Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Metabolic switching patterns provide definitive evidence of substrate-specificity and delineate subtle differences in positioning at the aromatase active site [Poster 06]

We found that extensive metabolic switching can be triggered in aromatization. Up to 20–35% of (19-³H₃)-labeled substrate is diverted to non-estrogenic product, namely 1β- and 2β-hydroxy androgens, negating usefulness for [³H]water assays. The strikingly substrate-specific metabolite patterns observed reflect positioning at the active site, and the large kinetic isotope effects involved. This switching is unusual in that it involves: 1) tritium labeling, 2) a multistep enzymic process, and 3) the potential unraveling of an enzymic mechanism.     

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues

In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-²H₅]-testosterone and [16,16,17-²H₃]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [²H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [²H₅]-Testosterone was converted into [²H₄]-oestradiol-17β, [²H₄]-oestrone and [²H₃]-6-dehydro-oestradiol-17 in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse.

On the basis of GC–MS characteristics (M⁺ m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M⁺). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [²H₄]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed.

In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [²H₃]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.     

Concerning the pathway from 19-oxoandrost-4-ene-3,17-dione to estrone

The conversion of a molecule of 19-oxoandrost-4-ene-3,17-dione [1a] to estrone [2a] by human placental aromatase requires a molecule of oxygen and of NADPH. An atom of this molecule of oxygen is incorporated into the extruded formic acid derived from C-19 of [1a]. It was proposed that the 0₂ is utilized for the enzymatic 2β-hydroxylation of [1a] and the released intermediate 2β-hydroxy-19-oxoandrost-4-ene-3, 17-dione [5a]aromatizes nonenzymatically. Should [5a] be an obligatory intermediate of estrogen biosynthesis, then all the oxygen of its 2β-hydroxyl must be incorporated into the extruded formic acid. We have previously synthesized [2β-¹⁸0;19-³H][5c] and proved that none of its 2β-¹⁸0 was incorporated in the formic acid extruded in the aromatization. On this basis we concluded that [5a] can not be an obligatory precursor of estrogen biosynthesis.

The trapping of radioactive androst-4-ene-2β,3β,17β,19-tetrol in a reductively terminated incubation of a mixture of radioactive androst-4-ene-3, 17-dione and [5a] with crude placental aromatase was interpreted as evidence in support of the intermediacy of [5a]. We confirmed that the tetrol can indeed be trapped in the reductively terminated incubations. However, considering that the crude placental enzyme preparation very likely contains numerous activated oxygen species capable of a variety of oxidation reactions, most of which may not be related to estrogen elaboration, and in view of our results quoted above, the origin and the eventual biosynthetic role of the parent compound of the tetrol remains to be determined.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Metabolism of some possible ecdysone precursors in Calliphora stygia

A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1-³H]-Cholesterol and [4-¹⁴C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1-³H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1-³H]-cholesterol sulphate, (22R)-22-hydroxy-[22-³H]-cholesterol, and 25-hydroxy-[26-¹⁴C]-cholesterol are not incorporated to a significant extent.     

Determination of δC values of valine in protein hydrolysate by gas chromatography–combustion isotope ratio mass spectrometry

A gas chromatographic–combustion isotope ratio mass spectrometric (GC–C-IRMS) method for the determination of [1-¹³C]valine enrichments in protein hydrolysates is described. Using a quick derivatization method, δ¹³C values of the N-methoxycarbonyl methyl ester of valine can be determined from baseline separated GC peaks. Evaluation studies with respect to precision, accuracy, linearity, reduction capacity of the CuO combustion furnace and isotope dilution as a result of derivatization, showed that our GC–C-IRMS system allows robust measurement of enrichments of [1-¹³C]valine in the range 0 to 1.5 MPE (S.D.±0.01 MPE, n=3). Therefore this method is suited to determine fractional synthetic rates (FSRs) of proteins as low as one-tenth of the FSR of human albumin, in studies using a primed, continuous (6 h) infusion with [1-¹³C]valine plasma enrichments of approximately 15 MPE and an hourly sampling schedule.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

Reactivity study of cyclopentadienyl osmium(II) bisphosphine azido complexes with activated alkynes and nitriles: Isolation of osmium triazolato and tetrazolato complexes by 1,3-dipolar addition

The cyclopentadienyl osmium(II) complexes [(η⁵-C₅H₅)Os(PPh₃)₂X] [X = Br (1), CH₃CN (2)] reacts with sodium azide (NaN₃) to yield the corresponding azido complex [(η⁵-C₅H₅)Os(PPh₃)₂N₃] (3). This undergoes [3+2] dipolar cycloaddition reaction with activated alkynes like dimethyl and diethyl acetylenedicarboxylate to yield triazolato complexes [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂(CO₂R)₂}] [R = –CH₂CH₃ (4) and –CH₃ (5)]. The complex 3 also reacts with nitriles such as tetracyanoethylene (TCE), fumaronitrile and p-nitrobenzonitrile to yield complexes of the type [(η⁵-C₅H₅)Os(PPh₃)₂{N₄C₂(CN)C(CN)₂}] (6), [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂HCN}] (7) and [(η⁵-C₅H₅)Os(PPh₃)₂{N₄C(C₆H₄–p-NO₂)}] (8). These complexes were fully characterized on the basis of microanalyses, FT-IR and NMR spectroscopic data. The molecular structure of the representative complex [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂(CO₂CH₂CH₃)₂}] (4) was determined by single crystal X-ray analysis.     

