 on pulmonary vascular resistance in the dog.

The effects of PGA1 and PGA2 were studied in the canine pulmonary vascular bed. Infusion of PGA1 into the lobar artery decreased lobar arterial and venous pressure but did not change left atrial pressure. In contrast, PGA2 infusion increased lobar arterial and venous pressure and the effects of this substance were similar in experiments in which the lung was perfused with dextran or with blood. These data indicate that under conditions of controlled blood flow PGA1 decreases pulmonary vascular resistance by dilating intrapulmonary veins and to a lesser extent vessels upstream to the small veins, presumably small arteries. The present data show that PGA2 increases pulmonary vascular resistance by constricting intrapulmonary veins and upstream vessels. The predominant effect of PGA2 was on upstream vessels and the pressor effect was not due to interaction with formed elements in the blood or platelet aggregation.     

Gastric and intestinal release of vasoactive intestinal polypeptide in the milk-fed lamb

Vasoactive intestinal polypeptide (VIP) has been proposed as the neurotransmitter of the atropine-resistant relaxation of gastric structures in the lamb. To examine this proposal VIP concentrations in plasma from arterial, gastric venous and intestinal venous blood were measured in healthy conscious lambs before, during and after teasing with, and sucking of milk. Basal arterial plasma VIP concentrations were undetectable (less than 3 pmol/l) and remained so during and after feeding. Before feeding VIP was detected in only 2 of 12 gastric venous plasma samples (5 and 13 pmol/l). During teasing with food there were increments in VIP of 19 +/- 4 pmol/l and during feeding of 27 +/- 5 pmol/l. VIP concentration in gastric venous plasma rapidly returned to fasting levels after cessation of sucking. In contrast VIP in the intestinal venous plasma did not rise during teasing or upon commencement of sucking but a peak increment of 34 +/- 6 pmol/l occurred at 5 min after cessation of feeding. The results are consistent with the hypotheses that VIP is released in anticipation of and during sucking from inhibitory neurones involved in relaxation of gastric structures and that intestinal release of VIP is a consequence of entry of digesta into the small intestine.     

Plasma [H+] regulation and whole blood [CO2] in exercising ponies

The major objective was to determine in ponies whether factors in addition to changes in blood PCO2 contribute to changes in plasma [H+] during submaximal exercise. Measurements were made to establish in vivo plasma [H+] at rest and during submaximal exercise, and CO2 titration of blood was completed for both in vitro and acute in vivo conditions. In 19 ponies arterial plasma [H+] was decreased from rest 4.5 neq/l (P less than 0.05) during the 7th min of treadmill running at 6 mph, 5% grade (P less than 0.5). A 5.6-Torr exercise hypocapnia accounted for approximately 2.9 neq/l of this reduced [H+]. The non-PCO2 component of this alkalosis was approximately neq/l, and it was due presumably to a 1.7-meq/l increase from rest in the plasma strong ion difference (SID). Despite the arterial hypocapnia, mixed venous PCO2 was 2.7 Torr above rest during steady-state exercise. Nevertheless, mixed venous plasma [H+] was 1.2 neq/l above rest during exercise, which was presumably due to the increase in SID. Also studied was the effect of submaximal exercise on whole blood CO2 content (CCO2). In vitro, at a given PCO2 there was minimal difference in CCO2 between rest and exercise blood, but plasma [HCO3-] was greater for exercise blood than for rest blood. In vivo, during steady-state exercise, arterial plasma blood. In vivo, during steady-state exercise, arterial plasma [HCO3-] was unchanged or slightly elevated from rest, but CaCO2 was 4 vol% below rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of prostaglandin of the renal vascular action of arginne vasopressin

To determine whether the renal vascular effect of arginine vasopressin (AVP) is modulated by renal prostaglandin E₂ (PGE₂) were determined during the infusion of AVP in dogs during control conditions and after the administration of the inhibitor of prostaglandin synthesis, indomethacin. During control conditions, intrarenal administration for 10 min of a dose of AVP calculated to increase arterial renal plasma AVP concentration by 75 pg/ml produced a slight renal vasodilation (p<0.01) and an increase in renal venous plasma concentration of PGE₂. Renal venous PGE₂ concentration during control and AVP infusion averaged 33 ± 7 and 52 ± 12 pg/ml (p<0.05), respectively. After administration of indomethacin, the same dose of AVP induced renal vasoconstriction (p<0.05) and failed to enhance renal venous PGE₂ concentration (9 ± 1 to 8 ± 1 pg/ml). Intrarenal administration of 20 ng/kg. min of AVP for 10 min induced a marked renal vasoconstriction (p<0.01) and increased renal venous plasma PGE₂. Renal venous PGE₂ during control and AVP infusion averaged 31 ± 10 and 121 ± 31 pg/ml (p<0.01), respectively. Administration of the same dose of AVP following indomethacin produced a significantly greater and longer lasting renal vasoconstriction (p<0.01) and failed to increase renal venous plasma PGE₂ (10 ± 1 to 9 ± 1 pg/ml). These results indicate that a concentration of AVP comparable to that observed in several pathophysiological conditions induces a slight renal vasodilation which is mediated by renal prostaglandins. The results also indicate that higher doses of AVP induce renal vasoconstriction and that prostaglandin synthesis induced by AVP attenautes the renal vasoconstriction produced by this peptide.     

