Inverse agonist activity of selected ligands of the cysteinyl-leukotriene receptor 1

Cysteinyl leukotrienes (CysLTs) are associated with several inflammatory processes, including asthma. Due to this association, considerable effort has been invested in the development of antagonists to the CysLT receptors (CysLT(1)R). Many of these molecules have been shown to specifically interact with CysLT(1)R, but little is known about their impact on the conformation of the receptor and its activity. We were especially interested in possible inverse agonist activity of the antagonists. Using a constitutively active mutant (N106A) of the human CysLT(1)R and the wild-type (WT) receptor coexpressed with the G(alphaq) subunit of the trimeric G protein, we were able to address this issue with ligands commonly used in therapy. We demonstrated that some of these molecules are inverse agonists, whereas others act as partial agonists. In cells expressing the CysLT(1)R mutant N106A exposed to Montelukast, Zafirlukast, or 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. On the other hand, 6(R)-(4-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BayU9773) and 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazole-5-YL)-butoxy]-phenyl ethanone] (LY171883) acted as partial agonists and alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV 5901) as a neutral antagonist. However, in cells expressing CysLT(1)R and G(alphaq), all antagonists used had inverse agonist activity. The decrease in basal inositol phosphate production by ligands with inverse agonist activity could be inhibited by a more neutral antagonist, confirming the specificity of the reaction. We demonstrate here that Montelukast, MK571, and Zafirlukast can act as inverse agonists on the human CysLT(1) receptor.     

Spectrophotometric assay for serum platelet-activating factor acetylhydrolase activity

We developed a spectrophotometric assay for serum platelet-activating factor acetylhydrolase (PAF-AH, EC 3.1.1.47.) activity using a platelet-activating factor (PAF) analogue with a 4-nitrophenyl group as substrate. PAF-AH hydrolyzes the sn-2 position of the substrate ?1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine, producing p-nitrophenyl succinate. This liberation was spectrophotometrically monitored and the activity determined from the change in absorption. The assay does not require radioisotopes and is applicable to an automatic analyzer. Utilizing this assay with an automatic analyzer, it is possible to measure the activities of thousands of samples in a few hours with excellent precision (CV 0.5%, n=30) and high correlation (r=0.979, n=100) with the results of a conventional radioisotopic assay. The assay should be particularly useful for clinical diagnostics.     

巴曲酶对大鼠局灶性脑缺血再灌注后血小板活化因子和血小板活化因子受体mRNA表达的影响

背景巴曲酶是目前比较公认的治疗缺血性脑血管病的理想药物之一,被广泛地应用于临床,因此对其在脑缺血再灌注损伤中的保护作用进行深入认识很有必要.目的探讨巴曲酶对大鼠局灶性脑缺血再灌注后血小板活化因子(PAF)水平及PAF受体基因(PAF-RmRNA)表达的影响.设计完全随机区组设计.地点和对象实验于2004-03/12在哈尔滨医科大学附属第二医院科研中心完成.选择40只健康Wistar雄性大鼠,体质量200～250 g,随机分为5组,每组8只.Ⅰ组假手术组;Ⅱ组为生理盐水组Ⅱa为缺血6h再灌注6 h组,Ⅱb为缺血6 h组;Ⅲ组为巴曲酶组Ⅲa为缺血6 h再灌注6 h组,Ⅲb为缺血6 h组.方法线栓法建立大鼠大脑中动脉闭塞(MCAO)及再通模型.应用RT-PCR技术检测MCAO及再通后缺血半暗带皮质PAF受体基因表达,同时用ELISA检测对应血浆PAF值.主要观察指标不同时间点各组缺血半暗带皮质PAF mRNA表达及血浆PAF值.结果生理盐水组中再灌组及缺血组PAF值均明显升高,Ⅱa,Ⅱb分别为(1 480±249)和(1 052±199)ng/L,而PAF-RmRNA表达降低,分别为0.44±0.06和0.48±0.05,分别与对应假手术组比较非常显著性意义(P＜0.01).巴曲酶组中再灌注及缺血组PAF值均降低,为(848±80)和(743±105)ng/L,PAF-RmRNA表达增强(0.63±0.08和0.67±0.06),与对应生理盐水组比较差异有显著性意义(P＜0.01).结论巴曲酶可降低脑缺血再灌注后血浆中PAF水平,并且可能对脑缺血再灌注缺血半暗带皮质组织PAF-RmRNA表达有影响,以期为预防性干预提供试验数据.     

