Vol. 265, No. 26, Issue of September 15, pp. 1555s15559,199O Printed in U.S. A.

Synthesis of Platelet-activating Factor by Endothelial Cells THE ROLE OF G PROTEINS*

(Received for publication, February 9,199O)

Ralph E. WhatleyS, Dan F. FennellO, Jeffery A. Kurrus, Guy A. Zimmermanli, Thomas M. McIntyre, and Stephen M. Prescott)) From the Departments of Medicine and Biochemistq, University of Utah School of Medicine and the Veterans Affairs Medical Center, Salt Lake City, Utah 84112

Production of the potent lipid autacoid, platelet-ac- tivating factor (PAF), is a stimulated response of the endothelium which has important physiologic conse- quences including mediating adherence of inflamma- tory cells to the endothelium. Consequently, an under- standing of the mechanisms that regulate PAF synthe- sis by the endothelium is important. To this end, we investigated the role of G proteins as a component of the signal transduction pathway that couples hormonal stimuli to PAF production. The addition of aluminum fluoride (AIF;) to endothelial cells resulted in produc- tion of PAF with a maximal effect at 20 mM fluoride and within 20-60 min of exposure. AlF; also aug- mented the production of PAF which occurs in response to hormonal agonists. In addition, submaximal concen- trations of AlF; converted an ineffective hormonal agonist (thrombin in bovine cells) to a maximally ef- fective agonist. The adherence of neutrophils to endo- thelial cells that had been exposed previously to AlF: was increased in a manner that paralleled PAF pro- duction. PAF production in response to AlF; was not consistently affected by pertussis or cholera toxin. In- troduction of guanosine 5’-0-(3-thiotriphosphate) (GTPrS) into permeabilized endothelial cells also re- sulted in PAF production, with reversal by guanosine 5’-0-(2-thiodiphosphate) (GDP@), consistent with an effect mediated by a G protein. G protein activation with AlF, or GTPrS resulted in entry of extracellular Ca2+ as determined using ‘%a’+ flux studies and Indo- 1 spectrofluorometry. Our data are consistent with the hypothesis that G proteins couple hormone-receptor binding to opening of a membrane calcium channel, a key step in the initiation of PAF production in endo- thelial cells.

* This work was supported in part by the Nora Eccles Treadwell Foundation: the Veterans Affairs Medical Research Proeram: Na- tional Institutes of Health Grants HL 34127, HL 35828, aid F32 HL 07529; and by Grant-in-aid (84-975) from the American Heart Asso- ciation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a research associate career development award from the Veterans Affairs Medical Research Program: To whom corre- spondence and reauest for reprints should be addressed: CVRTI, I$dg. 100, University of Utah-School of Medicine, Salt Lake City, UT 84112. Tel.: 801-581-8183.

3 Recipient of an American Lung Association training fellowship. 1 Recipient of Established Investigator Award 85-204 from the

American Heart Association. (1 Recipient of Established Investigator Award 87-225 from the

American Heart Association.

Endothelial cells respond to proinflammatory and vasoac- tive agents including hormones (l), oxidants (2-4), bacterial toxins (5, 6), and venom components (6, 7). There are early activation responses that are initiated within minutes of exposure, including secretion of procoagulants (8, 9), produc- tion and secretion of prostaglandins (lo), and synthesis of platelet-activating factor (11,12). These products presumably play a role in the maintenance of vascular homeostasis and comprise a salutory response to endothelial injury. We have shown that platelet-activating factor (I-0-alkyl-2-acetyl-sn- glycero-3-phosphocholine; PAFl) is involved in the interac- tion of circulating inflammatory cells with the endothelium (13, 14). Platelet-activating factor is not constitutively pro- duced by the endothelial cell but is rapidly synthesized upon appropriate stimulation (11, 12).

Hormones that elicit PAF synthesis include inflammatory agents such as histamine, thrombin, and bradykinin and vasoactive hormones such as angiotensin II and ATP (11,12). The ability to produce PAF is a characteristic of endothelial cells from throughout the vasculature although the magnitude of production and the spectrum of hormonal agonists that initiate it vary among vascular beds (12). Because these hormonal agents act via binding to specific cell surface recep- tors, it is presumed that the differences in responses of various vascular beds arise from quantitative or qualitative differences in receptors or in their coupling to effector mechanisms. In some vascular beds the relevant receptor is absent (15). In other cases there appears to be a qualitative difference in receptors. For example, high affinity thrombin binding sites are present on endothelial cells from human umbilical vein (16) and bovine pulmonary artery (1’7), but thrombin is a potent agonist for PAF and prostacyclin production only in the human cells (12, 17-20). The reason for this may be that there are different signal transduction mechanisms that cou- ple the receptor to intracellular events in the two cell types.

There are a number of potential mechanisms for transduc- tion of receptor binding to intracellular events. These include changes in intracellular ion concentrations (especially cal- cium), production of inositol phosphates, activation of protein kinases, and alterations in CAMP concentration. The signals for hormone-induced PAF production by endothelial cells have been partially characterized; an increase in cellular cal- cium and activation of protein kinase C are required for activation of phospholipase AS, which catalyzes the first step

’ The abbreviations used are: PAF, platelet-activating factor; HBSS, Hanks’ balanced salt solution; HEPES, 4-(2-hydroxyethyl)- 1.piperazinethanesulfonic acid; Indo-l AM, 1-[2-amino-5-(6-carbox- yindol-2-yl)-phenoxy]-2-(2’-amino-5’-methyl-phenoxy)-ethane- N,N,N’,N’-tetraacetic acid, pentaacetoxy-methylester; GTP-yS, guanosine 5’.0-(3-thiotriphosphate); GDPflS, guanosine 5’-O-(2- thiodiphosphate).

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in PAF synthesis (6). However, it is not known how an occupied receptor is coupled to these events. In other systems, guanine nucleotide-binding proteins (G proteins) are thought to couple receptor occupancy to specific membrane-associated events, including activation of phospholipases and membrane ion channels (21-25). Consequently, an attractive hypothesis is that initiation of PAF synthesis occurs in response to hormonal activation of a G protein.

Hormonal agonists that are effective in initiating arachi- donate and PAF production cause prolonged elevations in endothelial cell calcium, mediated by both release of calcium from intracellular stores and entry of extracellular calcium via recently described ligand-gated or receptor-operated cal- cium channels (6, 9, 26-29). These are voltage-dependent channels that open upon receptor occupancy (and not depo- larization) and allow entry of calcium (6, 27). Hormone- induced production of PAF is dependent upon this entry of extracellular calcium; hormonal stimulation in zero calcium buffer does not result in PAF production (6). Thus, a key event in this hormone-induced activation response is the entry of extracellular calcium. The mechanisms that couple hormone receptor binding to opening of the membrane chan- nel are not known, although in other cell types G proteins appear to mediate ion channel opening, including calcium channels (21-24).

