Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Interaction of phosphorylated FcepsilonRIgamma immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics.

To examine the characteristics of the interaction of the FcepsilonRIgamma ITAM with the SH2 domains of p72(syk), the binding of an 125I-labeled dual phosphorylated FcepsilonRIgamma ITAM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72(syk) SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcepsilonRIgamma ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

Pharmacology of a potent platelet-activating factor antagonist: Ro 24-4736.

Ro 24-4736, (5-(3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f] [1,2,4]triazolo[4,3-a][1,4]diazepin-2-yl]-2-propynyl)phenanthri din- 6(5H)-one), has been identified as a potent, selective, p.o.-active platelet-activating factor (PAF) antagonist with a long duration of action. In vitro, Ro 24-4736 competes with [3H]PAF for its receptor site on dog platelets with an IC50 of 9.8 +/- 1.0 nM and selectively inhibits PAF-induced aggregation of guinea pig, dog and human platelets with concentration dependence. Ro 24-4736 dose-dependently inhibits in vivo bronchoconstriction (ID50 of 0.006-mg/kg p.o.) and ex vivo platelet aggregation (ID50 of 0.004 mg/kg p.o.) induced by PAF in guinea pigs. Time course studies show complete blockade of PAF-induced platelet aggregation (ex vivo) up to 8 hr after a single p.o. dose of 0.03 mg/kg as well as a long duration of action in vivo (30 hr). The in vivo PAF antagonistic activity is specific because, even at high p.o. doses (up to 10 mg/kg), Ro 24-4736 shows no inhibitory activity toward the bronchoconstrictor effects of leukotriene D4 or histamine. In comparison with other PAF antagonists evaluated in this guinea pig model, Ro 24-4736 is markedly superior in terms of p.o. potency, bioavailability and p.o. duration of action. Studies were also performed with Ro 24-4736 in additional in vivo models. When administered p.o. to sensitized guinea pigs, the drug attenuates inhaled antigen-induced airway hyper-reactivity without effect on bronchoalveolar lavage leukocyte accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Solid-phase extraction on silica cartridges as an aid to platelet-activating factor enrichment and analysis.

Solid-phase extraction methods using pre-packed silica cartridges and various elution solvents have been developed and evaluated as chromatographic means to enrich biological lipid extracts for platelet-activating factor (PAF). The optimized procedure advanced selectively removed the major tissue/blood neutral lipids and non-choline-containing phospholipids from complex lipid mixtures and yielded thereby a choline phospholipid fraction markedly enriched in bioactive PAF. Some tested solid-phase extraction procedures, while capable of resolving choline phospholipids from other polar and non-polar species, were detrimental to PAF's bioactivity and evidenced considerable loss or degradation of this analyte. It is concluded that, with solvents of appropriate composition, strength and polarity, solid-phase extraction on silica cartridges has several unique advantages over conventional thin-layer and column chromatographic methods presently in use for PAF enrichment from biological sources.

Production and release of platelet-activating factor by the injured heart-muscle cell (cardiomyocyte).

The potential of the injured heart-muscle cell (cardiomyocyte) to produce platelet-activating factor (PAF) has been evaluated. Beating rat myocytes in primary monolayer culture were used as the study-object, and metabolic inhibition served as the injury stimulus. Coincident with the development of irreversible injury, myocytes evidenced a rapid, net production of PAF which was sustained for some 3-6 h and reached, maximally, low-ng levels within approximately 0.5 h of injury. In the presence of serum, much of the PAF was released from the injured myocytes, whereas under serum-free conditions the PAF remained largely intracellular. Cardiomyocyte-derived PAF was pro-aggregatory to platelets and displayed the chromatographic properties and chemical reactivity of authentic PAF. However, PAF itself did not elicit myocyte injury, and a specific PAF antagonist could not attenuate the myocyte damage associated with metabolic inhibition. These results suggest that the cardiomyocyte may constitute a cellular source of PAF during heart injury, although the cell itself is not directly responsive to PAF.

Nonesterified fatty acid accumulation and release during heart muscle-cell (myocyte) injury: modulation by extracellular "acceptor".

Long-chain nonesterified fatty acid (NEFA) accumulation in the heart muscle cell (myocyte) and NEFA release to the extracellular milieu are considered contributors to the pathogenesis of myocardial injury in a number of cardiovascular disease states. Reported here is a study of the factors which influence and control the interactions among NEFA formation, intracellular NEFA accumulation, and NEFA release to the extracellular compartment by the irreversibly injured myocyte. Under conditions of metabolic inhibition, neonatal rat myocytes in primary monolayer culture became virtually depleted of ATP within 8 h. The metabolically inhibited myocytes evidenced membrane phospholipid degradation and a resultant net accumulation of NEFA produced thereby in the extracellular medium. However, under conditions of nutrient deprivation, the injured myocytes retained the NEFA produced from phospholipid catabolism intracellularly and did not release it to the culture medium, although the extent of myocyte ATP depletion was the same as it had been from metabolic inhibition. Serum could elicit, in a concentration-dependent fashion, the quantitative release of NEFA from metabolically inhibited myocytes to the culture medium but did not influence the net production of NEFA by the injured cells. Similarly, NEFA release from nutrient-deprived myocytes incubated in serum-free, substrate-free medium or in physiological buffer could be induced by supplementing the medium or buffer with bovine serum albumin (BSA), and the extent of NEFA release, but not NEFA formation, was dependent upon the extracellular BSA concentration. No manipulations to media other than changing their serum content or supplementing them with BSA were found to influence the disposition of NEFA produced during phospholipid catabolism in the irreversibly injured, ATP-depleted myocyte. Therefore, although progressive metabolic compromise in the myocyte was correlated with increasing, net NEFA formation, the distribution of the NEFA between the intracellular and the extracellular compartments was not determined by the magnitude of ATP loss or by the nature or duration of at least two injury stimuli, metabolic inhibition and nutrient deprivation. Rather, the net release of NEFA from the ATP-depleted myocyte to the culture medium and the consequent reduction of intracellular myocyte NEFA overload were critically and causally dependent upon the presence and concentration of extracellular NEFA "acceptor". The influence of acceptor on the mobilization of NEFA from the injured myocyte has implications regarding the use of NEFA release as an index of myocyte pathology and could serve to modify the progression and extent of myocardial injury in vivo.

Radioligand competitive binding methodology for the evaluation of platelet-activating factor (PAF) and PAF-receptor antagonism using intact canine platelets.

High-affinity, stereoselective, and ligand-selective specific binding of 3H-labeled platelet-activating factor (PAF) to its receptor on the dog platelet is reproducible over wide mass and concentration ranges of [3H]PAF. The [3H]PAF specific binding can be competitively inhibited by low picogram amounts of nonlabeled PAF. These observations have led to the formulation of radioligand competitive binding methodology for the detection and estimation of PAF in a biological lipid sample and the quantitative evaluation of PAF-receptor antagonism. The methodology is predicated upon correlation between the ability of a PAF analog/biological lipid sample/(synthetic) substance to inhibit [3H]PAF specific binding to the washed canine platelet and the known inhibition of [3H]PAF specific binding by standard, nonradioactive PAF. Application of this methodology to lipid extracts of human saliva has uncovered the finding that subjects with upper respiratory infection and chronic allergies have high saliva PAF contents. Pharmacologically active antiallergy agents known to inhibit PAF-induced pathology in animal models of disease were demonstrated, with the methology advanced, to act as PAF-receptor antagonists, and their potencies were quantified. These investigations indicate that the system proposed, in its ease, economy, sensitivity, specificity and capacity, has practical value for detecting and estimating PAF in biological lipid extracts and for evaluating PAF-receptor antagonism.

Amphiphile-induced heart muscle-cell (myocyte) injury: effects of intracellular fatty acid overload.

Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)-induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5-(tetradecyloxy)-2-furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high-energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile-induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA "wash-out." Although the development and severity of amphiphile-induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid beta oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile-induced bioenergetic abnormalities appeared to constitute a state of "metabolic anoxia" underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.

Specific binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) to the intact canine platelet.

Binding of 3H-labeled 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF) to the intact, washed canine platelet has been defined and characterized as being specific and receptor-mediated. Under the conditions described, specific binding to 2 X 10(7) canine platelets reached saturation within 10 min at a [3H]PAF concentration of approximately 0.4 nM. Non-specific binding was accountable for, at most, some 30% of the total PAF bound at equilibrium. Above approximately 0.4 nM [3H]PAF, total binding and non-specific binding increased in parallel. Since no involvement of PAF ligand in dog platelet intermediary metabolism during the binding incubation could be demonstrated, non-specific PAF binding may reflect a partitioning of the molecule into a cellular compartment (perhaps the platelet membranes). Equilibrium analysis revealed that the canine platelet has one class of specific binding sites with a Kd of 0.63 +/- 0.02 nM PAF, a Bmax of 222 +/- 10 fmol/10(7) platelets, and, at most, 1.33 +/- 0.06 X 10(3) binding sites/platelet. [3H]PAF specific binding to the canine platelet is ligand-selective and stereo-selective, as demonstrated by the relative abilities of non-labeled PAF and various PAF analogs/metabolites to inhibit [3H]PAF specific binding in a concentration-dependent manner. The extents to which PAF and PAF analogs were able to displace specifically-bound [3H]PAF from the canine platelet correlated well with their physiological (i. e., pro-aggregatory) effects. These data offer the first quantitative description of canine platelet high-affinity PAF binding sites/receptors and link receptor-mediated PAF binding to canine platelet physiology.