Feeding of xenobiotic ω-phenylalkanoic acids remarkably changes the chemistry and toxicity of the defensive secretion of Oxytelus sculpturatus Grav. (Coleoptera: Staphylinidae: Oxytelinae)

Feeding experiments with 12-phenyl[2,2-²H₂]dodecanoic acid and the correponding methylester resulted in the formation of 2-phenylethanol (probably produced via β-oxidation) and high amounts of 2-phenylethylesters of free fatty acids from the defensive secretion of Oxytelus sculpturatus rove beetles. The extraordinarily high amount of the metabolites occurring in the glands after administration of methyl-12-phenyl[2,2-²H₂]dodecanoate had a significant effect on the toxicity of the toluquinone-saturated defensive secretion mixture. Analogous experiments with 11-phenyl-[2-²H]undecanoic acid revealed a less efficient incorporation of this precursor leading to esters of 1-phenylethanol and 4-phenylbutan-2-ol and traces of 10-phenyl-1-decene probably formed via oxidative decarboxylation.     

Synthesis of [6″-H]-, (6″-H)- and (2-H)-maltotriose

To prepare labeled precursors for biosynthetic studies, methods for the specific introduction of tritium and deuterium into the reducing and the terminal glucose unit of maltotriose were developed. Thus [6″-³H]- and (6″-²H)-maltotriose (17) and (18) were prepared via selective methoxytritylation, deprotection and subsequent modified Pfitzner-Moffatt oxidation, followed by reduction with sodium borotritiide or sodium borodeuteride, respectively. A simple two step procedure utilizing the Lobry de Bruyn/van Ekenstein transformation gave (2-²H)maltotriose (20).     

Synthesis, crystal structures and magnetic studies of dicyanamide bridged two new 1D copper(II) complexes

Two new dicyanamide bridged 1D polynuclear copper(II) complexes [Cu(L¹){μ_1,5-N(CN)₂}]_n (1) [L¹H = C₆H₅C(O)NHNC(CH₃)C₅H₄N] and [Cu(L²){μ_1,5-N(CN)₂}]_n (2) [L²H=C₆H₅C(O)CHC(CH₃)NCH₂CH₂N(CH₃)₂] have been synthesised and structures of both the complexes and their crystal packing arrangements have been established by X-ray crystallography. For complex 1, a tridentate hydrazone ligand (L¹H) obtained by the condensation of benzhydrazide and 2-acetylpyridine is used, whereas a tridentate Schiff base (L²H) derived from benzoylacetone and 2-dimethylaminoethylamine is employed for the preparation of complex 2. Variable temperature magnetic susceptibility measurement studies indicate there are weak antiferromagnetic interactions with J values −0.10 and −1.41 cm⁻¹ for 1 and 2, respectively.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Evidence for the DNA binding and adduct formation of estrone and 17β-estradiol after dimethyldioxirane activation

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E₁) and 17β-estradiol (E₂) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957–1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E₁ and E₂ after activation are able to bind DNA. This paper reports that after DMDO activation, [³H]E₁ and [³H]E₂ were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E₁–DNA and E₂–DNA adducts was detected by ³²P-postlabeling analysis.     

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C₂₁ to C₁₉ steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-³H]progesterone and [1,2-³H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the K_m values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the V_max values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the K_m values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the V_max values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the K_m values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the K_m values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the V_max values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of K_m values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.     

Metabolism of 19-methyl substituted steroids and a proposal for the third aromatase monooxygenation

The article summarizes the results of recent studies on the metabolism of 10-ethylestr-4-ene-3,17-dione, 10-[(1R)-1-hydroxyethyl]-,and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione, in placenta. These compounds are the 19-methyl analogs of androstenedione, 19-hydroxyandrostenedione, and 19-oxoandrostenedione, respectively. No conversion of 10-ethylestr-4-ene-3,17-dione to either estrogens or oxygenated metabolites was detected. Both 10-[(1R)-1-hydroxyethyl]- and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione were oxygenated to 10-(1,1-dihydroxyethyl)estr-4-ene-3,17-dione and isolated following in situ dehydration as 10-acetylestr-4-ene-3,17-dione. Evidence for the involvement of aromatase in these conversions is discussed. No conversion of 10-acetylestr-4-ene-3,17-dione to either estrogens or other oxygenated products was detected. These results lead us to propose a new mechanism for the third aromatase monooxygenation. We propose that the third oxygenation is initiated by 1β-hydrogen abstraction at C₁ of 19,19-dihydroxyandrostenedione, followed by homolytic cleavage of the C₁₀−C₁₉ bond with concurrent formation of a Δ^1(10),4−3-ketosteroid and a C₁₉ carbon radical, and terminated by oxygen rebound at C₁₉.     