In this manuscript we present evidence that G proteins mediate hormone-induced PAF production and adherence of neutrophils to cultured endothelial cells, a process that is likely of pathophysiologic significance (13,14,30). In addition, we have shown that the mechanism by which G proteins initiate PAF biosynthesis is by mediating entry of extracel- lular calcium, with a subsequent elevation of intracellular calcium concentrations.

EXPERIMENTAL PROCEDURES

Materials-PAF (>99% pure) was purchased from Avanti Polar Lipids, Inc. (Birmingham, AL). Calcium ionophore A23187, brady- kinin, pertussis toxin, cholera toxin, 3-0-methylglucose, GTP$S, and GDP&S were purchased from Sigma. Adenosine 5’-triphosphate, 3- 0-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester (caged ATP), and guanosine 5’-(3-thiotriphosphate), 3:0-(l-(4,5-dimethoxy-2-nitro- phenvl)ethvl) thioester (caged GTPrS) were purchased from Molec- - _ - ular Probes (Eugene, OR). PHIAcetate (3.6 Ci/mmol), [35S]guanosine (y-thiohriphosphate (1000 Ci/mmol), [a-“‘Plnicotinamide adenine dinucleotide (NAD), 3-0-[“H]methylglucose (79 Ci/mmol), and [4”Ca2’]chloride were purchased from Du Pont-New England Nuclear. L652,731 and L659,989 were generously supplied by John C. Chabala, PhD, of Merck, Sharp and Dohme Research Laboratories (Rahway, NJ). Indo-l AM was purchased from Calbiochem. Medium Ml99 and antibiotic solutions were purchased from M. A. Bioproducts (Walk- ersville, MD) or KC Biologicals (Lenexa, KS). Hanks’ balanced salt solution (HBSS) was purchased from M. A. Bioproducts; HEPES, from Calbiochem; fetal bovine serum, from HvClone Laboratories (Logan, UT); collagenase, from Cooper Biomedical, Inc. (Malvern, PA): precoated plates of Silica Gel 60 from Merck (Darmstadt. Federal Republic*of Germany). Purified human thrombin was a gift from Dr. John Fenton (Albany, NY). Glass beads (Glas-shot 82701 MS/MH, from Ferro Corp., Jackson, MS) were obtained from a local distributor of sand-blasting equipment.

Cell Culture-Primary cultures of endothelial cells from human umbilical vein were grown to confluence on gelatin-coated dishes (24- or 35-mm wells) as described (19). Bovine pulmonary artery endothe- lial cells were harvested, grown in 35-mm wells (12), and were subcultured by trypsinization into 24- and 35mm wells. Human umbilical vein endothelial cells were studied in primary culture or after a single passage. Bovine pulmonary artery cells were used in primary culture or within six passages. Measurements of Indo-l fluorescence were made in endothelial cells on glass coverslips. In all experiments, the monolayers were tightly confluent at the time of study. Endothelial cells used in these studies have been characterized by microscopic morphologic criteria, immunofluorescent staining for von Willebrand factor, uptake of fluorescently labeled acetylated low

density lipoprotein, angiotensin-converting enzyme activity, and prostaglandin I, synthesis.

Assay of Platelet-actiuating Factor Production-Platelet-activating factor synthesis was measured by the incorporation of [“HIacetate into PAF by a modification of the method of Mueller et al. (31), as described previously (11, 12). Briefly, culture medium was removed from confluent monolayers of endothelial cells and replaced with 1 ml of HBSS (1.3 mM Ca*‘, pH 7.4) that contained 25 &i of carrier- free [3H]acetate, and the appropriate concentration of agonist or sodium fluoride. In experiments using fluoride, sodium fluoride was added to the incubation buffer from a stock solution immediately prior to the incubation. Control incubations were performed by adding the same amount of sodium chloride. Incubations performed in cal- cium-free buffer used HBSS that contained no calcium. The incuba- tions were performed at 25 “C and were stopped at the appropriate times by the addition of 0.5 ml of 50 mM acetic acid in methanol. After the addition of the acidified methanol, the cells were scraped from the surface of the dish. The lipids were extracted (32) and were then separated by thin layer chromatography in CHC13/MeOH/gla- cial acetic acid/H*0 (50:25:8:4). The incorporation of [3H]acetate into a lipid that comigrated with authentic PAF was determined by scintillation spectroscopy. We demonstrated previously (12) that we routinely have greater than 90% recovery and that the radiolabeled product is identical to authentic PAF by comigration in the TLC system, identical elution volume on high performance liquid chro- matography (33), characteristic degradation by phospholipases (11, 12), and bioactivity (12,34). To confirm that the radiolabeled product was authentic platelet-activating factor, bovine pulmonary artery endothelial cell monolayers were exposed to aluminum fluoride (20 mM fluoride; 1 pM aluminum) in the presence of 25 &i/ml [“HI acetate for 20 min; the incubations were terminated and the endothe- lial cell lipids extracted (without carrier PAF) and separated by thin layer chromatography as above. The radiolabeled endothelial cell lipid that comigrated with a PAF standard was scraped from the plate and extracted (32). This product was identified as platelet-activating factor by its characteristic response to phosphohpases and by its bioactivity. 1) The radiolabeled lipid was degraded (>95%) by treat- ment with phopholipase A, (Crotallls adamantus) (11, 12), confirming that [3H]acetate had been incorporated at the sn-2 position of the phosoholipid. 2) Less than 5% of this radiolabeled lioid product was degraded by treatment with phospholipase A, (R&opus arrhizus) (11, 12) compared with >75% degradation of the I-acyl analog of PAF, l-acyl-2-[“HJacetyl-sn-glycero-3-phosphocholine (synthesized by the method of Gupta et al. (35)), confirming that the product is not the l-acyl analog of PAF. 3) The radiolabeled lipid had bioactivity characteristic of platelet-activating factor, causing adherence of “‘iridium-labeled neutrophils to gelatin-coated plastic dishes (12), which was completely inhibited by pretreatment of the neutrophils with the platelet-activating factor receptor antagonist L659,989. The amount of platelet-activating factor present was determined by com- parison with a standard curve of neutrophil binding in response to known concentrations of platelet-activating factor. Maximal stimu- lation of the endothelial cells resulted in approximately 2.8 pmol of PAF per 10” cells, and the specific radioactivity of the [“HIPAF recovered was approximately 3.6 Ci/mmol. These values are similar to measurements that we and others have made in hormonally stim- ulated endothelial cells (12, 36).

