Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca⁺⁺-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha₁-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha₁-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha₁-adrenergic actions seems to occur at an early step of the alpha₁-adrenergic action. TPA (10^?11–10^?6M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha₁-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca⁺⁺ ionophore A-23187.     

Roles of alpha1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha₁-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the α₁-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from β in the initial stages to later α₁. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to β-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced β-adrenergic sensitivity is probably related to the increased number of β-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of α₁-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished α₁-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the α₁-adrenergic signal, rather than to changes at the receptor level.     

Structural changes of alpha2- and ovomacroglobulins

The plasma ₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human 2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as ₂-macroglobulin.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Synthesis, structure and properties of TTF-carboxylate bridged paddlewheel dirhodium complexes, Rh2(BuCO2)3(TTFCO2) and Rh2(BuCO2)2(TTFCO2)2

Reaction of tetrathiafulvalene carboxylic acid (TTFCO₂H) with paddlewheel dirhodium complex Rh₂(Bu^tCO₂)₄ yielded TTFCO₂-bridged complexes Rh₂(Bu^tCO₂)₃(TTFCO₂) (1) and cis- and trans-Rh₂(Bu^tCO₂)₂(TTFCO₂)₂ (cis- and trans-2). Their triethylamine adducts [1(NEt₃)₂] and cis-[2(NEt₃)₂] were purified and isolated with chromatographic separation, and characterized with single crystal X-ray analysis. Trans-[2(NEt₃)₂] is not completely separated from a mixture of cis- and trans-[2(NEt₃)₂], but its single crystals were obtained from a solution of the mixture. A three-step quasi-reversible oxidation process was observed for 1 in MeCN. The first two steps correspond to the oxidation of the TTFCO₂ moiety and the last one is the oxidation of the Rh₂ core. The oxidation of cis-2 is observed as a two-step process with very similar E_1/2 values to those of the first two processes for 1. Both 1⁺ and cis-2²⁺ in MeCN at room temperature show isotropic ESR spectra with a g value of 2.008 and a_H = 0.135 mT for two equivalent H atoms and a_H = 0.068 mT for one H atom. The redox and ESR data of cis-2 suggest that the intramolecular interaction between the TTF moieties is very small.     

Crystal structures of MoCl2(O)(O2)(OPMePh2)2 and mixtures of MoCl2(O2)(OPPh3)2 and MoCl2(O)(O2)(OPPh3)2: allylic alcohol isomerization studies using MoCl2(O)(O2)(OPMePh2)2

Adding one equivalent of H₂O₂ to compounds of stoichiometry MoCl₂(O)₂(OPR₃)₂, OPR₃ = OPMePh₂ or OPPh₃, leads to the formation of oxo-peroxo compounds MoCl₂(O)(O₂)(OPR₃)₂. The compound MoCl₂(O)(O₂)(OPMePh₂)₂ crystallized with an unequal disorder, 63%:37%, between the oxo and peroxo ligands, as verified by single-crystal X-ray diffractometry, and can be isolated in reasonable yields. MoCl₂(O)(O₂)(OPPh₃)₂, was not isolated in pure form, co-crystallized with MoCl₂(O)₂(OPPh₃)₂ in two ratios, 18%:82% and 12%:88%, respectively, and did not contain any disorder in the arrangement of the oxo and peroxo groups. These complexes accomplish the isomerization of various allylic alcohols. A mechanism of this reaction has been constructed based on ¹⁸O isotopic studies and involves exchange between the alcohol and metal bonded O atoms.     

