Glomeruli from ischemic rat kidneys produce increased amounts of platelet activating factor

The production of Platelet Activating Factor (PAF-acether) by glomeruli isolated from rats subjected to a 60 min renal ischemia has been studied. PAF-acether has been purified by Sep-Pak columns and measured by its ability to release serotonin from previously loaded rabbit platelets. Glomeruli from ischemic kidneys had higher amounts of PAF-acether activity than glomeruli from sham operated rats. These data suggest a role for PAF-acether in the renal functional alterations induced by renal ischemia.     

Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82 X 10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87 X 10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.     

Effect of platelet-activating factor on arachidonic acid metabolism in renal epithelial cells

We studied the effects of platelet-activating factor (PAF-acether) on phospholipase activity in renal epithelial cells. When platelet-activating factor was added to renal cells prelabeled with [3H]arachidonic acid, it induced the rapid hydrolysis of phospholipids. Up to 26% of incorporated [3H]arachidonic acid was released into the medium from renal cells. After the addition of PAF-acether, the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were observed. The amount of [3H]arachidonic acid released were comparable to the losses of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In renal cells biosynthetically labeled by incorporation of [3H]choline into cellular phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, the range of concentrations of PAF-acether-induced hydrolysis of labeled phosphatidylcholine were approximately equal to the amounts of lysophosphatidylcholine produced. We also observed a transient rise of diacylglycerol after the addition of platelet-activating factor to these cells. To test for action of phospholipase C, the accumulations of [3H]choline, [3H]inositol and [3H]ethanolamine were determined. The radioactivities in choline and ethanolamine showed little or no change. An increase in inositol was detectable within 1 min and it peaked at 3 min. These results indicate that platelet-activating factor stimulates phospholipase A2 and phosphatidylinositol-specific phospholipase C activity in renal epithelial cells. These phospholipase activities were Ca2+ dependent. Moreover, PAF-acether enhanced changes in cell-associated Ca2+. These results suggest that the increased Ca2+ permeability of cell membrane stimulates phospholipases A2 and C in renal epithelial cells. Prostaglandin biosynthesis was also enhanced in these cells by platelet-activating factor.     

Acute effects of intratracheal administration of platelet-activating factor in baboons

The bronchomotor effect of intratracheal administration of PAF-acether (60 micrograms X kg -1) was investigated in 37 curarized baboons mechanically ventilated with constant volume and frequency. PAF-acether caused an immediate bronchoconstriction as assessed by a marked increase in peak inspiratory pressure with no change in static pulmonary compliance and chest X-rays. There was a concomitant fall in arterial PO2 and a significant increase in ventilated unperfused lung zones. A decrease of circulating platelets and leucocytes was also observed. Local anesthesia with lidocaine and atropine did not prevent PAF-acether-induced bronchoconstriction although both markedly reduced the bronchial response to histamine. Albuterol significantly reduced the bronchial response to PAF-acether. Pretreatment with aspirin (80 mg X kg -1 iv) did not prevent the bronchoconstriction caused by PAF-acether, and intravenous or intratracheal arachidonic acid caused no bronchial response. Thus the role of cyclooxygenase metabolites of arachidonic acid in PAF-acether-induced bronchoconstriction is unlikely. In conclusion, an acute bronchoconstriction probably not triggered by stimulation of irritant receptors of the airways and associated with aggregation of platelet takes place subsequent to intratracheal administration of PAF-acether. These data suggest that PAF-acether might play a role in the pathogenesis of human asthma.     

Inhibition of PAF-acether induced weal and flare reaction in man by a specific PAF antagonist

It has been suggested that PAF-acether may be an important mediator in asthma and a PAF antagonist may therefore have a potentially important role. PAF-acether has been shown to induce a dual inflammatory response in the skin of man and we investigated the effect of BN 52063, a PAF antagonist, on this response. At a dose of 80 mg, BN 52063 orally inhibited the inflammatory response to a sub-cutaneous injection of PAF-acether 400 ng. A late cutaneous response was not observed in any of the subjects. BN 52063 was also demonstrated to be well tolerated at doses of 20, 40, 80 and 120 mg with no significant side effects.     

Inhibition of PAF-acether induced weal and flare reaction in man by a specific PAF antagonist

It has been suggested that PAF-acether may be an important mediator in asthma and a PAF antagonist may therefore have a potentially important role. PAF-acether has been shown to induce a dual inflammatory reponse in the skin of man and we investigated the effect of BN 52063, a PAF antagonist, on this response. At a dose of 80mg, BN 52063 orally inhibited the inflammatory response to a sub-cutaneous injection of PAF-acether 400ng. A late cutaneous response was not observed in any of the subjects. BN 52063 was also demonstrated to be well tolerated at doses of 20, 40, 80 and 120mg with no significant side effects.     