Measurement of Aluminum-Samples (tissues, cultured cells, serum, and media) were digested for 24 h in 12 N HNOa. A portion of this digest was diluted to 0.5% in deionized water and neutralized with NaOH. Samples were microinjected with an autosampler into a Varian AA-1275 atomic absorption spectrophotometer equipped with a GTA-95 graphite tube atomizer. Spectrophotometer parameters included h = 309.3 nm; slit width = 0.5 nm; ashing temperature = 1400 “C; maximum atomizing temperature = 2800 “C; atmosphere = nitrogen gas. The amount of aluminum present in the samples was determined by comparison with standards.

To determine the concentration of aluminum in cultured endothe- lial cells it was necessary to measure the volume of the cells in the monolayer. This was determined by measuring the water volume of the monolayer using radiolabeled 3-0-methylglucose. The monolayer was washed and incubated in 10 mM 3-0-methylglucose in HBSS, 10 mM HEPES containing 0.5 &i/ml [“H]3-0-methylglucose for 90 min. The labeling medium was removed, and the monolayers were washed twice with ice-cold buffer. The monolayers were then scraped into a scintillation vial, and the amount of label present was deter- mined by scintillation spectroscopy. The water volume of the cell was

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then calculated by comparison with the amount of label/unit volume of the labeling buffer.

Membrane Labeling with f2P]NAD and Pertussis Toxin-The ability of pertussis toxin to [“‘PIADP-ribosylate an endothelial cell membrane protein was determined using the method of Clark et al. (37). Human umbilical vein endothelial cells were pretreated with concentrations of pertussis toxin ranging from 0 to 100 rig/ml in standard medium for 1 h at 37 “C. The medium was removed, and the monolayers were washed three times with ice-cold HBSS. The monolavers were then scraned into buffer A (20 mM HEPES. 2 mM MgC&,“l mM EDTA, pH’ 7.4), and the cells were disrupted by sonication with a probe sonicator (15 s x 100 watts). The disrupted cells were centrifuged (120,000 x g, 30 min), and the supernatant (cytosolic fraction) was removed. The pellet (crude membrane frac- tion) was resuspended in buffer A and centrifuged. The resulting pellet was resusDended in buffer and the protein content measured. The membranes (5 pg of protein) were incbbated for 20 min at 37 “C with 10 mM thvmidine. 1 mM ATP. 0.5 mM GTP. 5 UM [“‘PINAD, and 5 rg/ml pertussis toxin (preactivated by incubation ‘in i0 rni dithiothreitol for 20 min, 37 “C). Control incubations contained no pertussis toxin. The incubation was terminated by dilution with buffer A, the membranes were recovered by centrifugation, washed twice with buffer A, and solubilized in sodium dodecyl sulfate-poly- acrylamide gel electrophoresis sample buffer. Membrane proteins were separated using sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (10%). The gel was dried, and a “2P-labeled protein of approximately 40 kDa was identified by autoradiography. The labeled bands were cut from the gel, and the amount of ‘*P present was quantified using liquid scintillation spectroscopy.

Measurement of Neutrophil Adherence to Endothelial Cells-Hu- man neutrophils were isolated, labeled with “‘iridium, and their adherence to human umbilical vein endothelial cell monolayers was measured as described (13). In most experiments, endothelial cell monolayers were exposed to buffer (HBSS, pH 7.4) containing sodium fluoride or an agonist. Thrombin (2 units/ml, added to the HBSS) was used as a positive control (13, 14). For those experiments using fluoride, control incubations contained an equivalent amount of so- dium chloride or sodium iodide. The incubation buffer was removed, and the monolayers were washed with HBSS, 0.5% human serum albumin. Radiolabeled neutrophils were then placed on the monolayer and incubated for 5 min. At the end of the incubation, the neutrophil- containing buffer was removed, and the monolayers were washed gently. Calculation of the fraction of adherent neutrophils has been described (13).

Introduction of GTPyS and Caged Compounds into Cells-Glass beads with an average diameter of 200 pm were prepared by a brief washing in concentrated HCl followed by five rinses in distilled water. The beads were then stirred in a methanol wash, washed with chlo- roform, and allowed to air dry. After each wash, the beads rapidly settled to the bottom of the beaker, and the wash solution was decanted. Culture medium was removed from confluent monolayers, and the cells were washed with HBSS containing 10 mM HEPES, pH 7.4 (HBSS/HEPES). The same buffer, containing the substance to be loaded into the cell, was then placed on the monolayer (1 ml). Beads were sprinkled over the monolayer (150 mg of heads/24-mm dish or 300 mg/35-mm dish) sufficient to cover the monolayer with a single layer of beads. The dishes were then gently agitated and placed in an incubator for 10 min. After the incubation, the beads were removed by tilting the culture dish on a slight incline (30 degrees), and the monolayer was washed with buffer as the beads were removed by a suction pipette at the lower end of the plate. Once the beads were removed from the monolayer, complete medium was placed on the monolayers. In some experiments, the monolayers were placed in buffer or medium that also contained the agent present during the loading phase. Inspection of the monolayer immediately after treat- ment revealed that the cells were retracted, leaving areas of denuded plate. Within 2-3 h, the cells resumed a normal, confluent mor- Dholoev and were functionallv normal. as reDorted.’ A As& of ‘“Cc? Flux into &dothelial Cells-Culture medium was removed, and the monolayers were washed twice with 1 ml of HBSS. These monolayers were incubated at 25 “C for the indicated times with 1 ml of HBSS ([Ca’+] = 1.3 mM, pH 7.4) containing 5 &i/ml ““CaCL and the indicated concentrations of agonist. Control dishes were incubated identically but without an agonist. At the indicated times, the incubation buffer was removed and the culture dish im-

‘Fennell, D. F., Whatley, R. E., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1990) Arteriosclerosis, in press.

mersed in three sequential washes of ice-cold HBSS (without calcium) containing 0.1% EDTA to remove any remaining extracellular *“Ca”. The monolayers were solubilized in 1 ml of 1 M NH,OH and placed in scintillation vials for measurement of Ya” content.