Characterization of brain D2 dopamine receptors

In order to characterize and partially purify solubilized dopamine receptors, canine brain striatum microsomes were solubilized with 1% digitonin, and enriched by either gel permeation chromatography, preparative vertical column isoelectric focusing, or sucrose gradient ultracentrifugation. Chromatography on Sephacryl S-300 in buffer (contaning 0.05% Triton X-100) yielded a Stokes radius of 5.8 nm. Isoelectric focusing of the solubilized, radiolabelled receptor produced peaks of [³H]spiperone radioactivity corresponding to isoelectric values of 5.0 and 7.8, of which the former has been shown elsewhere to be the intact D₂ dopamine receptor. Sucrose density gradient ultracentrifugation, again in buffer containing 0.05% Triton X-100, indicated a hydrodynamic mol. wt of 136,000 Daltons, which corresponds closely to the value of 123,000 Daltons estimated using radiation inactivation. Such molecular characterization will aid in the distinction of dopamine receptor subtypes.     

Inotropic effect of PGE1 and PGE2 on isolated rat atria. Influence of adrenergic mechanisms

The effects of a wide range of PGE₁ and PGE₂ concentrations on the isometric developed tension of isolated rat atria beating spontaneously or paced at a fixed rate, were explored. PGE₁ only produced a negative inotropic effect (NIE), whereas PGE₂ elicited a biphasic inotropic action; negative at low concentrations and positive (PIE) at higher ones. Phenoxybenzamine and phentolamine failed to modify either the NIE or the PIE, but subthreshold exogenous norepinephrine abolished the NIE, suggesting a presynaptic inhibitory effect of PGEs on the adrenergic neurotransmitter release. Auricles pretreated with subthreshold norepinephrine react with a PIE to PGE₁, but not to PGE₂. On the contrary in the presence of subthreshold methoxamine the PIE of PGE₂ was increased whereas the action of PGE₁ was not modified.     

Photophysical properties of Eu(SiW11O39)2 and Eu(BW11O39)2

The photophysical properties (absorption, emission, and excitation spectra; luminescence quantum yields; luminescence decay lifetimes ) of K₁₃[Eu(SiW₁₁O₃₉)₂] and K₁₅[Eu(BW₁₁O₃₉)₂] in aqueous solution and in the solid state are reported. Both complexes exhibit broad and very intense O → W charge transfer bands in the U.V. region and weak and narrow f → f Eu₃₊ bands in the visible. At 77 K the luminescence emission of both complexes, which consists of ⁵D_O → ⁷F_J bands split by the local crystal field, can be pumped very efficiently via both the O → W CT and the f → f Eu³⁺ levels, whereas at 298 K only pumping via the f → f Eu³⁺ is efficient. The values of the luminescence decay lifetimes in H₂O and D₂O solution are quite similar, showing that no water molecule is coordinated to the central Eu₃₊ ion. The high resolution emission spectra are discussed in an attempt to define the coordination symmetry of Eu₃₊.     

Dynamic properties of the fluxional Rh2H(μ-PPh2)2(PPh3)3 complex

The rhodium dimer [Rh₂H(PPh₂)₂(PPh₃)₃]⁻ was prepared from RhCl(PPh₃)₃ and K₄Sn₉ in the presence of 2,2,2-cryptand in ethylenediamine/toluene solvent mixtures. The [K(2,2,2-crypt)]⁺ salt was isolated and characterized via NMR and X-ray diffraction studies. The solid state structure reveals a binuclear, diphenylphosphido-bridged, 32 electron Rh(I)-Rh(I) complex with edge-shared tetrahedral and square planar Rh centers with overall C_s point symmetry. 1-D and 2-D ¹H, ³¹P, and ³¹P{¹H} NMR experiments were used to characterize the complex.     

Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species

The activity of prostacyclin (PGI₂), PGE₁ or PGD₂ as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD₂ was a weak inhibitor in dog, rabbit and rat plasma whereas PGE₁ and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE₁ or PGD₂. Compound N-0164 abolished the inhibition by PGD₂ of human platelet aggregation but did not inhibit the effects of PGE₁ or prostacyclin. The results suggest that prostacyclin and PGE₁ act on similar sites on platelets which are distinct from those for PGD₂.     