Pyrido[2,1-b]quinazolinecarboxamide derivatives as platelet activating factor antagonists.

A series of N-[(heteroaryl)alkyl]pyrido[2,1-b]quinazolines were evaluated for their ability to inhibit the binding of radiolabeled platelet activating factor (PAF) to its receptor on dog platelets. The most potent compounds in this series were found to be pyrido[2,1-b]quinazoline-8-carboxamides possessing a four- or six-carbon chain between the carboxamide nitrogen atom and a 3-pyridinyl or 5-pyrimidinyl moiety. Since earlier metabolism studies with pyridoquinazolinecarboxamides suggest that the carboxamide moiety is labile to hydrolysis in vivo, attempts were made to find isosteric replacements for this group. The substitutions examined led to a loss of activity; however, insertion of a methyl group on the carbon atom alpha to the carboxamide nitrogen led to an enantioselective enhancement of potency. (R)-2-(1-Methylethyl)-N-[1-methyl-4-(3-pyridinyl)butyl]-11-oxo-11H- pyrido[2,1-b]quinazoline-8-carboxamide (34) was more potent than the corresponding S enantiomer in the PAF binding assay and was also shown to be more resistant to degradation by amidases present in whole liver homogenates obtained from guinea pig, dog, and squirrel monkey. The corresponding rac-2-(1-methylethyl)-N-[1-methyl-4-(3-pyridinyl)butyl]-11-oxo-11H- pyrido[2,1-b]quinazoline-8-carboxamide (33) was found to inhibit transient PAF-induced thrombocytopenia and decreases in blood pressure in guinea pigs after intravenous or oral administration and to have a duration of action of greater than 5 h after an oral dose of 200 mg/kg. Compound 33 thus represents the prototype of a new class of orally active PAF antagonists.

Retained inhibition of the beta receptor by propranolol after preincubation and washout.

Mouse thymus cells possess a B2-adrenergic receptor and, when centrifuged through a solution of propanolol at 5 degrees and washed three times, will produce reduced amounts of cyclic AMP during a 5 minute challenge with isoproterenol at room temperature. Depending on the concentration and nature of the antagonist, the retained inhibition may be greater or less than that seen when it is added with the agonist. This retained inhibition is readily reversed at 37 degrees even in the absence of the agonist. These effects can also be demonstrated with rat erythrocytes and with their subsequently prepared ghosts making it most likely that they result from the specific and tight binding of the antagonist to the beta receptor.

The effect of dihydroxy-2-aminotetraline (DATs) on dopamine and beta type adenylate cyclases.

A number of DATS have been studied for agonist activity with dopamine (DA) and beta (beta) type adenylate cyclases and the most potent ones were found among the 6,7-DATs and the 5,6-DATs, respectively. None of the compounds tested possessed antagonist activity. The observed activities were not expected on the basis of structural analysis of model compounds such as apomorphine and 6,7-dihydroxy-1-benzyltetrahydroisoquinoline. A new view was constructed which suggests that the nitrogens of the DAT compounds were positioned better than those of the model compounds with regard to their binding sites.

A structure-activity analysis of the preferred conformation of the benzyl substituent of tetrahydropapaveroline at the beta receptor.

The effect of restricting the motion of the 3,4-dihydroxybenzyl substituent of tetrahydropapaveroline (THI) on its ability to stimulate the beta adenylate cyclase of the rat erythrocyte was investigated. A number of protoberberines and aporphines were tested as models of two classes of compounds where the benzyl substituent is rigidly but differently fixed. Unexpectedly, the beta agonist activity was associated with both structural types. This could be explained by the assumption that a phenylalanyl or tyrosyl residue of the receptor protein is free to rotate and bind with either position of the benzyl moiety as fixed in the protoberberine and aporphine structures.