Measurements of Intracellular Calcium Concentrations Using Indo- l-Measurements of the time course of changes in intracellular calcium activity were performed using the calcium-sensitive dye Indo- 1 as described (6, 38). Confluent cultures of endothelial cells grown on coverslips were exposed to the acetomethoxy ester of Indo-l (10 pM in standard culture medium) for 1 h at 37 “C. The cells were then placed in dye-free medium for 20 min. The coverslip was placed in the perfusion chamber of a custom-designed spectrofluorometer (365 nm excitation). Fluorescence was measured at 410 and 500 nm, and the ratio of the signals, 410 nm/500 nm, reflects the intracellular calcium concentration (6, 38).

Measurements were made by perfusing the endothelial cell mono- layers first with control buffer (Na+, 132 mM; K’, 4 mM; Ca*+, 1.3 mM; M?‘, 0.5 mM; Cl-, 140 mM; glucose, 5 mM; HEPES, 10 mM; pH 7.4) to measure basal levels of [Ca’+] followed by perfusion with the same buffer containing an agonist or fluoride. Only one interven- tion was performed on each monolayer except in the case of a lack of a response. In such cases a positive control (bradykinin) was subse- quently tested to exclude technical artifacts as a cause of the lack of response. In control experiments it was found that fluoride did not affect the fluorescence of a solution of Indo-1.

Measurements of intracellular calcium changes in cells loaded with caged GTP-# exploited the characteristic that the wavelength at which photolysis of the caged compounds occurs is approximately 360 nm, which is also within the excitation band width used for Indo- 1 spectrofluorometry (365 f 10 nm). Endothelial cell monolayers on glass coverslips were permeabilized with glass beads and loaded with the caged compounds (0.5 mM caged GTPrS in the permeabilization buffer). The beads were then removed and the cells allowed to recover for 3 h, at which time the monolayer had resumed a normal mor- phology. During the hour prior to fluorometry, the cells were exposed to Indo-l AM (10 pM) as described above. The coverslip was then placed in the perfusion well of the spectrofluorometer, and a single microscopic field (approximately 25 cells) was excited at 365 nm. After obtaining a suitable baseline signal (410 and 500 emission) the field was moved to a previously unilluminated area of the coverslip, allowing simultaneous photolysis of the caged compound and meas- urement of the Indo-l signal. Control experiments used endothelial cell monolavers that had been exposed to caged GTPyS but not permeabilized (no beads), monolayers that had been loaded with caged ATP, and monolayers that had been loaded with caged GTPyS but had also been illuminated previously.

Statistical Analysis-Student’s t test was used to determine statis- tical significance.

RESULTS

Aluminum fluoride activates G proteins by binding to the a-subunit and mimicking the effect of GTP (39). We exposed endothelial cells to aluminum fluoride in the absence of a hormonal agonist and measured the synthesis of platelet- activating factor. We observed PAF accumulation with a maximal effect occurring between 20 and 60 min for human umbilical vein endothelial cells (Fig. la) and bovine endothe- lial cells (not shown). Maximal accumulation of PAF occurred with 20-25 mM fluoride (Fig. lb); at higher concentrations of fluoride PAF production was decreased. Precipitation of cal- cium fluorophosphate from the buffer occurred at the higher fluoride concentrations, which may explain the decreased PAF accumulation as extracellular calcium is required for PAF production (6).

We next examined whether aluminum fluoride would affect hormone-induced production of PAF in endothelial cells. Alu- minum fluoride enhanced the PAf production that occurred in human umbilical vein endothelial cells exposed to the weak agonists, histamine and bradykinin (Fig. 2~). In addition, aluminum fluoride increased PAF accumulation by 2-6-fold in response to the fully effective agonist, thrombin, compared with thrombin alone (Fig. 2b). Exposure to aluminum fluoride did not potentiate PAF production in response to the calcium

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b.

0 20 40 60 80 100 20 40 60 60

Tlmo (minutes) [NSF] (mM)

FIG. 1. The effect of aluminum fluoride on PAF production by endothelial cells. a, time course. Human umbilical vein endothelial cells were exposed to aluminum fluoride (10 pM Al 3+ 25 rnlvl NaF) in HBSS (1.3 mM , [Ca”], pH 7.4) containing 25 &i/ml l’H]acetate. Incubations were performed at 25 “C and terminated at the times indicated. The incorporation of 13H]acetate into PAF was determined. Each point (e) represents the mean of determinations in two separate dishes (106cells/dish). Control incubations (0) were performed identically except that 25 mM NaCl was substituted for the NaF. Although there was some variability in the time course of PAF accumulation in different experiments, maximal production typically occurred within IO-30 min and persisted for up to 60 min. b, concentration response. Bovine pulmonary artery endotheiial cells were exposed to varying concentrations of fluoride in buffer (HBSS, 1.3 mM [Ca’+], 10 pM Al”“, pH 7.4) containing 25 &$‘rnI [3HJacetate. The incubations were performed at 25 “C and terminated after 10 min. Incorporation of V’HJacetate into PAF was determined; each point (a) represents the mean of determinations made in two separate monolayers (lo6 ceils/ dish). Results shown are typical of determinations made in endothelial cells from four separate isolates. Production of PAF typically declined at concentrations of fluoride above 25 mM, although the amount of the decrease was variable and appeared to arise from formation of calcium fluorophosphate.

8. b.

6 ?

~ 25 mM NaCl

FIG. 2. Aluminum fluoride augments human endothelial cell production of PAF in response to hormonal stimulation. a, human umbilical vein endothelial cells (10” cells/dish) were exposed to the indicated agonist (10e6 M bradykinin, 10m5 M histamine) in buffer (HBSS, 10 mM HEPES, 10 @M Ala*, pH 7.4) containing 25 &i/ml [%Jacetate and 25 mM NaF or 25 mM NaCI. At 10 min, the reactions were stopped, and the incorporation of THjacetate into PAF was determined. Each group represents the mean of two dishes. b, human umbilical vein endothelial cells (10’ cel~s/dish) were exposed to thrombin (0.2 units/m1 (0) or 2 units/ml (0)) in buffer (HBSS, 10 mM HEPES, 10 yM At”‘, pH 7.4) containing 25 &i/ml 13H]acetate and the indicated concentration of sodium fluoride. At IO min, the reactions were stopped, and the incorporation of [3H]acetate into PAF was determined. Each group represents the mean of determinations made in two separate monolayers ( lo6 cells/dish).

ionophore A23187 (not shown). The enhancement of hor- mone-induced PAF accumulation was found consistently with all receptor-media~d agonists tested in both bovine and hu- man endothelial cells from multipie isolates, although the magnitude of the enhancement was variable. To define more clearly the ability of aluminum fluoride to augment receptor- mediated PAF synthesis, we tested the effect of fluoride on responses to thrombin in bovine pulmonary artery endothelial cells. In contrast to human cells, bovine endothelial cells do not synthesize PAF or prostag~andin IB in response to throm- bin, even though both species have affinity binding sites for thrombin (16, 17). As reported previously (12), exposure of bovine pulmonary artery endothelial cells to thrombin did not

cause PAF production. However, when cells were exposed to thrombin in the presence of 10 mM fluoride, PAF accumula- tion was increased to 74% of that seen with bradykinin, the most potent agonist in bovine endothelial cells (Fig. 3). The enhancement of thrombin-induced PAF accumulation was most apparent at suboptimal concentrations of fluoride (5-10 mM fluoride) at which thrombin and fluoride appeared to be synergistic in causing PAF accumulation (Fig. 3).