New preparations and molecular structures of cis-MCl2(Ph2PCH2PPh2)2, M = Ru,Os and trans-RuCl2(Ph2PCH2PPh2)2

The title compounds were made by reacting bis(diphenylphosphino)methane (dppm) with reduced solutions of OsCl₆^4? and Ru₂OCl₁₀^4?. The crystal and molecular structures of these compounds have been determined form three-dimensional X-ray study. The cis-isomers crystallize with one CHCl₃ per molecule of the complex. All three compounds crystallize in the monoclinic space group P2₁/n with unit cell dimensions as follows: Cis-OsCl₂(dppm)₂·CHCl₃: a = 13.415(4) Å, b = 22.859(4) Å, c = 16.693(3) Å, β = 105.77(3)°, V = 4926(3) ų, Z = 4. cis-RuCl₂(dppm)₂·CHCl₃: a = 13.442(3) Å, b = 22.833(7) Å, c = 16.750(4) Å, β = 105.53(2)°, V = 4953(3) ų, Z = 4. trans-RuCl₂(dppm)₂: a = 11.368(7) Å, b = 10.656(6) Å, c = 18.832(12) Å; β = 103.90(6)°, V = 2213(7) ų; Z = 2. The structures were refined to R = 0.044 (R_w = 0.055) for cis-OsCl₂(dppm)₂·CHCl₃; R = 0.065 (R_w = 0.079) for cis-RuCl₂(dppm)₂·CHCl₃ and R = 0.028 (R_w = 0.038) for trans-RuCl₂(dppm)₂. The complexes are six coordinate with stable four-membered chelate rings. The PMP angle in the chelate rings is ca. 71° in each case.     

Relations between fatty acids and oestrogen binding properties of pure rat alpha1-foetoprotein

A delipidation procedures based on treatment with charcoal at pH 3 has been applied to highly purified rat alpha₁-foetoprotein preparations. The oestrogen binding properties of the delipidated proteins have been studied with an equilibrium dialysis technique, and compared with the properties of the untreated foetal protein, as well as those of preparations reconstituted from the defatted α₁-foetoprotein and the removed lipids. An important increase has been evidenced for the binding levels of oestrone, oestradiol-17β and diethyl-stilboestrol by the delipidated α₁-foetoprotein. A reversal of this effect has been obtained by incubating the delipidated protein either with the lipids extracted from the purified α₁-foetoprotein or with a potent competitor of the rat α₁-foetoprotein-oestrogen interaction, designated as ‘L’, previously demonstrated and isolated from whole rat sera, and tentatively characterized as a mixture of fatty acids. Scatchard analysis of the oestrone and oestradiol-17β binding parameters show that the enhanced fixation of the hormones after defatting is primarily due to a two-fold increase of the apparent number of binding sites/ mol α₁-foetoprotein. The results are interpreted in terms of the probable, at least partial, identity between the lipids closely associated with the pure α₁-foetoprotein and the fatty acid mixture ‘L’ isolated from whole sera. The possible biological role of a complex interplay between oestrophilic α₁-foetoproteins, phenolsteroids and fatty acids in the control of eostrogen levels during development is discussed brieftly.     

Interaction of clonidine and clonidine analogs with human platelet α2-adrenergic receptors

Several new clonidine analogs were synthesized and their ability to inhibit [³H] phentolamine binding to human platelet α₂-adrenergic receptors was tested. The order of potency and calculated dissociation constants for clonidine and its analogs were as follows: clonidine (0.020 ± 0.005 μM) >p-aminoclonidine (0.100 ± 0.010 μM) > hydroxy-phenacetyl-aminoclonidine (0.20 ± 0.03 μM) >p-dansyl clonidine (1.00 ± 0.20 μM) >t-boc-tyrosine clonidine (1.80 ± 0.60 μM). Thus, p-amino substitution reduces α₂-adrenergic affinity in the platelet system. The effects of clonidine and its p-amino analogs on platelet adenylate cyclase were also evaluated. This enzyme is inhibited by epinephrine acting via α₂-adrenergic receptors. Both clonidine and p-aminoclonidine cause slight inhibition of basal adenylate cyclase and reverse the inhibition induced by epinephrine. These observations indicate that clonidine is a partial agonist for platelet α₂-adrenergic receptors.     

