Phenylpiperazine derivatives with strong affinity for 5HT1A, D2A and D3 receptors

Four 7-[3-(4-phenyl-1-piperazinyl)propoxy]coumarins were synthesized. The affinities of these compounds for DA (D_2A, D₃) and 5HT_1A receptors were evaluated for their ability to displace [³H]-raclopride and [³H]-8-OH-DPAT respectively from their specific binding sites. The affinities of the target compounds were all in the nanomolar range and followed the order 5-HT_1A > D₂ > D₃.     

Desensitization of PGE1 receptors in neuroblastoma-glioma hybrid cells

Prostaglandin E₁ receptor sites were measured in homogenates of NG108-15 neuroblastoma-glioma hybrid cells after exposure of intact cells to PGE₁. Scatchard analysis of competitive binding studies showed that incubation of NG108-15 cells in the presence of 2.5 μM PGE₁ for 16 h resulted in a loss of PGE₁ receptors and an increase in the dissociation constant of the remaining receptors. Thus, cells challenged with PGE₁ not only lose adenylate cyclase activity, but also lose PGE₁ receptors and decreased the affinity of the remaining receptors for PGE₁.     

PGD2 formation in the vasculature: Characteristics of rat tail vein prostaglandin endorperoxide-D isomerase

Rat tail vein homogenates, microsome and high speed supernatant fractions were incubated with [1-¹⁴C]prostaglandin endoperoxide (PGH₂) and products separated and identified by radio-thinlayer chromatography. PGI₂ synthase was localized to the microsomal fraction, but exhibited low activity compared to rat tail arteries prepared in the same manner. PGH-D isomerase was maximally active in the presence of reduced glutathione at pH 7.5–8.0, exhibited a K_m for PGH₂ of 33 μM, and was inhibited sulfhydryl-directed reagents. The similarities of this enzyme to PGD synthase of the rat cerebral microvasculature are discussed.     

pH-dependent modulation of agonist interactions with [3H]-ketanserin-labelled S2 serotonin receptors

The effects of varying the pH on the properties of S₂ serotonin receptors labelled by [³H]-ketanserin were examined. Between pH 7.0 and 8.2 the agonist affinities, as determined by competition experiments, increased dramatically. Serotonin, 5-methoxytryptamine, tryptamine, bufotenine and quipazine, demonstrated 15,16,8,6 and 5-fold increases in apparent affinity between pH 7.0 and 8.2. On the other hand the antagonists, ketanserin, cinanserin, and spiperone demonstrated little or no affinity changes between pH 7.0 and 8.2. The largest shift in affinity for an antagonist occurred with spiperone, which displayed a two-fold shift. Although changing pH is a rather non-specific manipulation, the selective affect on agonist interaction with S₂ receptors indicates further investigation of this pH effect may aid in discovering the difference in receptor interactions between serotonin agonists and antagonists.     

The quasi-relativistic Xα-SW molecular orbital method in organo-f-element chemistry: applications to UCl4, (η5-C5H5)4U and (η5-C5H5)2UCl2

The advantages and disadvantages of using the Xα scattered-wave molecular orbital method with quasi-relativistic corrections for the calculation of the electronic structure of organo-f-element compounds is discussed. Application of the method in organouranium chemistry is discussed via comparative calculations on UCl₄, (η⁵-C₅H₅)₄U, and (η⁵-C₅H₅)₂UCl₂. It is found that the η⁵-C₅H₅^? ligand is a better donor to U than is Cl^? and that the valence orbitals of (η⁵-C₅H₅)₂UCl₂ are energetically closer to those of (η⁵-C₅H₅)₄U than UCl₄. The calculational results are in excellent accord with experimental photoelectron spectroscopic studies.     

Butyrophenone analogues in the carbazole series: Synthesis and determination of affinities at D2 and 5-HT2A receptors

We describe a practical and efficient route for synthesis of 2-aminomethyl-1,2,3,9-tetrahydro-4H-carbazol-4-ones using an effective Fisher indole methodology. The most active compounds, 4b (QF 2003B) and 4c (QF 2004B), with pKi (5-HT_2A/D₂) ratio of 1.28 show an antipsychotic profile according to Meltzer's classification.     

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Localization of 5-HT1A serotonin receptors in the cerebellum of young rats

Previous studies have shown that functional 5-HT_1A receptors are present in the cerebellum only for the early postnatal period in rats. In order to investigate further the possible physiological significance of such a transient expression of 5-HT_1A receptors during maturation of the cerebellum, anatomical studies were performed for identifying which cell type(s) are endowed with these receptors in 8-day-old rats. Autoradiography (using [¹²⁵I]BH-8-MeO-N-PAT) with dry films and emulsion-coated coverslips, and radioimmunohistochemistry (using specific polyclonal anti-5-HT_1A receptor antibodies) of vermis sections revealed that 5-HT_1A receptors were mainly concentrated in the molecular layer of the anterior part of the lobule X and the posterior part of the lobule IXB. X-Irradiation on the 5th postnatal day yielded an agranular cerebellum whose density of 5-HT_1A sites was higher than that in age-paired control animals. These data indicate that 5-HT_1A receptors are not located on granule cells, but probably on glial cells in the molecular layer of the immature cerebellum. This location further supports the possible implication of glial 5-HT_1A receptors in some trophic action of 5-HT during CNS maturation.     

