Development and application of an LC-MS/MS method for measuring the effect of (partial) agonists on cAMP accumulation in vitro.

Cyclic-adenosine monophosphate (cAMP) plays an important role in cell signalling and is widely used as a marker for receptor activation and as a target for treating various diseases. In this paper we present the development and validation of a new method for the determination of cAMP and ATP (adenosine triphosphate) and other nucleotides in a biological system by combining zwitterionic hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The HILIC-MS/MS method was developed for the simultaneous quantitative analysis of cAMP and ATP, and was validated by assessment of linearity (over a range from 0.5 to 100nM for cAMP and 50 nM to 50 microM for ATP (r(2)>0.999)), resolution, limit of detection (0.5 and 50 nM for cAMP and ATP, respectively) and reproducibility. Furthermore, the method was validated and applied in vitro to determine cAMP accumulation in biological samples. The effect of several dopamine D(2) (partial) agonists and antagonists on cAMP accumulation was assessed by determination of the cAMP/ATP ratio in cells transfected with the human dopamine D(2L) receptor. Quinpirole, dopamine and ropinirole produced agonist effects on cAMP accumulation, with a potency of quinpirole>ropinirole>dopamine. Lisuride, terguride and bifeprunox were found to be partial agonists with efficacies of lisuride>terguride>bifeprunox. As expected, haloperidol, (-)-sulpiride and LY-741626 were antagonists. These results demonstrate that the present analytical method was robust, fast, sensitive, and selective. Moreover, it showed utility in determining cAMP/ATP in biological systems and the ability to study the effect of (partial) agonists and antagonists which makes it a useful tool for drug discovery.

No evidence for binding of clozapine, olanzapine and/or haloperidol to selected receptors involved in body weight regulation.

The underlying mechanisms of antipsychotic (AP)-induced weight gain are unknown, but both central and peripheral AP target receptors could potentially be involved. This study used radioligand binding assays to compare the binding affinities of clozapine, olanzapine and haloperidol for candidate receptors potentially involved in AP-induced weight gain. Selected candidates derived from known pathways involved in body weight regulation included receptors classified as anorexigenic (bombesin receptor subtype 3, calcitonin gene-related peptide receptor, cholecystokinin receptor, melanocortin-4 receptor, neurotensin receptor 1) or orexigenic (cannabinoid receptor 1, galanin 1 receptor, melanin-concentrating hormone receptor (MCHR), neuropeptide Y1 receptor) as well as receptors involved in physiological actions related to digestion and fluid homeostasis (angiotensin II type 1 receptor, bradykinin B2 receptor, endothelin receptor, neurokinin 1 receptor, vasoactive intestinal polypeptide receptor 1). Clozapine, olanzapine and haloperidol exhibited negligible affinities to all of these receptors except for the MCHR (Ki=501 nM; haloperidol). With respect to other candidates from (neuro)transmitter systems already suggested to be involved in AP-induced weight gain, the binding profile of olanzapine resembled that of clozapine, with high affinity (Ki<10 nM) for serotonin (5-HT) 5-HT2A, 5-HT2C and 5-HT6, muscarinic M1 and histamine H1 receptors. In contrast, the binding profile of haloperidol was substantially different (high affinity only for the dopamine D1 receptor). In conclusion, we have not identified a novel binding site of the two investigated atypical AP that could contribute to the induced weight gain.

Acute effects of oxidized low density lipoprotein on metabolic responses in macrophages.

The immediate effects of oxidized low density lipoprotein (OxLDL) on the metabolic activity of cultured macrophages (RAW 264.7) were studied using a microphysiometer. Administration of OxLDL acutely induced a concentration-dependent increase in metabolic activity, with an EC50 of 16 +/- 3 microg/ml OxLDL and a maximal effect of 35% +/- 4% (mean +/- SEM; n=5). A biphasic response was measured after administration of 75 or 100 microg/ml OxLDL consisting of an initial sharp increase, followed by the induction of a long-lasting hypoactivity of 80% of the control value. Incubation of cells with polyinosinic acid (polyI; 100 microg/ml) for 30 min prior to OxLDL administration could completely block the effect of 25 microg/ml OxLDL. In addition, polyI acted as a full antagonist on the decrease of the biphasic response of cells generated by 75 and 100 microg/ml OxLDL. Macrophages used in this study possessed a specific binding site for OxLDL, with a dissociation constant (KD) of 9 +/- 2 microg/ml and a maximal binding of 610 +/- 32 ng 125I-OxLDL/mg cell protein. Binding of 125I-OxLDL to macrophages could be completely competed for by unlabeled OxLDL, by polyI for 58%, and by AcLDL for 46%. In conclusion, OxLDL can acutely activate the metabolic state of macrophages by a receptor-mediated process in a concentration-dependent fashion, which could be antagonized by polyI. Metabolic responses to OxLDL may underlie the changes observed in macrophages in the early atherosclerotic plaque.