Interestingly, we found that PAF production in endothelial cells exposed to fluoride alone was equivalent to that seen in cells exposed to both fluoride and aluminum (1 PM) (not shown). Such a phenomenon has been observed in other G protein-dependent systems (40, 41) and has been attributed

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FIG. 3. Aluminum fluoride-induced PAF synthesis in thrombin-treated bovine endothelium. Bovine pulmonary endo- thelial cells (10’ cells/dish) were incubated at 25 “C in buffer (HBSS, 10 mM HEPES, 10 pM Al”‘, pH 7.4) containing 25 &i/ml [“HIacetate and the indicated agonist (thrombin, 2 units/ml; fluoride, 10 mM; bradykinin, lo-’ M). At 10 min, the reactions were stopped, and the incorporation of [“HIacetate into PAF was determined. Each group represents the mean and standard error of determinations made in three separate monolayers (lo6 cells/dish). The results shown are typical of results using four separate isolates. Although the magnitude of the PAF accumulation differed from isolate to isolate, synergy between thrombin and fluoride was observed in each case. *, p < 0.001 compared with fluoride alone; t, p < 0.001 compared with thrombin alone.

to the ubiquitous presence of aluminum in buffers and biologic materials. Alternatively, PAF production in response to flu- oride could be mediated via a non-G protein mechanism, such as inhibition of a phosphatase. To examine the lack of de- pendence on aluminum, we measured its concentration in our reagents and the cultured cells using atomic absorption spec- troscopy. Our buffers contained less than 1 PM aluminum. However, significant (micromolar) concentrations were found in the culture medium, and cultured cells had a concentration of approximately 10 PM. Although aluminum may be distrib- uted inhomogeneously through the cell, this concentration is adequate for activation of G proteins in the presence of fluoride (42).

The preceding experiments demonstrated a clear effect of aluminum fluoride, which we interpreted as an effect on G proteins. To exclude other mechanisms, we activated G pro- teins by introducing a nonhydrolyzable analog, GTP+. This required a technique for transient permeabilization, which we achieved with glass beads (43). This technique allows incor- poration of small molecules (51000 daltons) into 100% of the cells, and they rapidly resume normal appearance and func- tion.’ We first confirmed that this technique allowed the effective incorporation of [3”S]GTPyS. We next permeabil- ized human endothelial cells, loaded them with increasing concentrations of GTP+, and measured PAF accumulation. We found increasing accumulation of PAF associated with increasing concentration of GTPrS (Fig. 4). In most experi- ments, the amounts of PAF produced were equivalent to or within 80% of the amount of PAF produced in response to thrombin stimulation alone in the immediate postpermeabil- ization period. Maximal accumulation of PAF occurred within l-2 min after introduction of GTP$S (Fig. 5). This contrasts with the accumulation that occurs with thrombin stimulation at which peak accumulation was seen at 5-10 min. These data suggest that the activation of a G protein by GTPrS may bypass the initial step of transmembrane signaling, the bind- ing of hormone to receptor, resulting in a more rapid response.

To assess further whether a G protein is involved in endo- thelial cell PAF accumulation and to eliminate the possibility that the GTP analog was working via a cell surface purine

FIG. 4. GTP+ causes PAF production in endothelial cells. Human umbilical vein endothelial cells ( lo6 cells/dish) were perme- ahilized with glass beads in buffer (HBSS, 10 mM HEPES, pH 7.4) containing 25 FCi/ml [“HIacetate and the indicated concentration of GTPrS. Permeabilization was initiated with the addition of glass beads. Control incubations used monolayers that had been perme- abilized in buffer containing thrombin (2 units/ml). At 5 min, the reactions were stopped, and the incorporation of [“HIacetate into PAF was determined. Each group represents the mean and range of determinations made in separate monolayers (10’ cells/dish). *, p < 0.05 compared with no agonist control.

FIG. 5. Production of PAF is accelerated in endothelial cells loaded with GTPrS compared with those stimulated with a receptor-mediated agonist. Human umbilical vein endothelial cells (lo6 cells/dish) were permeabilized with glass beads in buffer containing 1 mM GTPrS (0) or 2 units/ml thrombin (0) and 25 &i/ ml [3H]acetate. At the indicated time, the incubation was stopped, and the incorporation of [3H]acetate into PAF was determined. Each point represents the mean of PAF accumulation in two separate monolayers (lo6 cells) using cells from a single isolate.

receptor, we utilized GDPPS, a GDP analog that inactivates G proteins (39). PAF production in GTPTS-loaded cells was inhibited by increasing concentrations of GDPPS (Fig. 6), consistent with an effect of GTPrS on a G protein.

We next examined the effect of pertussis and cholera toxins, which alter specific G proteins, on hormone-induced PAF accumulation. We found no consistent alteration in thrombin- induced PAF accumulation in endothelial cells treated with either of these bacterial toxins. In particular, there was no effect in cells exposed to varying concentrations of pertussis toxin (0.1-10 pg/ml) in preincubations ranging from 30 min to 4 h (data not shown). Using [32P]NAD labeling, others have found that bovine endothelial cells have a membrane substrate for pertussis toxin (37, 44). We also found that human umbilical vein endothelial cell membranes have a 40- kDa substrate for pertussis toxin-mediated [“‘P]ADP-ribosyl- ation. In addition, labeling of this substrate was inhibited by 67% in membranes from cells that had been preincubated with pertussis toxin (0.1 pg/ml for 1 h, 37 “C), similar to the observations of others in bovine endothelial cells (37). We conclude that substrates for pertussis toxin-mediated ADP- ribosylation are present in these cells, and the conditions of

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FIG. 6. Reversal of GTPrS-induced PAF production by GDPOS. Human umbilical vein endothelial cells (lo6 cells/dish) were bead nermeabilized in buffer containing 100 UM GTPrS, the indicated concentration of GDPPS, and 25 PCijrnl [‘H]acetatk. At 5 min, the incubation was stopped, and the incorporation of [“HIacetate into PAF was determined. Each point represents the mean and range of PAF produced in two separate monolayers (lo6 cells) using cells from a single isolate. *, p < 0.02 compared with monolayer treated with 100 GM GTPyS + 0 GDPBS.

pertussis toxin treatment used in our experiments are suffi- cient to cause ADP-ribosylation of those substrates. These data indicate that activation of a toxin-insensitive G protein is associated with production of PAF by endothelial cells.