Contribution of prostaglandins to the dilation that follows isometric forearm contraction in human subjects: effects of aspirin and hyperoxia.

In 11 healthy volunteers, we evaluated, in a double-blind crossover study, whether the vasodilation that follows isometric contraction is mediated by prostaglandins (PGs) and/or is O2 dependent. Subjects performed isometric handgrip for 2 min at 60% maximal voluntary contraction (MVC), after pretreatment with placebo or aspirin (600 mg orally), when breathing air or 40% O2. Forearm blood flow was measured in the dominant forearm by venous occlusion plethysmography. Arterial blood pressure was also recorded, allowing calculation of forearm vascular conductance (FVC; forearm blood flow/arterial blood pressure). During air breathing, aspirin significantly reduced the increase in FVC that followed contraction at 60% MVC: from a baseline of 0.09 +/- 0.011 [mean +/- SE, conductance units (CU)], the peak value was reduced from 0.24 +/- 0.03 to 0.14 +/- 0.01 CU. Breathing 40% O2 similarly reduced the increase in FVC relative to that evoked when breathing air; the peak value was 0.24 +/- 0.03 vs. 0.15 +/- 0.02 CU. However, after aspirin, breathing 40% O2 had no further effect on the contraction-evoked increase in FVC (the peak value was 0.15 +/- 0.02 vs. 0.16 +/- 0.02 CU). Thus the present study indicates that prostaglandins make a substantial contribution to the peak of the vasodilation that follows isometric contraction of forearm muscles at 60% MVC. Given that hyperoxia similarly reduced the vasodilation and attenuated the effect of aspirin, we propose that the stimulus for prostaglandin synthesis and release is hypoxia of the endothelium.     

Inhibition of vesicular stomatitis virus replication by prostaglandin A1 in Aedes albopictus cells

Cyclopentenone prostaglandins (PGs) exhibit antiviral activity against RNA and DNA viruses in mammalian cell lines, and this effect has been associated with the induction of a heat shock protein (hsp70). We investigated the effect of prostaglandin A1 (PGA1) on the replication of vesicular stomatitis virus (VSV) in Aedes albopictus (mosquito) cells. PGA1 was found to inhibit VSV replication dose dependently. Virus yield was reduced to 50% (3 microg PGA1/ml) and to 95% with 8 microg PGA1/ml. Even with the dramatic reduction of virus production observed in cells treated with PGA1, VSV-specific protein synthesis was unaltered. Treatment of cells with PGA1 (5 microg/ml) stimulated the synthesis of a polypeptide identified as a heat-shock protein (hsp) by immunoblot analysis. PGA1 induced hsp70 synthesis in uninfected cells. However, in VSV-infected cells the induction of hsp70 by PGA1 was reduced. This is the first report of antiviral effects of PGs affecting the replication of VSV in a mosquito cell line.     

A DIRECT METHOD FOR DETERMINING DOPAMINE SYNTHESIS AND OUTPUT OF DOPAMINE METABOLITES FROM BRAIN IN AWAKE ANIMALS

Abstract— A direct method for measuring the rate of dopamine (DA) synthesis and the DA metabolites by the brain of awake monkeys ( Macaca arctoides ) is described. The method utilizes a coupling of a measure of cerebral blood flow with the mass spectrometrically determined difference in the concentrations of the metabolite under study in plasma obtained from arterial and internal jugular bulb blood. For homovanillic acid (HVA) a consistent and highly significant veno-arterial (V-A) difference of 2.2 ± 0.4 ng/ml of plasma ( P < 0.0005) was found. When this V-A difference was coupled with a measure of cerebral blood flow it was determined that, in the awake monkey, the average output of HVA by brain was 113.4 ± 19.1ng/100g brain min⁻¹. There were large individual variations, however, between animals (range = 38-194 ng/100g brain min⁻¹). In contrast to HVA, no consistent V-A difference for dihydroxyphenylacetic acid (DOPAC) was found; i.e. the concentrations of DOPAC in plasma obtained from arterial and internal jugular bulb venous blood were essentially identical. These data indicate that, in contrast to the rat, in this non-human primate HVA is the major metabolic product of brain DA. Since HVA is the major metabolite of DA, production of HVA under steady state conditions gives a measure of DA synthesis by whole brain; i.e. the rate of DA synthesis by whole brain in the awake monkey is 113.4 ± 19.1ng/100g brain min⁻¹. It is suggested that this technique may be of value in both basic and applied types of studies.     