Design and synthesis of an orally active GPIIb/IIIa antagonist based on a phenylpiperazine scaffold.

The design and synthesis of an orally active LMW non-peptide GPIIb/IIIa antagonist, based on a N,N'-bisphenylpiperazine scaffold, is described. The optimal compound showed a high in vitro binding potency (pIC50 = 8.7) in combination with potent oral antithrombotic activity (30-40% inhibition of thrombus growth at 0.3-3 mg/kg) with a duration of action of > 90 min. in a hamster cheek pouch model.

Dual effects of endothelin-1 on extracellular acidification by A7r5 smooth muscle cells.

The effect of endothelin-1 on metabolic activity of A7r5 rat aortic smooth muscle cells was studied. Endothelin-1 (pEC(50) 7.5) elicited an increase in the rate of extracellular medium acidification of the A7r5 cells. The ETA receptor antagonist BQ-123 blocked the endothelin-1 effect completely (pA(2) 7.6). Ca2+ channel blockers affected the endothelin-1 induced response in different ways: diltiazem and nifedipine partially blocked the endothelin-1 induced response, whereas verapamil did not influence this endothelin-1 induced effect. However, upon removal of verapamil an endothelin-1 dependent rise in extracellular acidification occurred, apparently reflecting the lifting of the verapamil blockade of an endothelin-1 induced process. Thus, this study shows that the complex integrated cellular responses upon ET-1 receptor activation are reflected in metabolic activity.

Regulation of plasminogen activation in rat cell lines.

The regulation of plasminogen activators (PA) and their inhibitors (PAI) in the rat cell lines: HTC and L2 was studied. HTC plasminogen activator inhibitor type 1 (PAI-1) production was stimulated by dexamethasone, serum factors and insulin; that of tissue-type plasminogen activator (tPA) by cAMP raising agents. Retinoic acid, butyrate, phorbol ester and endotoxin did not affect net PA/PAI activity elaborated by HTC. L2 cells produced tPA, which production was stimulated by retinoic acid, phorbol myristate acetate, butyrate and cAMP; serum factors blunted their response, whereas in the synthetic serum substituting medium Ultraculture and with cocktail Ultroser the action of tPA stimulators was enhanced.

Polar secretion of von Willebrand factor by endothelial cells.

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the polarity of von Willebrand factor (vWF) secretion. Endothelial cells cultured under these conditions allow direct measurements of substances released at both the apical and basolateral surface. The constitutive secretion of vWF was compared to the release of vWF from their storage granules after stimulation (regulated secretion). The basal, constitutive release of vWF occurs into both the apical and subendothelial direction. The rate of accumulation of vWF to the subendothelial direction is about three times higher than the amount of vWF secreted into the lumenal medium per unit of time. However, upon stimulation of confluent endothelial cell monolayers with phorbol myristate acetate, endothelial cells predominantly secrete vWF at the lumenal surface. Under these conditions, vWF does not accumulate in the collagen matrix. Thus, endothelial cells are able to organize themselves into a polarized monolayer, in such a way that vWF secreted by the regulated pathway accumulates at the lumenal site, whereas resting endothelial cells release vWF predominantly at the opposite, basolateral surface.

Storage and secretion of von Willebrand factor by endothelial cells.

Endothelial cells synthesize and store von Willebrand factor. We have studied the storage and secretion of von Willebrand factor in cultured human umbilical vein endothelial cells. In particular, we were interested in the nature of the storage compartment and the effects of perturbation on the storage and secretion processes. The storage compartment for von Willebrand factor was isolated from homogenates of endothelial cells. By an immunostaining technique the isolated vesicles stained for von Willebrand factor. The staining pattern was similar to that of Weibel-Palade bodies in intact endothelial cells. We concluded that the storage compartment containing von Willebrand factor is identical to the Weibel-Palade body. The von Willebrand factor of the isolated storage vesicles is predominantly constructed of polypeptide chains with a Mr of 220 kD. On the other hand, von Willebrand factor continuously secreted by endothelial cells is constructed of both a 220 kD and a larger precursor (apparent Mr of 275 kD) subunit. The storage vesicles contain von Willebrand factor that supports ristocetin-induced platelet aggregation. Thus, endothelial cells store fully processed, biologically active von Willebrand factor within Weibel-Palade bodies. Short-term (less than 1 h) treatment of endothelial cells with the perturbing phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) results in release of cellular stored von Willebrand factor. 24-48 h after exposure to PMA the endothelial cell distribution of von Willebrand factor is changed distinctly. While the contents of the von Willebrand factor storage sites in the cells are gradually restored within 48 h, enhanced amounts of von Willebrand factor are secreted into the medium. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increase after exposure to phorbol ester, as determined by immunofluorescence microscopy. Phorbol ester treated cells release stored von Willebrand factor 48 h after they have been stimulated. PMA decreases the von Willebrand factor contents of the extracellular matrix; the deposition of von Willebrand factor in the subendothelium is blocked by PMA, whereas the degradation of matrix von Willebrand factor is not affected. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution.

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short-term (less than 1 hr) treatment of endothelial cells with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long-term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse-chase experiments revealed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.

Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets.

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus-induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

von Willebrand factor in cultured human vascular endothelial cells from adult and umbilical cord arteries and veins.

Endothelial cells were cultured from various human arteries and veins, obtained from adult individuals and from umbilical cords. We compared the storage and secretion of von Willebrand factor by endothelial cells from umbilical veins with that of endothelial cells cultured from a number of adult vessels, including aorta, arteria iliaca, vena saphena magna and vena cava. There were no differences in the way the cultured endothelial cells handled the von Willebrand factor they synthesized. Endothelial cells from the various vessels responded to stimuli in secreting stored von Willebrand factor. The cells also responded to thrombin and ionophore A23187 in producing enhanced amounts of prostacyclin. Thus, cultured umbilical vein endothelial cells have properties that are very similar to those of cultured endothelial cells of various other origins. It is concluded that foetal venous cells provide a representative model for studies of endothelial cell von Willebrand factor biosynthesis and prostacyclin production.

Cigarette smoke impairs endothelial cell prostacyclin production.

Production of prostacyclin by endothelial cells is considered to be important in rendering the vessel wall nonthrombogenic. Cigarette smoking is an important risk factor in the pathogenesis of atherosclerosis. Here we show that the incubation of cultured human endothelial cells with a cigarette smoke condensate impaired the basal prostacyclin release. Also, the enhanced release of prostacyclin provoked by phorbol myristate acetate was inhibited by cigarette smoke condensate. Furthermore, cigarette smoke condensate impaired the thrombin-induced prostacyclin production. The production of prostacyclin from exogenous arachidonate was not affected by cigarette smoke condensate, indicating that cigarette smoke condensate constituents exert their inhibitory properties on the level of arachidonate mobilization from cellular phospholipids, rather than on cyclooxygenase or prostaglandin synthetase. The effects noted for cigarette smoke condensate could not be attributed to the cigarette smoke constituents nicotine and cadmium. While inhibiting the endothelial cell prostacyclin production significantly, cigarette smoke condensate did not cause cell death or impairment of secretory function, as measured by the release of von Willebrand factor. This in vitro study shows that impairment of an endothelial cell function is related to a risk factor for atherosclerosis.

Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from the platelet membrane glycoprotein IIa.

To define the role of membrane components that function in endothelial cell physiology and to characterize them biochemically, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained producing antibodies which bound to endothelial cells and also to platelets. The antibody of one of these clones, CLB-HEC 75, was studied in more detail. This antibody is directed against a single protein which is synthesized constitutively by endothelial cells and is expressed on the surface of both endothelial cells and platelets. The CLB-HEC 75 antigen was isolated from Nonidet P-40-solubilized endothelial cells and platelets by immunoprecipitation and exhibited an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 145,000 in the presence of 2-mercaptoethanol. Two-dimensional polyacrylamide gel electrophoresis and crossed immunoelectrophoresis revealed that the mobility of the CLB-HEC 75 antigen relative to platelet glycoproteins Ib, IIa, IIb, and IIIa fits previously defined criteria for platelet membrane glycoprotein IIa. The CLB-HEC 75 antigen isolated from endothelial cells co-migrated under all conditions tested with the antigen from platelets. These results indicate that endothelial cells share a plasma membrane protein indistinguishable from platelet membrane glycoprotein IIa. This protein may be a component involved in the interaction of endothelial cells with their environment including coagulation factors, platelets, and the subendothelial matrix. CLB-HEC 75 may serve as a useful tool for studying these processes.

Comparison of secretion and subcellular localization of von Willebrand protein with that of thrombospondin and fibronectin in cultured human vascular endothelial cells.

Cultured human vascular endothelial cells synthesize von Willebrand protein, thrombospondin and fibronectin. These proteins are secreted in the culture medium and incorporated into the extracellular matrix. We have compared the subcellular localization and the secretion of these proteins in response to stimulants in cultured human umbilical vein endothelial cells. Density gradient centrifugation using colloidal silica showed that the storage and secretion organelle with von Willebrand protein did not contain thrombospondin or fibronectin. Indirect immunofluorescence microscopy indicated that thrombospondin and fibronectin are not located in the rod-shaped organelles containing von Willebrand protein. Thrombin, ionophore A23187 and phorbol myristate acetate did not affect secretion of thrombospondin and fibronectin, while von Willebrand protein secretion was stimulated upon incubation of cells with these agents for 30 min. Prolonged incubation of cultured endothelial cells after a 1-h treatment with phorbol myristate acetate resulted in an increased secretion of von Willebrand protein into the conditioned medium; in contrast, accumulation of thrombospondin and fibronectin in endothelial cell-conditioned medium was decreased. These findings indicate that, unlike in platelets, these major endothelial proteins are not located in the same subcellular compartments. Von Willebrand protein is distinguished from thrombospondin and fibronectin both by its unique subcellular localization and its secretion rate in response to stimuli.