We have demonstrated previously that endothelial cell PAF production mediates adherence of neutrophils to the endothe- lium (13, 14). Consequently, we hypothesized that activation of endothelial cell G proteins would cause increased adherence of neutrophils as a result of PAF production. To test this, we examined the effect of fluoride-induced activation of endothe- lial cell G proteins upon neutrophil binding. Neutrophil ad- herence to human umbilical vein endothelial cell monolayers that had been exposed to fluoride was dramatically increased compared with control monolayers (Fig. 7). Furthermore, pretreatment of endothelial cells with fluoride potentiated neutrophil binding when the endothelium was subsequently stimulated with thrombin (13, 14) similar to the effect of fluoride on PAF accumulation (Fig. 2%). Increased adherence of neutrophils was dependent upon the concentration of flu- oride and the time of preexposure to the fluoride-containing buffer (Fig. 7, a and b). Importantly, the increase in neutrophil adherence had the same fluoride time and concentration dependence as fluoride-induced PAF production by endothe- lial cells. Maximal adherence was seen after exposure of the endothelial cells to 20-25 mM fluoride (Fig. ‘7~). Although we frequently observed minor variability in the time course of fluoride-induced PAF accumulation, when PAF synthesis and neutrophil binding were measured in parallel in the same experiment, they had the same time course (Fig. 7b). In addition, treatment of neutrophils with the specific competi- tive PAF receptor antagonists L652,731 or L659,989 (14) inhibited their binding to fluoride-treated endothelial cells (not shown). These data are consistent with the hypothesis that activation of an endothelial cell G protein causes PAF production that mediates neutrophil adherence to the endo- thelium.

We next investigated the mechanism by which aluminum fluoride results in PAF production in endothelial cells. We have found previously that a prolonged elevation in intracel- lular calcium concentration is sufficient to cause PAF synthe- sis in these cells (6). PAF accumulation in response to recep- tor-mediated agonists arises from a prolonged entry of extra- cellular calcium (6) via receptor-operated calcium channels (6, 26, 27). Consequently, we hypothesized that aluminum

fluoride causes a prolonged elevation in intracellular calcium. To test this, we first examined the calcium dependence of aluminum fluoride-induced PAF accumulation. As expected, we found that exposure of endothelial cells to aluminum fluoride in calcium-free buffer resulted in PAF accumulation only slightly above basal levels (not shown). We next exam- ined the possibility that aluminum fluoride mediated entry of extracellular calcium by examining 45Ca2’ uptake. When endothelial cells were exposed to aluminum fluoride, there was an increase in ‘?a’+ entry which was dependent upon the concentration of aluminum fluoride (Fig. 8). We next examined the effect of aluminum fluoride on intracellular calcium concentrations. Aluminum fluoride caused an eleva- tion in cellular calcium concentration with the magnitude dependent upon the concentration of fluoride (Fig. 9a). In addition, similar experiments in calcium-free buffer demon- strated that the increase in intracellular calcium was depend- ent upon extracellular calcium (not shown). Thus, the mech- anism for fluoride-induced PAF accumulation is a prolonged elevation in intracellular calcium concentration which is me- diated by entry of extracellular calcium. To determine if a similar mechanism is responsible for the enhancement of hormonal stimulation seen in the presence of fluoride, we examined the effect of fluoride on thrombin-induced calcium entry in bovine cells. As noted above, in bovine cells thrombin causes PAF accumulation that is only slightly above unstim- ulated controls. Similarly, thrombin resulted in a transient (approximately 1-min) elevation in intracellular calcium which arose from an intracellular source (Fig. 9b). Although this elevation may be prolonged slightly by a small amount of extracellular calcium entry (Fig. 9b), this is considerably less than that seen with fully effective agonists (6, 27). However, exposure of endothelial cells to thrombin in the presence of increasing concentrations of fluoride increased the magnitude and duration of the thrombin-induced elevation in intracel- lular calcium (Fig. 9c). In addition, if bovine cells were preex- posed to a suboptimal concentration of fluoride (5 mM) and then exposed to thrombin (2 units/ml), there was a similar increase in the magnitude and duration of the thrombin- induced calcium elevation which was dependent upon the duration of pretreatment with fluoride (Fig. 9d).

Finally, to confirm that the aluminum fluoride-induced calcium entry was due specifically to activation of a G protein, we examined the effect of GTP on calcium entry. To do this, we first loaded endothelial cells with caged GTPyS or with a control compound using glass bead permeabilization. Changes in intracellular calcium concentration were then measured using Indo-l spectrofluorometry. Illumination of a field of cells (approximately 25 cells) which had been loaded with caged GTPrS resulted in a marked rise in intracellular cal- cium which occurred within 1 min and could be reproduced by moving to a previously unilluminated field of cells (Fig. 10). Illumination of a previously illuminated area of cells resulted in no change in cellular calcium, indicating that the response was due to photolysis of the caged compound (Fig. 10). Control cells that had been treated identically except that the caged GTPyS was not present during permeabilization showed no change in calcium when illuminated (not shown). Controls loaded with caged ATP and controls exposed to the caged GTPyS but not permeabilized (no beads) also showed no change in cellular calcium (not shown).