The effects of N-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes

Human leukocytes generate platelet-activating factor (PAF-acether), a lipid mediator of inflammation, from membrane alkyl phospholipids through the release of arachidonic acid or other fatty acids at the 2-position and subsequent acetylation. Because it was previously demonstrated that fish oil fatty acids suppress human leukocyte arachidonic acid release and metabolism, separate experiments were conducted to investigate the effects of dietary fish oil supplementation and in vitro incubation with fish oil fatty acids on calcium ionophore-stimulated PAF-acether generation in human monocytes. In subjects on their regular diets, a 4-hr incubation of monocyte monolayers with an optimally effective concentration of arachidonic acid of 1 micrograms/ml resulted in a 64% increase of calcium ionophore-induced net PAF-acether generation from 7.75 +/- 0.78 ng/10(6) cells for untreated monolayers to 12.70 +/- 1.21 ng/10(6) cells (mean +/- SEM). Treatment of monolayers with eicosapentaenoic acid (EPA) at the optimal concentration of 1 micrograms/ml decreased net PAF-acether generation by 28%. However, treatment of monocyte monolayers with docosahexaenoic acid did not appreciably affect net PAF-acether generation. The changes in PAF-acether release with each fatty acid added in vitro paralleled those in total PAF-acether generation; the percentage PAF-acether release remained unaffected. Three weeks of dietary supplementation with 18 g MaxEPA daily, providing 3.2 g EPA did not affect the PAF-acether generation of calcium ionophore-stimulated human monocyte monolayers. However, 6 weeks of dietary supplementation resulted in a 47% decrease of net total PAF-acether generation and a concomitant 59% decline in net PAF-acether release; the percentage release of PAF-acether was not affected. Thus, whether added to the diet or introduced in vitro, fish oil-derived fatty acids suppress PAF-acether generation by human monocyte monolayers.     

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1 microM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 +/- 39% and synthesis by 87 +/- 27% whereas at 0.1 microM a decrease of IL1 release of 52 +/- 9% and synthesis of 46 +/- 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase of inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1 microM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocytes functions, possibly via specific binding sites.     

The role of beta 2-adrenergic vascular receptors in the peripheral vasodilation caused by 17 beta-estradiol in anesthetized pigs

It has been previously shown in anesthetized pigs that intravenous infusion of 2 microg/h of 17beta-estradiol primarily dilated renal, iliac and coronary circulations, while higher doses of the hormone were required to cause vasodilation also in the mesenteric vascular bed. In the same experimental model, a tonic beta2-adrenoceptor mediated vasodilation, which could be argued to attenuate the vasodilator effect of 17beta-estradiol, has been described. The present study was planned to investigate the role of beta2-adrenergic receptors in the hemodynamic responses of renal and mesenteric vascular beds to 17beta-estradiol. Changes in flow caused by intravenous infusion of 2 microg/h of the hormone at constant heart rate and aortic blood pressure in the left renal and superior mesenteric arteries were assessed using electromagnetic flowmeters. In six pigs, infusion of 17beta-estradiol caused an increase in renal blood flow, which averaged 12.1% of the control values, without affecting mesenteric blood flow. In the same pigs, after hemodynamic variables had returned to the baseline values, blockade of beta2-adrenergic receptors with butoxamine caused an increase in aortic blood pressure and an increase in renal and mesenteric resistance. The subsequent infusion of 17beta-estradiol elicited increases in renal and mesenteric blood flow which respectively averaged 19.6% and 12.8%. Therefore, the present study in anesthetized pigs have shown that the vasodilator responses of the renal and mesenteric circulations to 17beta-estradiol were attenuated and even masked by a tonic beta2-adrenoceptor mediated vasodilation. This indicates that some vasodilator effects elicited by normally used replacement doses of the hormone may not be apparent.     

Beneficial effects of prostaglandin E2 in endotoxic shock are unrelated to effects on PAF-acether synthesis

The effects of endotoxic shock on the synthesis of PAF-acether by the stomach, duodenum and lung were examined in the rat. Furthermore, the effect of pretreatment with prostaglandin E₂ on endotoxin induced PAF-acether synthesis and changes in vascular permeability were examined. Administration of endotoxin resulted in significant increases in PAF-acether synthesis in all tissues studied. Such increases were apparent within 5–15 minutes of the administration of endotoxin, corresponding to the time when significant hypotension, hemoconcentration and increases in gastrointestinal vascular permeability were first observed. Pretreatment with prostaglandin E₂ resulted in a significant reduction of endotoxin-induced hypotension, hemoconcentration and changes in vascular permeability in the gastrointestinal tract. However, prostaglandin pretreatment did not significantly alter endotoxin-induced PAF-acether release from the gastrointestinal tissues studied. These results demonstrate that prostaglandin E₂ can significantly attenuate several of the systemic and gastrointestinal manifestations of endotoxic shock. The mechanism responsible for these beneficial actions appears to be unrelated to effects of prostaglandin E₂ on PAF-acether synthesis.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1μM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 ± 39% and synthesis by 87 ± 27% whereas at 0.1μM a decrease of IL1 release of 52 ± 9% and synthesis of 46 ± 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase or inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1μM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocyte functions, possibly specific binding sites.     

Partial characterization of the embryo-derived platelet-activating factor in mice

The platelet-activating factor (PAF) produced by mouse embryos showed similar kinetics of action and dose-response curve, in a bioassay, as did 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether). The activity of the embryo-derived PAF was not affected by inhibitors of the ADP (pyruvate kinase with phosphoenol pyruvate) or cyclo-oxygenase (indomethacin) pathways of platelet activation. Chlorpromazine, an inhibitor of the PAF-acether pathway of platelet activation, caused a significant inhibition of the effects of embryo-derived PAF. Phospholipases A2, C and D significantly inhibited the activity while lipase had no effect, suggesting a phospholipid structure. All the embryo-derived PAF was found in the chloroform fraction after chloroform:methanol (2:1 v/v) extraction, as was PAF-acether. Both factors migrated at a similar rate (Rf 0.10-0.12) on silica thin-layer chromatography (chloroform:methanol:water; 65:35:4 by vol.). The embryo-derived PAF therefore displays chemical, biochemical and physiological properties similar to those of PAF-acether.     

Physiological responses to low dose infusions of atrial peptide in conscious dogs

Mongrel dogs prepared with chronic catheters in their femoral artery and vein and urinary bladder received 60 minute infusions of atrial peptide ranging from 5 to 100 ng/kg/min. Infusion of atrial peptides caused dose dependent increases in plasma atrial peptide concentration with doses of 25 ng/kg/min or less increasing plasma concentrations to levels observed in normal animals during stimulation of endogenous atrial peptide secretion. Atrial peptide infusion at doses of 10 ng/kg/min and above caused significant decreases in mean arterial pressure which were not accompanied by statistically significant changes in heart rate. Atrial peptide infusion at doses of 25 ng/kg/min and above increased urinary sodium excretion and urine flow rate. Atrial peptide infusion was without effect on plasma vasopressin, ACTH and corticosterone concentrations. However, atrial peptide infusion resulted in dose dependent decreases in plasma aldosterone concentration and plasma renin activity, but the decreases were only significant with the high physiologic (25 ng/kg/min) and pharmacologic doses (50 & 100 ng/kg/min). These data show that atrial peptide infusions in conscious dogs have minimal effects when infused in small doses that mimic endogenous atrial peptide release. At higher doses, significant effects on the cardiovascular, renal and endocrine systems can be observed but their physiological significance is unclear.     

Effects of indomethacin on local blood flow regulation in canine heart and kidney.

In two series of experiments we studied the effects of indomethacin on (a) coronary reactive hyperemia and, (b) renal blood flow, autoregulation, and reactive dilation. Coronary blood flow was measured in closed-chest dogs. Reactive hyperemia was induced by coronary occlusion for 5 and 15 sec. Indomethacin, an inhibitor of prostaglandin synthesis, was infused intra-arterially in doses of 90-200 mg over periods ranging from 30-120 min. Coronary reactive hyperemia was not affected by indomethacin. The canine renal vascular bed was studied under conditions of natural flow, controlled flow, and controlled pressure. Intra-arterial infusion of 90 mg of indomethacin over a 30- to 60- min period caused increased renal vascular resistance and an attenuation of reactive dilation (induced by stopping renal blood flow for 90 sec). Indomethacin slightly attenuated the autoregulatory response to decreasing perfusion pressures, but did not affect the respone to increasing pressures. Thus the study fails to provide evidence for participation of the prostaglandins in regulation of coronary blood flow and suggests only minimal participation of prostaglandings in renal blood flow regulation.     

Effect of platelet-activating factor on the release of PGF-2 alpha and PGE-2 by separated cells of human endometrium

Synthetic platelet-activating factor (PAF-acether, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) caused a dose-dependent increase in the synthesis of prostaglandin (PG) E-2 by an enriched glandular, but not stromal, fraction of human endometrium removed in the secretory phase of the menstrual cycle. PAF-acether did not alter the synthesis of PGF-2 alpha by this fraction and consequently there was a significant reduction in the ratio of the amounts of PGF-2 alpha/PGE-2 released by the cells into the culture medium. The preimplantation human embryo secretes PAF, and these findings may have relevance to the local maternal recognition of pregnancy.     

Effect of felodipine on renal hemodynamics and excretion in the dog

The effects of felodipine on renal hemodynamics and excretion were evaluated in the anesthetized dog. Unilateral renal arterial infusion of felodipine produced ipsilateral increases in the absolute and fractional excretion of sodium and water which were greater than those of potassium; these effects occurred in the absence of changes in mean arterial pressure, renal blood flow, or glomerular filtration rate. There were no significant effects on renal hemodynamic or excretory function in the contralateral kidney. The unilateral renal arterial infusion of isotonic saline or vehicle produced no significant effects on renal hemodynamic or excretory function in either ipsilateral or contralateral kidney. Felodipine, a calcium antagonist with vasodilator antihypertensive properties, in doses which do not affect systemic or renal hemodynamics in the dog, increased urinary flow rate and sodium excretion by decreasing renal tubular water and sodium reabsorption. As a vasodilator antihypertensive agent, felodipine possesses potentially advantageous diuretic and natriuretic properties.     

Transient increase of cytosolic free calcium in cultured human vascular endothelial cells by platelet-activating factor

The effect of platelet-activating factor (PAF-acether) on cytosolic free calcium, [Ca2+]i, in adherent human vascular endothelial cells in culture was directly determined using a new fluorescent calcium indicator, fura-2. It was found that PAF-acether but not lyso PAF-acether induced a rapid and transient increase in [Ca2+]i in endothelial cells. Restimulation with PAF-acether after the first challenge did not cause further response, while the cells were able to respond to thrombin. In the absence of extracellular calcium, PAF-acether evoked a similar transient increase, suggesting that PAF-acether raises [Ca2+]i mainly by discharging calcium from intracellular pools. PAF-acether-induced rise in [Ca2+]i was completely blocked by a specific antagonist, BN 52021. These results suggest the receptor-mediated increase in [Ca2+]i as an early event in PAF-acether activation of human vascular endothelial cells.     

Role of extrarenal and intrarenal converting enzyme inhibition in renal vasodilator response to intravenous captopril

Participation of intrarenal converting enzyme (ICE) in mediation of the renal vasodilator response to captopril (C) was studied in 7 anesthetized dogs. Blood pressure (BP), renal blood flow (RBF) and femoral blood flow (FBF) were measured and vasoconstrictor responses were elicited by i.a. injections of angiotensin (A) I to the renal and femoral vascular beds. The latter responses served as indices of intrarenal and skeletal muscle converting enzyme activity, respectively. Successive infusions of C were given i.a. to the kidney at 0.4, 0.8 and 1.6 μg/kg/min for 30 min each. RBF and renal vascular resistance (RVR) were unaffected by any of these doses of C. The % changes in RBF caused by A-I were reduced from 45 to 23, 20 and 17% by these successive doses of C, respectively; however, the decrements were not significantly different from each other. When C was administered i.v., 0.5 mg/kg, after the highest i.a. dose had been given, there was no further decrease in the response to A-I, suggesting maximal blockade of ICE obtainable by C. BP, RBF and RVR were further affected by the i.v. administration of C. BP decreased from 146 to 136 mm Hg (P<0.05), RBF increased from 240 to 290 ml/min (P<0.01) and RVR decreased from 32 to 24 mm Hg/ml/min/g (P<0.01). These results suggest that ICE plays a minor role in the renal vasodilator response to C, and implicate an influence of circulating peptides on the kidney.     

Prostaglandin D2, another renal prostaglandin?

Prostaglandin (PG) D₂ was biosynthesized by rabbit renal papillae incubates in vitro. Quantification of the renal prostaglandins by gas chromatography-mass spectroscopy demonstrated that the concentration of PGD₂ generated by renal papillae was to the amount of PGE₂ or about 1 μg/g tissue/30 min. Infusion of the sodium salt of PGD₂ into the renal artery of the dog produced a dose related increase in renal blood flow and urine flow, free water clearance, sodium excretion and potassium excretion without changes in systemic hemodynamics. At low doses PGD₂ increased renal blood flow to all cortical zones. Higher concentrations of PGD₂ produced a shift in the intrarenal distribution of blood flow toward the juxtamedullary nephrons.