Plasma platelet-activating factor acetylhydrolase activity in critically ill patients

OBJECTIVE: Platelet-activating factor (PAF) is a potent proinflammatory mediator in systemic inflammation and sepsis and is inactivated by the enzyme PAF-acetylhydrolase (PAF-AH). Recently, a large phase III clinical trial using recombinant PAF-AH to treat patients with severe sepsis was performed but failed to reduce 28-day mortality rate. To get more information on the activity of PAF-AH in sepsis, we repeatedly measured its activity in plasma in critically ill patients compared with healthy controls. DESIGN: Retrospective cohort study. SETTING: Intensive care unit. PATIENTS: Two hundred thirty-one patients who were admitted to an operative intensive care unit within 1 yr were enrolled and evaluated daily for American College of Chest Physicians/Society of Critical Care Medicine criteria. PAF-AH activity was measured as the release of [H]-acetate from [H]-acetyl-PAF. INTERVENTIONS: Analysis of plasma samples. MEASUREMENTS AND MAIN RESULTS: At the day of admission, PAF-AH activity of patients was below controls but markedly increased over time. Higher activities were seen in patients with severe sepsis or septic shock compared with those without organ failure. With respect to the clinical outcome, lower values were found in nonsurvivors only as long as they had not developed organ failure. In severe sepsis/septic shock, values of nonsurvivors exceeded those of survivors. PAF-AH activity was positively correlated with plasma levels of inflammatory mediators such as neopterine and tumor necrosis factor-alpha but not with acute phase reactants such as C-reactive protein, interleukin-6, or PCT. In addition, parenteral nutrition with lipid emulsions was seemingly associated with low PAF-AH activity compared with enteral nutrition. CONCLUSION: The data indicate severity- and time-dependent changes in PAF-AH activity and may help to explain the failure of recombinant PAF-AH treatment strategies that were not based on activity measurements.     

Protective effects of specific platelet-activating factor receptor antagonists in experimental glomerulonephritis

The present study was undertaken to evaluate the hypothesis that enhanced production of platelet-activating factor (PAF) contributes to the renal hemodynamic alterations and impaired glomerular permselectivity that characterize the heterologous phase of nephrotoxic serum nephritis. Intravenous administration of nephrotoxic gamma globulin to normal rats led to a rapid decline in glomerular filtration rate within 60 min from 1.08 +/- 0.08 to 0.45 +/- 0.12 ml/min/100 g b.wt., P less than .001, and in effective renal plasma flow rate from 2.56 +/- 0.15 to 1.35 +/- 0.21 ml/min/100 g b.wt., P less than .02. Concomitantly, the fractional excretion of protein rose from 3.4 +/- 0.6 X 10(-5) to 11.6 +/- 2.3 X 10(-5), P less than .03. Pretreatment with the specific PAF receptor antagonists WEB 2086 or WEB 2170 significantly ameliorated the impairment in glomerular filtration rate and in effective renal plasma flow rate induced by nephrotoxic globulin. Filtration rate declined by 0.63 +/- 0.12 ml/min/100 g b.wt. 60 min after administration of nephrotoxic globulin, but fell only 0.30 +/- 0.14 ml/min/100 g b.wt. in rats pretreated with WEB 2086, P less than .05 and 0.34 +/- 0.11 ml/min/100 g b.wt. in rats pretreated with WEB 2170, P less than .01. The decline in effective renal plasma flow rate 60 min after administration of nephrotoxic globulin was 1.21 +/- 0.30 ml/min/100 g b.wt., but only 0.86 +/- 0.23 ml/min/100 g b.wt. in rats pretreated with WEB 2086, NS, and 0.52 +/- 0.21 ml/min/100 g b.wt. in rats pretreated with WEB 2170, P less than .003.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of platelet-activating factor receptor protects mice from osteoporosis following ovariectomy