DISCUSSION

Endothelial cells play a central role in vascular physiology, producing several potent vasoactive and inflammatory media- tors, including arachidonate metabolites, procoagulants, en-

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FIG. 7. Activation of endothelial cells with fluoride induces increased neutrophil adherence. a, concentration response. Human umbilical vein endothelial cells were exposed to the indicated concentration of sodium fluoride (0) or control salts (sodium iodide (W) or sodium chloride (0)) in buffer (HBSS, pH 7.4) for 10 min. The incubation buffer was removed, and the monolayers were washed with HBSS, 0.5% human serum albumin. Radiolabeled neutrophils were then placed on the monolayer and incubated for 5 min. At the end of the incubation, the buffer containing nonadherent neutrophils was removed, and the monolayers were washed gently. Calculation of the fraction of adherent neutrophils was then determined. In two experiments not shown, we found that these concentrations of fluoride did not stimulate neutrophils to bind to endothelial-free surfaces (gelatin matrices) during a 5-min incubation, excluding “carryover” of fluoride and direct activation of neutrophils as the mechanism of binding. b, time course. Human umbilical vein endothelial cells were exposed to 25 mM sodium fluoride (0) or control (25 mM sodium chloride (0)) in buffer (HBSS, pH 7.4) (solid lines). At the indicated time, the incubation buffer was removed, and the monolayers were washed twice with HBSS, 0.5% human serum albumin. Radiolabeled neutrophils were then placed on the monolayer and incubated for 5 min. At the end of the incubation the neutrophil-containing buffer was removed, and the monolayers were washed gently. The fraction of adherent neutrophils was then determined as in a above. PAF accumulation (dashed lines) was measured in parallel assays, using cells from the same isolate, exposed to 25 mM sodium fluoride (0) or control (25 mM sodium chloride (0)).

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FIG. 8. Fluoride causes 45calcium uptake by endothelial cells. Bovine pulmonary artery endothelial cells (lo6 cells/dish) were incubated at 25 “C in the presence of “Ca’+ (5 pCi/ml) in HBSS (1.3 mM Ca”). The incubations contained increasing concentrations of sodium fluoride. After 10 min the incubation buffer was removed, and the dishes were rapidly washed in three sequential baths of ice- cold Ca”-free HBSS containing 0.1% EDTA. The cells were solubi- lized with 1 M ammonium hydroxide and the cellular “Ca*’ content determined by scintillation spectroscopy. Each point (0) represents the mean of measurements made in two separate monolayers.

dothelium-derived relaxing factor, and platelet-activating fac- tor. Production of these mediators by endothelial cells may be initiated by pathologic agents including venom components

(6, 7), bacterial toxins, and oxidants (2-6). However, hor- monal agents may also initiate production of these mediators via binding to specific receptors (1, 7, 9-10, 16, 17), and it is likely that these responses to hormonal agents have a prom- inent physiologic role. The endothelial cell has specific hor- monal receptors, which vary among different vascular beds, and it is uniquely poised at the interface between circulating blood and tissues. Presumably, in the steady state, responses to circulating hormonal regulators maintain vascular tone, facilitate movement of water and solutes, and preserve the nonthrombogenic nature of the endothelium. However, under pathologic conditions, the endothelial cell must respond, in a regulated manner, by production of various vasoactive and proinflammatory agents mentioned above. An example of this process is production of platelet-activating factor; the endo- thelial cell synthesizes this unusual and potent lipid autacoid when appropriately stimulated by pathologic agents or specific hormones (4, 6, 11, 12, 19). Production of platelet-activating factor by the endothelial cell is an early (within minutes) activation response that mediates adherence of inflammatory cells (neutrophils) to the endothelium (13, 14). This is one component of a rapid endothelial activation response that includes production and secretion of prostacyclin, endothe- lium-derived relaxing factor, von Willebrand factor, and other mediators (1, 6, 9, 45). Importantly, in most cases, these processes occur in response to common initiators, including hormones such as bradykinin, histamine, and thrombin (1, 6,

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FIG. 9. Fluoride causes elevations in cellular calcium concentrations. Bovine endothelial cells grown on coverslips were labeled with Indo-l AM (10 pM in standard culture medium) for 1 h at 37 “C. The labeling medium was then removed and replaced with dye-free medium for 20 min. The coverslips were placed in the perfusion chamber of a custom spectrofluorometer (excitation 365 nm) and fluorescence was measured at 410 and 500 nm. The ratio of 410 nm/500 nm, expressed here as arbitrary units, is proportional to the intracellular calcium concentration. a, concentration response. The cells were initially perfused with control buffer and then with the same buffer containing the indicated concentration of sodium fluoride (arrow). b, thrombin response. Cells were perfused with buffer and then with the same buffer containing the indicating concentration of calcium and 2 units/ ml thrombin (arrow). c, fluoride augmentation of the thrombin response. Cells were perfused with control buffer (1.3 mM Ca”) and then with the same buffer containing the indicated concentration of fluoride and 2 units/ml of thrombin (arrow). cl, time dependence of the fluoride enhancement. Cells were perfused with buffer containing 5 mM fluoride for the times indicated. The buffer was then changed to buffer containing 2 units/ml thrombin (arrow).

FIG. 10. Photolysis of caged GTP-yS causes elevations in cellular calcium concentrations. Bovine endothelial cells grown on coverslips were permeabilized with glass beads and loaded with caged GTP-yS (0.5 mM) for 10 min in complete darkness. The permeahilization buffer was removed and replaced with growth medium; the cells were allowed to recover in an incubator for 3 h. Indo-l AM (10 pM) was added to the medium for the final hour of recovery. The medium was then removed and replaced with dye-free medium for 20 min. The coverslips were placed in the chamber of a custom spectrofluorometer (excitation 365 nm), perfused with buffer (1.3 mM Ca’+), and fluorescence was measured at 410 and 500 nm from a field of approximately 25 cells. The changes in the 410/500 ratio were recorded after movement of the coverslip to previously unilluminated fields (A) or to fields of cells that had been previously illuminated (B). Results shown are typical of three separate experiments using different isolates. The magnitude and duration of the response were essentially identical in each case; the time between initiation of illumination and the onset of the rise in cell calcium varied between 30 and 80 s.

9, 11, 12, 19, 45). At one level, regulation of this response endothelial cells (16, 17), but thrombin-induced production of arises from differences in the number of receptors present on prostacyclin or platelet-activating factor is minimal in bovine the endothelial surfaces due to constitutive differences or to cells (12, 17, 18) compared with human cells, where it is a differences in processing or turnover of receptors (15). How- maximally effective agonist (18, 19). This paradox demon- ever, there appears to be another level of regulation as well. strates that transduction of hormone receptor binding to This is best demonstrated by the fact that high affinity intracellular processes is regulated and complex. thrombin receptors are present on both human and bovine To understand the activation and injury responses of the

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15558 G Proteins Mediate PAF Production

endothelium, it is critical to understand how hormonal agents initiate intracellular processes such as platelet-activating fac- tor synthesis. Presumably, hormone binding to a cell surface receptor initiates one or more signal transduction mecha- nisms. In endothelial cells, it is known that turnover of phosphatidylinositol polyphosphates is associated with hor- mone receptor binding (44, 46, 47). Furthermore, in these cells hormone-mediated entry of extracellular calcium occurs via receptor-operated calcium channels (6, 27-29). The pre- dominant type of calcium channel in endothelial cells is a channel that opens in response to hormone receptor binding, and the magnitude of calcium entry is dependent upon mem- brane potential (27). The prolonged elevation of intracellular calcium is required for endothelial cell production of platelet- activating factor, eicosanoids, and other mediators (6,9, 48).