Supramolecular assemblies of new 2-D complexes: Syntheses, crystal structures, spectroscopic and magnetic properties of {[MnAu2(CN)4(NITpPy)2(H2O)2]}n and {[Co(N(CN)2)2(NITpPy)2(H2O)2]}n

Two new complexes, {[MnAu₂(CN)₄(NITpPy)₂(H₂O)₂]}_n (1) and {[Co(N(CN)₂)₂(NITpPy)₂(H₂O)₂]}_n (2), have been synthesized and characterized. The single-crystal X-ray analysis for the complexes 1 and 2 demonstrates that each M(II) (M = Mn or Co) ion assumes a distorted octahedral MN₄O₂ coordination polyhedron. Four nitrogen atoms come from the cyanide groups and the pyridyl rings in a common plane, and two oxygen atoms come from the H₂O molecules in trans-positions. The structures of complexes 1 and 2 illustrate that aurophilicity and/or hydrogen bonding interactions play important roles in increasing dimensionality. Magnetic investigations on complexes 1 and 2 show the presence of weak antiferromagnetic interactions.     

Solution-mediated syntheses and characterizations of two new zincophosphites [C6H14N2]0.5[Zn(H2PO3)2] and [C4H12N2]0.5[(CH3)2NH2][Zn2(HPO3)3]

Two new zincophosphites [C₆H₁₄N₂]_0.5[Zn(H₂PO₃)₂] 1 and [C₄H₁₂N₂]_0.5[(CH₃)₂NH₂][Zn₂(HPO₃)₃] 2 have been solvothermally synthesized in mixed solvents of N,N-dimethylformamide (DMF) and 1,4-dioxane (DOA), respectively. Single-crystal X-ray diffraction analysis reveals that compound 1 exhibits a neutral inorganic chain formed by ZnO₄ and HPO₂(OH) units. Interestingly, the left- and right-handed hydrogen-bonded helical chains are alternately formed via the hydrogen-bonds between two adjacent chains. Compound 2 exhibits a layer structure with 4- and 12-MRs formed by ZnO₄ and HPO₃ units, in which two kinds of organic amine molecules both act as countercations to compensate the overall negative electrostatic charge of the anionic network.     

Basal level of human platelet prostaglandins: PGE1 is more elevated than PGE2

Radioimmunoassays of platelet prostaglandins E₁ and F_1α in platelet rich plasma or platelet suspension, demonstrate that both PGE₁ and PGF_1α are present at higher concentrations than prostaglandins E₂ and F_2α. Gas chromatography — mass spectrometry determinations of prostaglandins E₁ and E₂ in resting washed platelets confirm this difference. Lastly, there is a greater incorporation of [1-¹⁴C] acetate into prostaglandins E₁ and F_1α compared to that into prostaglandins E₂ and F_2α.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Syntheses and characterization of the W(II) and W(III) N2 complexes, [WCl2(PMe3)3]2N2 and [WCl3(PMe3)2]2N2

Refluxing WCl₄(PMe₃)₃ under a nitrogen atmosphere in the presence of two equivalents of sodium amalgam leads to a reduction to the W(II) complex [cis,mer-WCl₂(PMe₃)₃]₂N₂ (1), which can be converted to [mer,trans-WCl₃(PMe₃)₂]₂N₂ (2) via appropriate oxidation/chlorination. Structural data have been obtained for both complexes, and demonstrate significantly increased steric crowding in 1 due to PMe₃/PMe₃ interactions. The N-N bond distances in the two compounds are similar, at 1.279(4) and 1.243(18) Å, respectively.     

Solubilization and functional reconstitution of human neuropeptide FF2 receptors

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF₂ receptor expressed in Chinese hamster ovary (CHO) cells by the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors.