Characterization of prostaglandin (PG)-binding sites expressed on human basophils. Evidence for a prostaglandin E1, I2, and a D2 receptor.

Recent data suggest that prostaglandins (PGs) are involved in the regulation of basophil activation. The aim of this study was to characterize the basophil PG-binding sites by means of radioreceptor assays using 3H-labeled PGs. Scatchard analysis for pure (greater than 95%) chronic myeloid leukemia (CML) basophils revealed two classes of PGE1-binding sites differing in their affinity for the natural ligand (Bmax1 = 217 +/- 65 fmol/10(8) cells; Kd1 = 0.5 +/- 0.2 nM; Bmax2 = 2462 +/- 381 fmol/10(8) cells; Kd2 = 47 +/- 20 nM; IC50 = PGE1 less than PGI2 less than PGD2 less than PGE2 less than PGF2 alpha) as well as two classes of PGI2 (iloprost)-binding sites (Bmax1 = 324 +/- 145 fmol/10(8) cells; Kd1 = 0.5 +/- 0.3 nM; Bmax2 = 2541 +/- 381; Kd2 = 27 +/- 6 nM; IC50 = PGI2 less than PGE1 less than PGD2 less than PGE2 less than PGF2 alpha. In addition, CML basophils exhibited a single class of PGD2-binding sites (Bmax = 378 +/- 98 fmol/10(8) cells; Kd = 13 +/- 4 nM; IC50: PGD2 less than PGI2 less than PGE1 less than PGE2 less than PGF2 alpha). In contrast, we were unable to detect specific saturable PGE2-binding sites. Primary and immortalized (KU812) CML basophils revealed an identical pattern of PG receptor expression. Basophils (KU812) expressed significantly (p less than 0.001) lower number of PGE1 (PGI2)-binding sites (Bmax1: 9% (20%) of control; Bmax2: 36% (50%) of control) when cultured with recombinant interleukin 3 (rhIL-3), a basophil-activating cytokine, whereas rhIL-2 had no effect on PG receptor expression. Functional significance of binding of PGs to basophils was provided by the demonstration of a dose-dependent increase in cellular cAMP upon agonist activation, with PGE1 (ED50 = 1.7 +/- 1.1 nM) and PGI2 (ED50 = 2.8 +/- 2.3 nM) being the most potent compounds. These findings suggest that human basophils express specific receptors for PGE1, PGI2 as well as for PGD2.     

Evaluation of prostaglandin-receptors in human and rat liver: interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50:2.5 +/- 1.7, (6.1 +/- 2.1) microM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9 +/- 0.9 (2.0 +/- 0.8) microM. The Scatchard analysis on [3H]PGE1- as well as on [3H]iloprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3 +/- 5.2 (21.3 +/- 4.3) fmol/mg protein and a Kd of 2.1 +/- 1.8 (1.9 +/- 0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4 +/- 18.2 (86.1 +/- 13.2) fmol/mg protein and a Kd of 10.5 +/- 2.9 (15.1 +/- 3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4 +/- 13.9 (35.9 +/- 8.2) fmol/mg protein and a Kd of 4.1 +/- 1.2 (1.7 +/- 1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3 +/- 42.1 (142.9 +/- 17.8) fmol/mg protein and a Kd of 16.3 +/- 4.9 (9.2 +/- 7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4 +/- 51.9 (38.8 +/- 7.4) fmol/mg protein and a Kd of 16.2 +/- 3.2 (2.5 +/- 1.2) nM. It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are able to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Tissue calmodulin levels in normal and Graves' thyroids