Our data demonstrate that G proteins play a role in PAF production by endothelial cells. First, we found that exposure of endothelial cells to aluminum fluoride or nonhydrolyzable GTP analogs initiated PAF production. Each of these agents binds to the a-subunit of the G protein, maintaining it in its active state. In each case these agents worked in a time- and concentration-dependent manner. The specificity of this ef- fect is demonstrated further by our observation that GDP&$ an inhibitor of G proteins, reversed the effect of the GTPrS (Fig. 6). Because little, if any, aluminum dependence could be demonstrated, we measured aluminum concentrations in cul- tured cells and found sufficiently high concentrations of alu- minum to activate G proteins in the presence of added fluo- ride. In addition, we found that neither pertussis toxin nor cholera toxin affected aluminum fluoride-induced PAF pro- duction, despite the presence of a substrate for pertussis toxin- mediated ADP-ribosylation in these cells. Although these toxins inactivate certain G proteins (G, and Gi), insensitive G proteins are described (49, 50); we conclude that the rele- vant G protein in endothelial cells is one that is toxin insen- sitive. A role for G proteins as a signal transduction mecha- nism in hormone-induced PAF production was demonstrated by the ability of aluminum fluoride to convert partially effec- tive hormonal agonists to fully effective agents. This is best shown by our experiments with bovine cells exposed to throm- bin. In previous studies, we and others have found a lack of response of bovine endothelium to thrombin (12, 17, 18, 20), which has been perplexing, as high affinity binding sites for thrombin can be found on bovine endothelial cells (17). Our data suggest that the difference arises from differences in the activation of G proteins that are coupled to the thrombin receptor.

We also found that G protein activation in endothelial cells resulted both in PAF biosynthesis and a physiologic conse- quence of the production as demonstrated by increased ad- herence of neutrophils to the endothelial cells (Fig. 7). This implicates G proteins in a process that is physiologically and pathologically relevant to vascular cell activation.

Because G proteins may regulate membrane ion channels in other cell types and because entry of extracellular calcium is necessary (and sufficient) to induce PAF accumulation, we next tested the hypothesis that the observed effects of G protein activation arise from an effect on the free cytosolic calcium concentration. The dependence of aluminum fluo- ride-induced PAF production on extracellular calcium, the aluminum fluoride-induced %alcium entry, and elevations in cellular calcium concentrations as measured by Indo-l spec- trofluorometry (Figs. 8 and 9) are all consistent with that hypothesis. Moreover, the elevations in cellular calcium con- centrations seen when a different activator of G proteins was used (photolysis of caged GTP-#) are also consistent with

the hypothesis that G protein activation mediates membrane calcium channel opening. Furthermore, these experiments confirm the specificity of the aluminum fluoride effect. In addition, our data demonstrate that G proteins couple hor- mone-receptor binding to calcium entry. As shown by 45calcium entry and Indo-l spectrofluorometry in bovine cells, thrombin was converted to a fully effective agonist from an agonist that was ineffective in initiating calcium entry (Fig. 9). The role of G proteins as a mechanism for coupling hormone receptor binding to calcium channel opening is attractive as each of these components is membrane associ- ated (51, 52), and such an arrangement (receptor-G protein- ion channel) has been proposed in other systems (21, 22, 24, 51). In the case of endothelial cells, the predominant mem- brane calcium channel appears to be a receptor-operated type of channel; these channels have not been purified and have been only partially characterized (27-29, 53-55). Conse- quently, although the interaction of the components (receptor, G protein, channel) has not yet been described at a molecular or electrophysiologic level, our data demonstrate a role for G proteins as the transducing component between receptor and channel. An alternative explanation for our findings is that the regulatory component, although activated by guanine nucleotide binding, is unlike conventional G proteins. For example, it is conceivable that the membrane calcium channel involved is regulated by guanine nucleotide binding without a G protein intermediary. As the relevant ion channel has not been characterized, such a mechanism, although unconven- tional, would be consistent with our observations.

In endothelial cells, hormone receptor binding results in activation of phospholipase C with the consequent hydrolysis of phosphatidylinositol and production of inositol phosphates (20,44); in some cell types this process appears to be regulated by G proteins (56,57). Inositol1,4,5-trisphosphate appears to cause transient elevations in cell calcium concentration by mediating the release of calcium from intracellular stores in certain cell types (47); other inositol phosphate isomers have been proposed as mediators of extracellular calcium entry (58). Consequently, it is possible that the effects of G protein acivation which we observe are mediated via phospholipase C activation and production of inositol phosphate second mes- sengers although we have no evidence that inositol phosphates cause entry of extracellular calcium in endothelial cells.

In summary, we have demonstrated that activation of G proteins initiates PAF production in endothelial cells, that G proteins also mediate hormone-induced PAF production, and that, when activated, G proteins can convert an ineffective hormone-receptor interaction to one that has a full intracel- lular effect. In addition, G protein activation is associated with increased adherence of neutrophils to endothelial cells, a physiologic consequence of PAF production. We also find that the mechanism for G protein-induced PAF accumulation is elevation in cellular calcium concentrations due to entry of extracellular calcium, although the precise mechanism of that process and the role of other potential mediators (e.g. inositol phosphate) remains undefined. Our data are consistent with the hypothesis that G proteins couple hormone-receptor bind- ing to membrane channel opening. Such an arrangement would be an attractive and efficient mechanism for stimulus response coupling and potentially an excellent model for investigating the interactions of these components at a mo- lecular level.

Acknowledgments-We thank John H. B. Bridge for assistance in the measurements of atomic absorption spectroscopy, for the use of his spectrometer, and for advice on measurement of ion fluxes.

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PrescottR E Whatley, D F Fennell, J A Kurrus, G A Zimmerman, T M McIntyre and S M

Synthesis of platelet-activating factor by endothelial cells. The role of G proteins.

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