Inhibition of the Prostaglandin Transporter PGT Lowers Blood Pressure in Hypertensive Rats and Mice

Inhibiting the synthesis of endogenous prostaglandins with nonsteroidal anti-inflammatory drugs exacerbates arterial hypertension. We hypothesized that the converse, i.e., raising the level of endogenous prostaglandins, might have anti-hypertensive effects. To accomplish this, we focused on inhibiting the prostaglandin transporter PGT (SLCO2A1), which is the obligatory first step in the inactivation of several common PGs. We first examined the role of PGT in controlling arterial blood pressure blood pressure using anesthetized rats. The high-affinity PGT inhibitor T26A sensitized the ability of exogenous PGE₂ to lower blood pressure, confirming both inhibition of PGT by T26A and the vasodepressor action of PGE₂ T26A administered alone to anesthetized rats dose-dependently lowered blood pressure, and did so to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto control rats. In mice, T26A added chronically to the drinking water increased the urinary excretion and plasma concentration of PGE₂ over several days, confirming that T26A is orally active in antagonizing PGT. T26A given orally to hypertensive mice normalized blood pressure. T26A increased urinary sodium excretion in mice and, when added to the medium bathing isolated mouse aortas, T26A increased the net release of PGE₂ induced by arachidonic acid, inhibited serotonin-induced vasoconstriction, and potentiated vasodilation induced by exogenous PGE₂. We conclude that pharmacologically inhibiting PGT-mediated prostaglandin metabolism lowers blood pressure, probably by prostaglandin-induced natriuresis and vasodilation. PGT is a novel therapeutic target for treating hypertension.     

Fibrinolytic activity of prostacyclin and iloprost in patients with peripheral arterial disease

We studied the effect of prostacyclin /PGI₂/ and its stable analog, iloprost, on blood fibrinolytic activity in 33 patients with peripheral arterial disease. Ten subjects /group A/ received three 5-hour infusions of iloprost on three consecutive days. The remaining 23 patients received three different 5-hour infusions /placebo, iloprost 2 ng/kg/min, PGI₂ 5 ng/kg/min/. Tissue plasminogen activator /t-PA/, total plasma fibrinolytic activity and euglobulin clot lysis time /ECLT/ were determined in patients before and after each infusion, both in freely flowing blood samples and following 10 min venous occlusion. In patients of group A, ECLT at rest was significantly shortened after all three iloprost infusions /on average by about 5–11%/. First and third infusions produced also shortening of ECLT after venostasis /by 21 and 32%/. Statistically significant rise in t-PA activity /by about 68% on average/ accompanied only the first infusion. In patients of the group B iloprost provoked significant fall in ECLT at rest /by about 19% on average/ only. PGI₂ shortened ECLT both at rest and after venous occlusion /by about 17% and 20% on average, respectively/ and led to a rise in t-PA activity after venous occlusion by about 33% on average. Our results indicate that prostacyclin and its stable analog, iloprost, enhance fibrinolytic activity in man by releasing or facilitating the release of tissue plasminogen activator from the vessel wall.     

Human cardiac plasma concentrations of atrial natriuretic peptide quantified by radioreceptor assay

The presence of high affinity receptors for atrial natriuretic peptide in bovine adrenal cortex has enabled the development of a sensitive, specific and rapid radioreceptor assay for this peptide in human plasma. In 18 normal subjects, venous plasma atrial natriuretic peptide concentration ranged from 6 to 65 pM. This plasma concentration was two-fold higher in right atrium as compared to venous blood in 12 patients investigated by cardiac catheterisation, confirming that the right atrium is the site of release of atrial natriuretic peptide into circulation. There was a further step up in plasma atrial natriuretic peptide concentration between pulmonary arterial and aortic plasma. This finding indicates that released hormone in man may undergo further activation in the lungs, or that there may be direct release from the left atrium.