Further evidence for lack of specific receptors for PGE2 in the rat ovary

Prostaglandin E₂ (PGE₂) seems to stimulate cAMP accumulation in ovaries of all mammals. While it acts through specific receptors in some species, our earlier observations (1) suggest absence of PGE₂ receptors in the rat ovary. In order to further substantiate this assumption we digested ovarian membranes from the bovine and the rat with various enzymes and measured cAMP after stimulation with PGE₂, NaF, and hCG. Pronase, trypsin, and phospholipase C abolished cAMP accumulation completely. Neuraminidase, β-galactosidase and phospholipase D did not interfere with cAMP formation. After treatment with phospholipase A₂ PGE₂-mediated cAMP accumulation was abolished in the bovine but not in the rat ovary. Formation of cAMP disappeared after hCG but not after NaF in both species. Furthermore specific binding of PGE₂ could not be demonstrated in phospholipase A₂-treated bovine ovaries. These findings are consistent with presence of specific PGE₂ receptors in the bovine and their absence in the rat ovary.     

Desensitization of CholecystokininB Receptors in GH3 Cells

Abstract: Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca²⁺],) was characterized in GH₃ cells, a pituitary tumor cell line, which are known to possess CCK_B receptor subtype. The CCK-8-induced [Ca²⁺], transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH- induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca²⁺], transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 n M ). The responsiveness to CCK-8 was also reduced by phorbol 12, 13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways.     

Homologous Desensitization of the D1 Dopamine Receptor

Preincubation of D384 cells, derived from the human astrocytoma cell line G-CCM, with dopamine resulted in a time-dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE1) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 microM. A comparison of dopamine concentration-response curves of control and dopamine-preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 less than 3,4-dihydroxynomifensine less than fenoldopam less than 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4-dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the beta-adrenergic receptor.     

Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors

Neuropeptide Y (NPY) and NPY receptors are widely distributed in the CNS, including the retina, but the role of NPY in the retina is largely unknown. The aim of this study was to investigate whether NPY modulates intracellular calcium concentration ([Ca²⁺]_i) changes in retinal neurons and identify the NPY receptors involved. As NPY decreased the [Ca²⁺]_i amplitudes evoked by 30 mM KCl in only 50% of neurons analyzed, we divided them in two populations: NPY-non-responsive neurons (Δ2/Δ1 ≥ 0.80) and NPY-responsive neurons (Δ2/Δ1 < 0.80), being the Δ2/Δ1 the ratio between the amplitude of [Ca²⁺]_i increase evoked by the second (Δ2) and the first (Δ1) stimuli of KCl. The NPY Y₁/Y₅, Y₄, and Y₅ receptor agonists (100 nM), but not the Y₂ receptor agonist (300 nM), inhibited the [Ca²⁺]_i increase induced by KCl. In addition, the inhibitory effect of NPY on evoked-[Ca²⁺]_i changes was reduced in the presence of the Y₁ or the Y₅ receptor antagonists. In conclusion, NPY inhibits KCl-evoked [Ca²⁺]_i increase in retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. This effect may be viewed as a potential neuroprotective mechanism of NPY against retinal neurodegeneration.     

Synthesis and crystal structure of the MgCl2(CH3COOC2H5)2· (CH3COOC2H5) adduct

The reaction of α-MgCl₂ with boiling ethyl acetate affords MgCI₂(CH₃COOC₂H₅)₂· (CH₃COOC₂H₅), which is obtained as crystals suitable for X-ray analysis only from the mother liquor. M=315.5, orthorhombic, space group P2₁22₁ (No. 18), a=25.077(3), b=8.616(1), c=7.345(1) Å, V=1587.0(3) ų, Z=4, D_x=1.32 g cm⁻³,λ A(Mo Kα)=0.71069 Å, μ=4.17 cm⁻¹, F(000)=664, T=298 K, observed reflections: 1667, R=0.059 and R_w=0.069. The structure is composed of polymeric chains of MgCl₂(CH₃COOC₂H₅₎₂ and the ethyl acetate molecules occupy a mutually trans position.     

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.     

P2X2, P2X2–2 and P2X5 receptor subunit expression and function in rat thoracolumbar sympathetic neurons

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.     

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.     