Calmodulin levels in normal human thyroids and Graves' disease thyroids were measured by specific radioimmunoassay in the presence of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The calmodulin levels in tissues from patients with Graves' disease treated with thionamide drugs were significantly higher than those in normal tissues from euthyroid patients with solitary cold nodules (normal: 484 +/- 50 ng/mg protein, mean +/- SE, n = 15; Graves': 901 +/- 54 ng/mg protein, n = 48, p less than 0.001). Such a rise in calmodulin levels in Graves' disease thyroids was also present even after the administration of 50 micrograms of T3 for 5 days before operation (828 +/- 137 ng/mg protein, n = 6, p less than 0.01). Calmodulin levels in Graves' disease thyroids were closely related to the cell height of follicular epithelium. Calmodulin levels in a columnar cell predominant group were significantly higher than those in a flat cell predominant or a cuboidal cell predominant group (columnar cell predominant: 1150 +/- 118 ng/mg protein, n = 13; flat cell predominant: 561 +/- 125 ng/mg protein, n = 3, p less than 0.05; cuboidal cell predominant: 596 +/- 40 ng/mg protein, n = 25, p less than 0.001). The increase in calmodulin content in Graves' disease thyroid could therefore possibly be attributed to the stimulation of the thyroid gland by the thyroid stimulating antibody. An immunofluorescence study demonstrated the presence of calmodulin immunoreactivity in the thyroid epithelial cells, particularly enriched in the apical border in the form of a granulated structure.     

Characterization of specific binding sites for PAF in the iris and ciliary body of rabbit

The protective effect exerted by BN 52021 a specific PAF-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAF in rabbit iris and ciliary body. Two classes of PAF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes: a high affinity site Kd1 congruent to 4.9 +/- 0.47 nM, Bmax1 congruent to 3.17 +/- 0.50 pmoles/mg protein, a low affinity sites Kd2 congruent to 11.6 +/- 0.33 nM, Bmax2 congruent to 12.46 +/- 2.3 pmoles/mg protein for iris. The specific binding was not affected by lyso-PAF the biologically inactive precursor and metabolite of PAF, up to 10(-6) M; inhibition by unlabelled PAF demonstrated a biphasic curve partially antagonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAF in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021.     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E-and prostglandin F-like material (304 +/- 20 and 582 +/- 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10(-4)-10(-6)M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of 3H-prostaglandin E2 (3H-PGE2) and 3H-prostaglandin F1 alpha (3H-PGF2 alpha) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca++ concentration in medium up to 2 mM, was heat labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal 3H-PG binding was found in the 27000 x g pellet. Scatchard analysis of 3H-PGE2 binding revealed the presence of a single population of binding sites with a Kd= 1.2 nM and a binding site concentration of 1-2 pmoles/g protein. A single population of binding sites for 3H-PGF2 alpha was also detected with a Kd= 1.7 nM and a similar binding site concentration. Non-radioactive PGE1 and PGE2 were almost equally effective to compete for 3H-PGE1 binding sites (ED50= 5 and 2 nM, respectively). Unlabeled PGF1 was relatively ineffective to compete for 3H-PGE2 binding (ED50 greater than 1000 nM) but displaced effectively 3H-PGF2 alpha binding (ED20=1.2 nM).     

Cooperativity among manganese-binding sites in the H+-ATPase of chloroplasts

Coupling factor, isolated from lettuce chloroplasts, contained several binding sites for Mn2+ ions. Three of these sites showed strong cooperative interactions having a Hill coefficient of 2.9 +/- 0.20 and a Kd of 14.7 +/- 0.44 microM. Three additional non-interacting Mn2+-binding sites were found with a Kd of 46.7 +/- 2.3 microM. Chemical modification with naphthylglyoxal of 1 arginyl residue/chloroplast coupling factor 1, which inhibited ATPase activity, inhibited the cooperativity among the sites but did not prevent Mn2+ binding to the enzyme. It is suggested that the cooperative interaction among the Mn2+-binding sites is an expression of the interaction among the active sites of the enzyme which is required for catalysis.     

The ontogeny of the rabbit hepatic glucose transporter

We delineated and characterized the fetal hepatic glucose transporter in the rabbit. Employing the 2-deoxy-D-glucose displaceable 3H-cytochalasin B binding assay we estimated the number and Kd of the GT per mg of liver protein. A gradual increase in the number was observed during development, the fetus (23.8 +/- 2.04 pmoles/mg) expressing a lesser amount when compared to the neonate (59.5 +/- 17 pmoles/mg; p less than 0.05) and adult (142 +/- 11 pmoles/mg; p less than 0.05). On the other hand the affinity of the glucose transporter was higher in the fetus (Kd 287 +/- 81 nM) when compared to either the neonate (988 +/- 222 nM, p less than 0.05) or the adult (706 +/- 101 nM, p less than 0.05). We conclude that the fetal hepatic GT is more efficient secondary to a higher affinity for glucose.     