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Binding activities by propiverine and its N-oxide metabolites of L-type calcium channel antagonist receptors in the rat bladder and brain

The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N → O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N → O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N → O) inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the K_i value for P-4(N → O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly K_d values for bladder (+)-[³H]PN 200–110 binding. DPr-P-4(N → O) had little inhibitory effects on the bladder (+)-[³H]PN 200–110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[³H]PN 200–110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-³H]scopolamine methyl chloride ([³H]NMS) binding in the rat bladder. The ratios of K_i values for (+)-[³H]PN 200–110 to [³H]NMS were markedly smaller for propiverine and P-4(N → O) than oxybutynin and DEOB. Propiverine and P-4(N → O) inhibited specific binding of (+)-[³H]PN 200–110, [³H]diltiazem and [³H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The K_i values of propiverine and P-4(N → O) for [³H]diltiazem were significantly smaller than those for (+)-[³H]PN 200–110 and [³H]verapamil. Further, their K_i values for [³H]verapamil were significantly smaller than those for (+)-[³H]PN 200–110. The K_i values of propiverine for each radioligand in the cerebral cortex were significantly (P < 0.05) smaller than those of P-4(N → O). In conclusion, the present study has shown that propiverine and P-4(N → O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.     

Organometallic chemistry of diphosphazanes. Rhodium(I) complexes of RN(PX2)2 (R = C6H5; X = OC6H5, OC6H4Br−p, R = CH3; X = OC6H5)

Reactions of [Rh(COD)Cl]₂ with the ligand RN(PX₂)₂ (1: R = C₆H₅; X = OC₆H₅) give mono- or disubstituted complexes of the type [Rh₂(COD)Cl₂{ν²−C₆H₅N(P(OC₆H₅)₂)₂}] or [RhCl{ν²−C₆H₅ N(P(OC₆H₅)₂)₂ }]₂ depending on the reaction conditions. Reaction of 1 with [Rh(CO)₂Cl]₂ gives the symmetric binuclear complex, [Rh(CO)Cl{μ−C₆H₅N(P(OC₆H₅)₂)₂} ₂, whereas the same reaction with 2 (R = CH₃; X = OC₆H₅) leads to the formation of an asymmetric complex of the type [Rh(CO)(μ−CO)Cl{μ−CH₃N(P(OC₆H₅)₂)₂}₂ containing both terminal and bridging CO groups. Interestingly the reaction of 3 (R = C₆H₅, X = OC₆H₄Br−p with either [Rh(COD)Cl]₂ or [Rh(CO)₂Cl]₂ leads only to the formation of the chlorine bridged binuclear complex, [RhCl{ν²−C₆H₅N(P(OC₆H₄Br−p)₂)₂}]₂. The structural elucidation of the complexes was carried out by elemental analyses, IR and ³¹P NMR spectroscopic data.     

Photosynthetic carbon metabolism in Panicum milioides, a C3-C4 intermediate species: Evidence for a limited C4 dicarboxylic acid pathway of photosynthesis

Panicum milioides, a naturally occurring species with C₄-like Kranz leaf anatomy, is intermediate between C₃ and C₄ plants with respect to photorespiration and the associated oxygen inhibition of photosynthesis. This paper presents direct evidence for a limited degree of C₄ photosynthesis in this C₃-C₄ intermediate species based on:

1. (a) the appearance of 24% of the total ¹⁴C fixed following 4 s photosynthesis in ¹⁴CO₂-air by excised leaves in malate and aspartate and the complete transfer of label from the C₄ acids to Calvin cycle intermediates within a 15 s chase in ¹²CO₂-air;

2. (b) pyruvate- or alanine-enhanced light-dependent CO₂ fixation and pyruvate stimulation of oxaloacetate- or 3-phosphoglycerate-dependent O₂ evolution by illuminated mesophyll protoplasts, but not bundle sheath strands; and

3. (c) NAD-malic enzyme-dependent decarboxylation of C₄ acids at the C-4 carboxyl position, C₄ acid-dependent O₂ evolution, and ¹⁴CO₂ donation from [4-¹⁴C]C₄ acids to Calvin cycle intermediates during photosynthesis by bundle sheath strands, but not mesophyll protoplasts.

However, P. milioides differs from C₄ plants in that the activity of the C₄ cycle enzymes is only 15 to 30% of a C₄ Panicum species and the Calvin cycle and phosphoenolpyruvate carboxylase are present in both cell types. From these and related studies (Rathnam, C.K.M. and Chollet, R. (1979) Arch. Biochem. Biophys. 193, 346–354; (1978) Biochem. Biophys. Res. Commun. 85, 801–808) we conclude that reduced photorespiration in P. milioides is due to a limited degree of NAD-malic enzyme-type C₄ photosynthesis permitting an increase in pCO₂ at the site of bundle sheath, but not mesophyll, ribulosebisphosphate carboxylase-oxygenase.