P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes

Previous work has established the presence of functional P2X₇ subunits in rat cerebellar astrocytes, which after stimulation with 3'- O -(4-benzoyl)benzoyl ATP (BzATP) evoked morphological changes that were not reproduced by any other nucleotide. To further characterize the receptor(s) and signaling mechanisms involved in the action of BzATP, we have employed fura-2 microfluorometry and the patch-clamp technique. BzATP elicited intracellular calcium responses that typically exhibited two components: the first one was transient and metabotropic in nature – sensitive to phospholipase C inhibition and pertussis toxin treatment –, whereas the second one was sustained and depended on the presence of extracellular calcium. The ionotropic nature of this latter component was corroborated by measurements of Mn²⁺ entry and macroscopic non-selective cation currents evoked by either BzATP (100 μM) or ATP (1 mM). The two components of the calcium response to BzATP differed in their pharmacological sensitivity. The metabotropic component was partially sensitive to pyridoxalphosphate-5'-phosphate-6-azo-(-2-chloro-5-nitrophenyl)-2,4-disulfonate, a selective antagonist of P2Y₁₃ receptors, while the ionotropic component was modulated by external magnesium and markedly reduced by brilliant blue G and 3-(5-(2,3-dichlorophenyl)-1 H -tetrazol-1-yl)methyl pyridine (A438079), thus implying the involvement of P2X₇ purinergic receptors. It is concluded that P2Y₁₃ and P2X₇ purinergic receptors are functionally expressed in rat cerebellar astrocytes and mediate the increase in intracellular calcium elicited by BzATP in these cells.     

Prostaglandin E2 formation by rat gastroduodenal tissue following intragastric acid perfusion

Rat gastroduodenal mucosa forms prostaglandin (PG) E2. However, little is known about regional differences in PGE2 formation or the effect of gastric hydrochloric acid (HC1) perfusion on regional PGE2 formation. In this study, the rats were divided into 3 groups. Group 1 received intravenous (i.v.), 1 Ml/h, and intragastric (i.g.), 8 ml/h, perfusions of saline simultaneously for 3 h. Group 2 received saline i.v. and 0.15 N HC1 i.g., 8 ml/h. Group 3 was injected with a bolus of asprin (ASA), 60 mg/kg, followed by ASA, 40 mg/kg/h i.v., and 0.15 N HC1 i.g.. The gastric aspirates were analyzed for volume and pH. Segments of gastroduodenal tissue from the fundus, corpus, antrum, and duodenum were minced and then incubated in 1 ml of 5 mM Tris buffer, pH 8.4, for 30 sec with mixing; the incubate was assayed for PGE2 by radioimmunoassay. Intragastric HC1 decreased the pH of aspirate without producing gastric mucosal lesions. However, when combined with i.v. ASA, ulcer formation was present in all animals (p less than 0.05). PGE2 was formed by isolated tissue from four different gastroduodenal regions. The duodenum formed significantly greater amounts than the fundus, antrum, or corpus, which were similar. Intragastric HC1 produced a trend toward increased PGE2 formation (pmol PGE2/mg tissue) in the fundus, 143 +/- 36 to 237 +/- 57; corpus, 87 +/- 13 to 200 +/- 57; antrum, 157 +/- 28 to 224 +/- 65; and duodenum, 235 +/- 56 to 338 +/- 51. However, statistical significance was not reached.     

Prostaglandin E2 and leukotriene C4 levels following different reperfusion periods in rat brain correlated with morphological changes

Prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) are the metabolites of arachidonic acid (AA) that increase in forebrain following global ischemia and reperfusion. These mediators are highly potent vasoconstrictors of cerebral arteries leading to enhanced vascular permeability that induces the formation of vasogenic edema. In this study, after developing and experimental animal model simulating the concept of ischemic penumbra in the rat, the levels of PGE₂ and LTC₄ produced in the forebrain were measured and the effects of these mediators in short duration and prolonged reperfusion were investigated and then correlated with nueropathological findings. We found statistically significant reduction both in PGE₂ and LTC₄-like activities after just 10 min ischemia (p<0.05, p<0.05). PGE₂-like activity significantly increased in the 4th and 60th min of reperfusion (p<0.05, p<0.05). In the 15th min of reperfusion, PGE₂ was found to be significantly reduced (p<0.005) that may be due to the formation of free oxygen radicals by activation of PG hydroperoxidase reaction that inhibits PGE₂ production in the cylooxygenase pathway. LTs were not significantly increased in any reperfused group. Inhibition of the lipoxygenase pathway of AA metabolism may occur as a result of 15-HPETE (15-hydroperoxyeicosatetraenoic acid) production. Pathologically, edema and degeneration of brain tissue were seen beginning from the 4th min of reperfusion that reached a peak in the 60th min of reperfusion which is in accordance with biochemical changes in the damaged tissue. It is concluded that by preventing the formation of AA metabolites in the early hours of ischemia and reperfusion, it could be possible to increase blood flow in the ischemic penumbra that should limit the infarct area.