Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O₂⁻ and CO₂⁻ was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O₂⁻ and correspondingly with O₂ of ferrocytochrome c) is by a factor of approx. 10³ slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O₂⁻/O₂ and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.     

The effects of prostacyclin PGI2 on prolactin secretion in vitro

Continuously superfused rat anterior pituitary cells were used to study the effects of exogenous prostaglandins (PGs) and thromboxanes (TXz) on the secretion of prolactin (PRL). No change in hormone release was observed upon superfusion with TXB₂ (10⁻⁵M) or the TX synthesis inhibitor, imidazole (1.5 mM). PGs A2, B2, d2, e1, e2, f1α, F2α, and endoperoxide analogs, U-44069 and U-46619, also had no effect on PRL secretion (all at 10⁻⁵M), In contrast 10⁻⁵M PGI2 was repeteadly found to stimulate PRL release to a level at least 125% above control, while producing no apparent change in the amount of hormone secreted in response to TRH. Somatostatin (SRIF), at a dose of 10⁻M, maximally inhibited TRH-induces PRL output, but failed to alter the PRL response to PGI₂. These studies indicate that PGI₂ may have a direct effect on the anterior pituitary to modify PRL secretion.     

The metabolism of prostaglandins F2α and E2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue,luteal tissue and conceptuses in vitro

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF_2a and PGE₂ and the degree of metabolism of the prostaglandins measured. Prostaglandins and metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Degradation of HeLa S3 cell chromatin by auromomycin and its chromophore

The antitumor protein agent auromomycin was found to degrade chromatin structure primarily by inducing strand scissions in linker regions. The reaction was stimulated by dithiothreitol. The chromophore form of the drug caused similar effects on chromatin, but it appeared to function at a more rapid rate. There was no evidence that auromomycin could cause breakage in core regions of chromatin.     

Bone remodelling induced by physical stress is prostaglandin E2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E₂ synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E₂ was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E₂ synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E₂. Prostaglandin E₂ added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E₂ the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis.The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis.We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.     

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.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.     

Differential effects of prazosin and rauwolsin on the release of prostaglandins I2 and E2 from the perfused mesenteric arterial bed of the rabbit following nerve stimulation

Sympathetic nerve stimulation of the perfused mesenteric arterial bed of the rabbit, , increase the secretion of prostaglandin (PG)I₂ and PGE₂. Prazosin (4.8 × 10⁻⁶), and α₁ adrenergic receptor antagonist, inhibited this inrease in release of PGI₂ but not of PGE₂ whereas rauwolsin (10⁻⁷ M), an α₂ adrenergic receptor antagonist, inhibited the increase in release of PGE₂ but not of PGI₂. Prazosin (10⁻⁶ M) completely blocked the vasoconstrictor response to nerve stimulation, and to norepinephrine and phenylephrine administration, suggesting there to be little of an α₂ adrenergic receptor component in this response. It is concluded that the increase in PGI₂ release follows the activation of α₁ adrenergic receptors and is therefore post-junctional in origin, whereas the increase in PGE₂ release follows the activation of α₂ adrenergic receptors and may be pre- and/or post-junctional in origin.Indomethacin (2.8 × 10⁻⁷, 5.6 × 10⁻⁷ and 1.12 × 10^{−6 M} did not affect the vasoconstrictor responses to nerve stimulation at 10 Hz, whereas rauwolsin (10⁻⁷ M) in the presence of indomethacin substantially increased them. These results indicate that PGE₂ does not regulate norepinephrine release following nerve stimulation at 10 Hz to rabbit mesenteric arteries, and that the inhibition of norepinephrine release following stimulation of α₂ pre-junctional receptors is independent of PG involvement.     

Thymosin β11: A peptide from trout liver homologous to thymosin β4

A peptide containing 41 amino acid residues has been isolated from trout liver and identified as a member of the β-thymosin family. Sequence analysis shows it to be 78% homologous to thymosin β₄, which is the peptide present in the thymus and other tissues of higher vertebrates, including, as reported here, livers of a species of reptile. Thymosin β₁₁ appears to replace the more prevalent thymosin β₄ in at least two species of bony fish, and represents the sixth structurally characterized member of this widely distributed family of peptides.     

Modification of chloroplast CF1 by fluoresceinisothiocyanate

Incubation of the chloroplast coupling factor with fluorescein isothiocyanate inhibits the Ca-ATPase activity of the enzyme and results in incorporation of fluorescein into the α and β subunits. High concentrations of ATP prevent the inhibition and reduce the incorporation of fluorescein into the α and β subunits. Ca and Mg ions increase fluorescein isothiocyanate inhibition and fluorescein incorporation into the α, β and γ subunits. It is suggested that fluorescein isothiocyanate modifies the catalytic site of the enzyme by blocking lysine residues in the α and β subunits which are involved in the binding of ATP.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.     

3H-cimetidine and the H2-receptor

The H₂-antagonist cimetidine is widely employed in biochemical and pharmacological studies of the H₂-receptor. These studies include the use of ³H-cimetidine in radioligand binding experiments. Confirming our previous finding as to the unsuitability of this ligand in these types of investigations, we now report data showing the lack of correlation between the displacement of specific ³H-cimetidine binding and histamine stimulated adenylate cyclase activity, and the displacement of specific binding by imidazoles devoid of H₂-receptor activity. Results are also presented which question the use of copper ions in ³H-cimetidine binding studies. Our conclusions are discussed in relation to the work carried out by a number of laboratories where ³H-cimetidine is reported to label the H₂-receptor.     