While platelet-activating factor (PAF) is produced in various diseases associated with bone resorption, its functions in bone metabolism remain unknown. Using PAF receptor-deficient mice, we evaluated the role of PAF in the development of bone resorption following ovariectomy, a model of postmenopausal osteoporosis. Through observations of bone mineral density and histomorphometric parameters, it was found that bone resorption was markedly attenuated in PAF receptor-deficient mice, indicating that PAF links estrogen depletion and osteoporosis in vivo. Osteoclasts expressed higher amounts of the enzymes required for PAF biosynthesis than osteoblasts. TNF-alpha and IL-1beta increased the acetyl-coenzyme A:lyso-PAF acetyltransferase activity in osteoclasts. Osteoclasts, but not osteoblasts, expressed the functional PAF receptor. PAF receptor stimulation prolonged the survival of osteoclasts in vitro. Furthermore, osteoclasts treated with a PAF receptor antagonist, and also those from PAF receptor-deficient mice, showed reductions in survival rate and Ca resorption activity. Consistently, in organ cultures, bone resorption was significantly suppressed by a PAF receptor antagonist treatment or genetic PAF receptor deficiency. Thus, these results suggest that, through the inflammatory cytokines, estrogen depletion enhances PAF production as a unique autocrine factor for osteoclast functions. Inhibition of PAF function might pave the way for a new strategy to prevent postmenopausal bone loss without disturbing osteoblast functions.     

Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice.

To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 microM), 4'-chlorodiazepam was the most effective agent, clonazepam was the least effective agent, and midazolam had an effect intermediate between those of the other two ligands. The peripheral benzodiazepine receptor ligands had a dose-dependent suppressive effect, and the 50% inhibitory concentrations were 0.01 microM for 4'-chlorodiazepam and 5 microM for midazolam. Concomitant use of PK 11195 (10 microM), an antagonist of the peripheral benzodiazepine receptor, reversed this suppressive effect with 4'-chlorodiazepam (10 microM) or midazolam (10 microM). PK 11195 showed this antagonistic effect in a dose-dependent manner. Intravenous 4'-chlorodiazepam (5 mg/kg of body weight) significantly suppressed LPS (100-micrograms)-induced TNF activity of sera (2 h postchallenge with LPS) from thioglycolate-treated mice. The present findings suggest that the peripheral benzodiazepine receptor plays an important role in modulating LPS-induced TNF activity in mouse macrophages.     

Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor

Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor-positive and -negative (PAF-R-positive and -negative) cells and PAF-R-deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R-positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R-deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R-expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.     

Functional validation of platelet-activating factor receptor sites characterized biochemically by a specific and reproducible [3H]platelet-activating factor binding in human platelets

In human platelet membranes, [3H]platelet-activating factor(PAF)-C18 binding sites exhibited high affinity (Kd 0.074 +/- 0.005 nM, n = 28 healthy volunteers), saturability, elevated stereoselectivity, marked pharmacological specificity and small intersubject variability. The maximal binding capacity was 215 +/- 12 fmol/mg protein. Saturation of [3H]PAF binding was obtained with 0.3 nM ligand, and its isotherm was compatible with a single class of binding sites. The stereoselectivity for [3H]PAF was clearly indicated by the low displacing potency of enantio-PAF-C16 (the synthetic enantiomer of PAF) that was 5000-fold less potent than PAF. Specific [3H]PAF binding attained 65% with 0.1 nM ligand and was displaced fully not only by cold PAF but also by RP 59227 (Ki = 6.2 +/- 1.3 nM, n = 7), a novel, potent and specific PAF receptor antagonist in a pure enantiomeric form and several other antagonists such as CV-6209, WEB 2086, L-652,731 and BN 52021. Various classical pharmacological agents did not interfere with the [3H]PAF binding. In intact platelets, [3H]PAF binding shared the same properties as those just described for membrane preparations. A functional role for these binding sites was suggested by the high correlation (r = 0.94, P less than .001) between the Ki values for several known PAF antagonists determined in [3H]PAF binding and the IC50 values obtained against PAF-induced aggregation in whole platelets. Thus, the present [3H]PAF binding in human platelet membranes may be a useful pharmacological tool to study possible changes in [3H]PAF binding parameters induced by pathological states for which PAF may be directly or indirectly responsible.     

Inverse agonist activity of atypical antipsychotic drugs at human 5-hydroxytryptamine2C receptors