Neurotoxicity of diallate and triallate when administered orally or topically to hens.

Two allylthiocarbamate herbicides, diallate and triallate, were evaluated for neurotoxicity by oral and topical dosing studies with mature white leghorn hens. Diallate was tolerated for 90 days at topical doses of 40 mg/kg/day and oral doses of 20 mg/kg/day. Reversible ataxia and narcosis occurred at diallate doses of 80 mg/kg/day and higher by either route of administration. Triallate did not elicit signs of neurotoxicity at 300 mg/kg/day topically or 400 mg/kg/day orally. The oral dose, however, resulted in gastrointestinal irritation and severe weight loss, such that dosing was terminated after 25 days. Triallate was tolerated at oral dosages of 90 mg/kg/day and topical doses up to 330 mg/kg/day.

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells.

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca2+ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160-168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g X ml-1), and a dense one with a peak density of 1.12 g X ml-1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca2+ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.

In vitro neurotoxic esterase assay using leptophos oxon analogs as inhibitors.

In comparing neurotoxic esterase (NTE) inhibition properties of a series of phenylphosphonates, it was discovered that certain compounds including leptophos inhibited mipafox-insensitive phenylvalerate hydrolases. This leads to erroneous values for NTE inhibition which can be corrected by a differential assay: the total amount of mipafox-insensitive activity is determined with O-(2,6-dichlorophenyl)O-methyl phenylphosphonate and subtracted from the apparent NTE determined with the test compound before calculating pI50's.

On the molecular and submolecular structure of the semiquinone cations of alloxazines and isoalloxazines as revealed by electron-paramagnetic-resonance spectroscopy.

The thermodynamically stable and, therefore, analytically most important alloxazine and isoalloxazine radical cations have been studied in detail by electron paramagnetic resonance (EPR) spectroscopy. Isotopic and chemical substitutions have been made as in earlier studies with the less stable neutral and anionic species. The experimental spectra have been calculated with the aid of a more sophisticated computer-simulation program than previously used. Excellent fits were obtained only when all of the following atoms were taken into account in the hyperfine coupling scheme: N-5 H, N-10 H or CH3, C-6 H, C-7 H, C-8 H or CH3 and C-9 H. An additional but small coupling constant was required for the fit. This latter coupling constant is assigned to the nitrogen atom(s) of the pyrimidine subnucleus of (iso)alloxazine radical cations. The EPR-active proton is attached to N-5 as we also found for the neutral flavosemiquinone. The alloxazine and isoalloxazine radical cations exhibit an identical hyperfine coupling scheme but differ especially in the pyrazine nucleus with respect to the spin density distribution. This suggests that the geometrical structure of the two kinds of radicals is somewhat different. The highest spin density is, however, located at N-5 of (iso)alloxazine as has been found for the other flavosemiquinone species. The hyperfine coupling constants are interpreted in terms of spin densities and comparison is made with the most recently available quantum chemical calculations. All monomeric flavosemiquinone species are compared with each other and their differences in the submolecular structure are discussed briefly.

Biotransformation and porphyringogenic action of hexachlorobenzene and its metabolites in a primary liver cell culture.

Hexachlorobenzene (HCB) is metabolized in a primary culture of chick embryo liver cells and causes porphyrin accumulation within 24 h after administration. The HCB-metabolites, pentachlorothiophenol (PCThP), pentachlorobenzene (PeCB) and pentachlorophenol (PCP) identified in liver cell culture are already known from long-term experiments with rats. The pattern of accumulated porphyrins is comparable with the pathological porphyrin pattern observed in oral feeding studies with warm blooded laboratory animals. Protein bound radioactivity was found in cell cultures treated with [14C] HCB. Addition of the monooxygenase-inhibitor piperonyl butoxide or ascorbic acid decreased the irreversible binding of 14C-metabolites. The results show that biotransformation of HCB fulfils an essential role in the onset of porphyria. Since none of the main HCB-metabolites could induce a pathological porphyrin pattern, a reactive intermediate capable of reacting with glutathione or thiol-groups of uroporphyrinogen decarboxylase (UROG-D) is believed to be responsible for the inhibition of UROG-D. The chick embryo liver cell system may be considered as a useful and sensitive system for studying the metabolism of xenobiotics in relation to their toxicity.