Corpus luteum susceptibility to prostaglandin F2α (PGF2α) luteolysis in hysterectomized prepuberal and mature gilts

The susceptibility of induced corpora lutea (CL) of prepuberal gilts and spontaneously formed CL of mature gilts to prostaglandin F_2α (PGF_2α) luteolysis was studied. Prepuberal gilts (120 to 130 days of age) were induced to ovulate with Pregnant Mare Serum Gonadotropin and Human Chorionic Gonadotropin (HCG). The day following HCG was designated as Day 0. Mature gilts which had displayed two or more estrous cycles of 18 to 22 days were used (onset of estrus = Day 0). Gilts were laparotomized on Day 6 to 9, their CL marked with sterile charcoal and totally hysterectomized. On Day 20, gilts were injected IM with either distilled water (DW), 2.5 mg PGF_2α or 5.0 mg PGF_2α. An additional group of prepuberal gilts was injected with 1.25 mg PGF_2α, a dose of PGF_2α equivalent, on a per kilogram body weight basis, to the 2.5 mg PGF_2α dose given to the mature gilts. The percentages of luteal regression on Day 27 to 30 for mature and prepuberal gilts given DW, 2.5 mg PGF_2α and 5.0 mg PGF_2α were 0.0 vs 4.4, 43.5 vs 96.8 and 47.7 vs 91.6, respectively; the percentage of luteal regression for the prepuberal gilts given 1.25 mg PGF_2α was 75.1. These results indicate that induced CL of the prepuberal gilt were more susceptible to PGF_2α luteolysis than spontaneously formed CL of the mature gilt and that pregnancy failure in the prepuberal gilt could be due to increased susceptibility of induced CL to the natural luteolysin.     

Vitamin K1 hydroquinone formation catalyzed by DT-diaphorase

Vitamin K₁ hydroquinone has been identified as a metabolite of vitamin K₁ biotransformation catalyzed by highly purified DT-diaphorase (NAD(P)H dehydrogenase, EC 1.6.99.2) isolated from livers of 3-methylcholanthrene induced rats. The hydroquinone was sufficiently stable to permit enzymatic reactions to be conducted under an atmosphere of air and quantitation of hydroquinone by high performance liquid chromatography. Based on kinetic data reported here, warfarin and probably dicoumarol at therapeutic levels do not appreciably affect DT-diaphorase catalyzed vitamin K hydroquinone formation.     

Reversible envelope effects during and after killing of Escherichia coli W by a highly-purified rabbit polymorphonuclear leukocyte fraction

The effects of a highly-purified, potently bactericidal fraction from rabbit polymorphonuclear leukocytes on the envelope of Escherichia coli (W) have been examined. This leukocyte fraction has equally enriched bactericidal, permeability-increasing and phospholipase A₂ activities, and is essentially devoid of lysozyme, myeloperoxidase and protease activities (Weiss, J., Franson, R.C., Beckerdite, S., Schmeidler, K. and Elsbach, P. (1975) J. Clin. Invest. 55, 33–42). Rapid killing of E. coli by this fraction is accompanied by two almost immediate alterations in the bacterial envelope: (1) a discrete increase in envelope permeability (measured by inhibition of bacterial leucine incorporation by normally impermeant actinomycin D), and, (2) hydrolysis of ¹⁴C-labeled fatty acid-prelabeled E. coli phospholipids. Both envelope effects are promptly reversed during further incubation at 37 °C, but not at 0 °C, with 40 mM Mg²⁺. Reversal is also produced by Ca²⁺ (40 mM) and trypsin (200 μg/ml), but 200 mM K⁺ causes only partial recovery and Na⁺ and hyperosmolar sucrose are ineffective. Upon addition of Mg²⁺, phospholipid degradation ceases abruptly and the labeled products of hydrolysis (free fatty acids and lysocompounds) disappear with a corresponding reaccumulation of radioactive diacylphosphatides. The time course of resynthesis of phospholipids coincides with that of restoration of the permeability barrier. Higher concentrations of the leukocyte fraction and prolonged incubation increase both the extent of phospholipid degradation and the time required for reversal of both envelope effects. These findings suggest that both the initiation of the increased permeability and its reversal are linked to respectively the breakdown and resynthesis of major E. coli membrane phospholipids, and thus depend on the fact that the biochemical apparatus of E. coli remains capable of biosynthesis despite loss of viability.Treatment of E. coli, exposed to the leukocyte fraction, with albumin results in extracellular sequestration of the products of hydrolysis and also restores the permeability barrier to actinomycin D, suggesting that the accumulation of lytic products of lipid hydrolysis within the bacterial envelope, rather than the loss of phospholipids per se, causes increased permeability.     

Continuous fluorometric assay of phospholipase A2 with pyrene-labeled lecithin as a substrate

1,2-Bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine was synthesized as a fluorogenic substrate for phospholipase A₂. It has a critical micellar concentration of 7.3 μm and gives only excimer fluorescent emission at 480 nm in aqueous micellar dispersion. When hydrolyzed by phospholipase A₂, the products give only monomer emission which is monitored best at 382 and 400 nm. Conditions were developed for an assay for phospholipase A₂ using this substrate. The assay was sensitive to as little as 8 ng of pure porcine pancreatic phospholipase A₂.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.