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Evaluation of prostaglandin-receptors in human and rat liver: Interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50: 2.5±1.7, (6.1±2.1) μM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9±0.9 (2.0±0.8) μM. The Scatchard analysis on [3H]PGE1- as well as on [3H]ilioprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3±5.2 (21.3±4.3) fmol/mg protein and a Kd of 2.1±1.8 (1.9±0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4±18.2 (86.1±13.2) fmol/mg protein and a Kd of 10.5±2.9 (15.3±3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4±13.9 (35.9±8.2) fmol/mg protein and a Kd of 4.1±1.2 (1.7±1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3±42.1 (142.9±17.8) fmol/mg protein and a Kd of 16.3±4.9 (9.2±7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4±51.9 (38.8±7.4) fmol/mg protein and a Kd of 16.2±3.2 (2.5±1.2) nM.It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are stable to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.     

Regulation of 1,4-dihydropyridine and beta-adrenergic receptor sites in coronary artery smooth muscle membranes.

The receptor sites for 1,4-dihydropyridine (DHP) calcium channel ligands were identified and pharmacologically characterized in partially purified canine coronary artery smooth muscle (CSM) membranes (purification factor for 1,4-DHPs 2.8 and 2.2 respectively) using Ca2+ channel agonist (-)-S-[3H]BAYK 8644 and antagonist (+)-[3H]PN 200-110 as radioligands. The beta-adrenergic receptors were identified with (-)-3-[125I]iodocyanopindolol (ICYP). Specific binding of 1,4-DHPs and ICYP to membrane fraction was saturable, reversible and of both high and low affinity. The Kd for 1,4-DHP Ca2+ channel agonist was 0.59 +/- 0.05 and for antagonist 0.35 +/- 0.06 nmol/l and for low affinity binding sites Kd = 9.0 +/- 0.18 and 18.0 +/- 1.1 nmol/l. The high affinity 1,4-DHP binding (Bmax = 265 +/- 21 and 492 +/- 12 fmol/mg protein), showed stereoselectivity, temperature-dependence as well as pharmacological specificity: isoprenaline- and GTP-sensitivity, positive modulation with dilthiazem and negative modulation with verapamil, that is, properties characteristic of 1,4-DHP receptor sites on L-type Ca2+ channels. The low affinity binding sites were characterized as nonselective, temperature independent, dipyridamol-sensitive and represented a nucleoside transporter. The proportion of high affinity binding sites identified in the CSM membranes was 1.85 : 1.0 in favour of the antagonist. Results obtained with [125I]omega Conotoxin GVI A demonstrated that CSM membrane fractions isolated from median layers of coronary artery were devoid of substantial contamination with fragments of neuronal cells.     

A bumetanide-sensitive, potassium carrier-mediated transport system in excitable tissues

The binding of [3H]-bumetanide to rat brain synaptosomes revealed the existence of two binding sites. The high affinity site (R1 = 46.6 fmoles/mg protein) binds bumetanide and furosemide with Kd1 of 13 nM and 1.5 microM respectively, while the low affinity site (R2 = 1.37 nmoles/mg protein) is characterized by Kd2 of 200 microM and 680 microM for bumetanide and furosemide, respectively. Bumetanide sensitive 86Rb uptake was 34 +/- 14.5, 38.3 +/- 1.4, 18.6 +/- 1.3 and 29.0 +/- 6.1% of total 86Rb uptake in synaptic plasma membrane vesicles, rat brain synaptosomes, Neuroblastoma N1E115 cell line and chick chest muscle cells, respectively. Furosemide and bumetanide inhibited 86Rb uptake to rat brain SPM- vesicles in a dose dependent fashion. Half maximal inhibition (IC50) was observed at 20 nM and 4 microM for bumetanide and furosemide, respectively. Bumetanide-sensitive transport was dependent on extravesicular sodium and chloride concentrations with a Km of 21 and 25 mM for the two ions, respectively. These results demonstrate the existence of a "loop diuretic" sensitive carrier-mediated K+ transport system in brain and other excitable cells.     