Clozapine is the prototype atypical antipsychotic drug, producing little or no extrapyramidal side effects, while improving negative symptoms of psychosis. Clozapine's high affinity for serotonin receptors has been hypothesized to confer the unique antipsychotic properties of this drug. Recently, we demonstrated that both typical and atypical antipsychotic drugs are inverse agonists at constitutively active 5-hydroxytryptamine2A (5-HT(2A)) receptors. To determine whether inverse agonist activity at 5-HT(2C) receptors plays a role in antipsychotic efficacy, typical and atypical antipsychotic drugs were tested for inhibition of basal inositol phosphate production in mammalian cells expressing rat or human 5-HT(2C) receptors. Atypical antipsychotic drugs (sertindole, clozapine, olanzapine, ziprasidone, risperidone, zotepine, tiospirone, fluperlapine, tenilapine) displayed potent inverse agonist activity at rat and human 5-HT(2C) receptors. Typical antipsychotic drugs (chlorpromazine, loxapine, thioridazine, prochlorperazine, perphenazine, mesoridazine, trifluperidol, fluphenazine, spiperone, haloperidol, pimozide, penfluridol, thiothixene) were devoid of inverse agonist activity, with the exception of loxapine. We review the evidence that loxapine has unique properties characteristic of both atypical and typical antipsychotic drugs. Several typical antipsychotic drugs (chlorpromazine, thioridazine, spiperone, thiothixene) displayed neutral antagonist activity by reversing clozapine inverse agonism. These data suggest that 5-HT(2C) inverse agonist activity is associated with atypical antipsychotic drugs with moderate to high affinity for 5-HT(2C) receptors, and imply that effects of atypical antipsychotic drugs on the 5-HT(2C) receptor may play a role in their unique clinical properties. These data also imply that dysfunction of brain 5-HT(2C) receptor systems may be one of the factors involved in the etiology of psychosis.     

Anti-inflammatory activity of pergolide, a dopamine receptor agonist.

Pergolide, a dopamine agonist effective in the treatment of Parkinson's disease, has been shown to have anti-inflammatory activity in the carrageenan paw edema assay in rats at p.o. doses greater than or equal to 0.3 mg/kg. Studies were done to investigate the mechanism of action and to determine the pharmacologic significance of this finding. Because pergolide elevates circulating glucocorticoids, the effect of pergolide on carrageenan-induced paw swelling was assessed in adrenalectomized rats. Pergolide retained its anti-inflammatory activity in adrenalectomized carrageenan-injected rats, thus eliminating corticosterone induction as a possible mechanism of action. Pergolide treatment also did not decrease thromboxane B2, prostaglandin E2 or leukotriene B4 production, ruling out direct effects on arachnoid acid inflammatory mediators. Interactions with the autonomic nervous system were suggested, in that an alpha adrenergic agonist (clonidine) mimicked the activity of pergolide in the carrageenan assay, and an alpha adrenergic antagonist (phenoxybenzamine) blocked the anti-inflammatory activity of pergolide in this assay. Dopamine receptor antagonists (haloperidol or sulpiride) partially inhibited the effect of pergolide in the carrageenan model. However, the peripherally restricted dopamine antagonist, domperidone, was ineffective, suggesting that a central dopamine receptor was involved in the effect. Experiments in chronic inflammation models such as lipoidal-amine induced arthritis in rats and picryl chloride-induced delayed type hypersensitivity in mice also revealed an anti-inflammatory effect of pergolide. Activity in the carrageenan system and the lipoidalamine model demonstrated that the anti-inflammatory effects of pergolide were separable from potential immunosuppressive effects. Multiple dose studies indicated that tolerance might develop to the anti-inflammatory effect of pergolide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnostic value of the serum platelet-activating factor acetylhydrolase activity in inflammatory bowel disease

Platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme hydrolyzing platelet-activating factor (PAF), a potent inflammatory mediator, but the relationship between this enzyme and inflammatory bowel disease (IBD) is not fully elucidated. The aim of the present study was to examine the usefulness of the serum PAF-AH activity in order to differentiate ulcerative colitis (UC) from Crohn's disease (CD). The serum PAF-AH activity was measured in 57 patients with IBD (39 UC and 18 CD patients) and 13 control subjects by a spectrophotometric method. The serum PAF-AH activity was thus found to be significantly lower in patients with CD (median 265.5 U/l) than in those with UC (355 U/l) or control subjects (374 U/l). This marker at a cutoff level of 386 U/l demonstrated a sensitivity of 46%, a specificity of 100%, and a positive predictive value of 100% regarding its ability to distinguish UC from CD. Moreover, the marker responded inversely to the changes in the disease activity of IBD. These results suggest that measuring the serum PAF-AH activity is a useful diagnostic modality for making a differential diagnosis between UC and CD.     

Synaptic activity becomes excitotoxic in neurons exposed to elevated levels of platelet-activating factor

Neurologic impairment in HIV-1-associated dementia (HAD) and other neuroinflammatory diseases correlates with injury to dendrites and synapses, but how such injury occurs is not known. We hypothesized that neuroinflammation makes dendrites susceptible to excitotoxic injury following synaptic activity. We report that platelet-activating factor, an inflammatory phospholipid that mediates synaptic plasticity and neurotoxicity and is dramatically elevated in the brain during HAD, promotes dendrite injury following elevated synaptic activity and can replicate HIV-1-associated dendritic pathology. In hippocampal slices exposed to a stable platelet-activating factor analogue, tetanic stimulation that normally induces long-term synaptic potentiation instead promoted development of calcium- and caspase-dependent dendritic beading. Chemical preconditioning with diazoxide, a mitochondrial ATP-sensitive potassium channel agonist, prevented dendritic beading and restored long-term potentiation. In contrast to models invoking excessive glutamate release, these results suggest that physiologic synaptic activity may trigger excitotoxic dendritic injury during chronic neuroinflammation. Furthermore, preconditioning may represent a novel therapeutic strategy for preventing excitotoxic injury while preserving physiologic plasticity.     

PMS-601, a new platelet-activating factor receptor antagonist that inhibits human immunodeficiency virus replication and potentiates zidovudine activity in macrophages

We assessed the anti-human immunodeficiency virus (anti-HIV) activity in vitro of new platelet-activating factor (PAF) receptor antagonists, as PAF and viral replication are thought to be involved in HIV neuropathogenesis. We found that PMS-601 inhibited proinflammatory cytokine synthesis and HIV replication in macrophages and potentiated the antiretroviral activity of zidovudine. These results suggest that PMS-601 is of potential value as an adjuvant treatment for HIV infection.     

3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([3H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [3H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [3H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [3H]52770 RP or [3H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [3H]PAF and [3H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [3H]PAF and [3H]52770 RP. Thus, [3H]52770 RP and [3H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum platelet-activating factor acetylhydrolase (PAF-AH) activity in more than 3000 healthy Japanese

BACKGROUND: A spectrophotometric assay for platelet-activating factor acetylhydrolase (PAF-AH) activity differs from the radioisotopic assay in its value because of a difference in substrate specificity. The spectrophotometric assay is more precise than the radioisotopic assay, providing information that is not clear with the radioisotopic assay. METHODS: We measured the serum PAF-AH activity in 3106 healthy Japanese, utilizing the spectrophotometric assay with an Hitachi 7170 automatic analyzer. We also measured the serum PAF-AH activity in 18 healthy volunteers to investigate the effect of diet and the change in activity in a day and over 6 weeks. Changes were examined at 0 (day 1), 1, 2, 4 and 6 weeks. RESULTS: The mean value for females was significantly lower than that of males at the 5% level and both male and female activity had a tendency to increase with advancing age. It is known that the PAF-AH is primarily associated with LDL in blood and the PAF-AH activity correlated with the total cholesterol (r=0.52, n=126) and the LDL cholesterol (r=0.60, n=126) concentrations. In the diet study, there was no observable effect on activity. No difference in PAF-AH activity was observed between serum and plasma sample types. The serum PAF-AH activity was stable at 7 degrees C for at least 7 days and at -20 degrees C for at least 2 months. CONCLUSIONS: The serum PAF-AH activity in women was lower than in men until the menopausal age was reached. We could use not only fresh fasting serum, but also plasma sample, non-fasting sample and stored sample to estimate the PAF-AH activity.     

Negative inotropic effect of platelet-activating factor: association with a decrease in intracellular sodium activity

Platelet-activating factor (PAF) is an autacoid whose cardiovascular actions include a potent negative inotropic effect. The mechanism of this decrease in myocardial contractility is still at issue, as both a decrease and an increase in trans-sarcolemmal Ca++ influx have been reported. Because changes in intracellular sodium activity (aiNa) are known to influence myocardial contractility, we investigated whether PAF affects aiNa. Thus, we have measured contractile responses to PAF (1 nM-1 microM) in isolated guinea pig right ventricular papillary muscles paced at constant rate, and recorded transmembrane action potential and aiNa with conventional and sodium-selective microelectrodes, respectively. Our findings suggest that PAF does not affect slow inward Ca++ current, because PAF neither affected nor prevented histamine-induced restoration of contractile responses in K+-depolarized papillary muscles. On the other hand, we found the negative inotropic effect of PAF to be associated with a shortening of the action potential duration and with a decrease in aiNa. The specific PAF antagonist compound CV-3988 inhibited all three electro-mechanical responses. Our findings imply that the decrease in contractile force caused by PAF may depend on the reduction in aiNa; as aiNa falls, intracellular Ca++ may be lost via the Na+/Ca++ exchange and contractility decreases. The shortening of the action potential duration by PAF may reflect a decrease in Na+ influx and the consequent reduction in aiNa.