Opioid peptides and catecholamines in human pheochromocytoma

Opioid peptides (OP) and catecholamines (CA) were measured in twelve human pheochromocytomas (PHEO). In all tumors the CA concentrations were much higher than those OP (range: 300-85,000 fold higher). Large intertumor variability in the levels of both substances was encountered (mean +/- S.E.M. = CA: 44.9 +/- 7.7 mumoles/g; OP: 38.1 +/- 17.1 nmoles/g; range = CA: 10.1-93.1 mumoles/g; OP: 0.7-181 nmoles/g). Norepinephrine (NE) was the main CA in seven of the 12 tumors. In four of these PHEO, NE accounted for 85% or more of the total CA. These "noradrenergic PHEO" derived from the right adrenals, were of smaller size (36 +/- 15g), had the lowest levels of OP (1.1 +/- 0.3 nmoles/g) and CA (28 +/- 10 mumoles/g), produced moderate to severe sustained hypertension (MBP: 160 +/- 11 mmHg) and the most severe and persistent clinical manifestations. Epinephrine (EPI) was the main CA in five of the 12 tumors. These PHEO had intermediate levels of OP (12 +/- 3 nmoles/g), and four of them were of left adrenal origin. Patients bearing these tumors were generally normotensive (MBP: 103 +/- 4 mmHg) and asymptomatic, with occasional paroxysmal crisis. The highest levels of OP (132 +/- 24 nmoles/g) were found in two tumors of extra-adrenal location and in one of right adrenal origin. The proportion of NE and EPI ranged between 60-80% and 20-40% respectively, of total tumor CA. The two extra-adrenal PHEO were the largest of this series (180 and 245g). These patients had mild hypertension (MBP: 118 +/- 7 mmHg) of sustained or paroxysmal course, and frequent symptomatic episodes. Differences in the synthesis, storage, metabolism and release of CA and OP in PHEO probably account for their variable tumoral content, as well as for the clinical heterogeneity produced by these tumors. It remains to be seen whether OP can contribute to the clinical manifestations of patients with pheochromocytomas.     

Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver.

Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p less than 0.05 to p less than 0.001) lower binding capacity and binding affinity for 3H-PGE1, 3H-PGE2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 +/- 7% for 3H-PGE2, 75.3 +/- 9% for 3H-PGE1 and 78.9 +/- 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p less than 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha.     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes.

We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.     

Binding of spermidine to transfer ribonucleic acid

The binding of spermidine to yeast tRNAPhe and Escherichia coli tRNAGlu2 at low and high ionic strength was studied by equilibrium dialysis. Once corrected for the expected Donnan effect, the binding at low ionic strength obeys the simple relationship of equivalent binding sites, and cooperative binding of spermidine to tRNA could not be detected. At low ionic strength (0.013 M Na+ ion), tRNAPhe (yeast) has 13.9 +/- 2.3 strong spermidine binding sites per molecule with Kd = 1.39 X 10(-6) M and a few weak spermidine binding sites which were inaccessible to experimentation; tRNAGlu2 (E. coli) has 14.8 +/- 1.6 strong spermidine binding sites and 4.0 +/- 0.1 weak spermidine binding sites with Kd = 1.4 X 10(-6) M and Kd = 1.23 X 10(-4) M, respectively. At high ionic strength (0.12 M monovalent cation) and 0.01 M Mg2+, tRNAPhe (yeast) has approximately 13 strong spermidine binding sites with an apparent Kd = 3.4 X 10(-3) M while the dimeric complex tRNAPhe X tRNAGlu2 has 10.4 +/- 1.2 strong spermidine binding sites per monomer with an apparent Kd = 2.0 X 10(-3) M. In the presence of increasing Na+ ion or K+ ion concentration, spermidine binding data do not fit a model for competitive binding to tRNA by monovalent cations. Rather, analysis of binding data by the Debye-Hückel approximation results in a good fit of experimental data, indicating that monovalent cations form a counterion atmosphere about tRNA, thus decreasing electrostatic interactions. On the basis of equilibrium binding analyses, it is proposed that the binding of spermidine to tRNA occurs predominantly by electrostatic forces.