Nonspecific cytotoxic effects of antigen-transformed lymphocytes: II. Inhibition by drugs

High concentrations of prednisolone (10^?5M) failed to inhibit the nonspecific cytotoxic effects of human lymphocytes that had already transformed in response to PPD. In contrast, prednisolone added at the beginning of lymphocyte culture caused a significant inhibition of subsequent cytotoxicity at concentrations as low as 10^?8M. A single concentration of prednisolone (10^?6M) caused progressively less inhibition the later it was added in the lymphocyte culture period, and it is suggested that there is a steroid-sensitive phase in the early stages of development of nonspecific cytotoxicity after stimulation of lymphocytes with antigen. This steroid-sensitive phase could not be attributed to a difference in lysosome activity, since chloroquine caused the same degree of inhibition at the beginning as at the end of culture. In addition, studies with cycloheximide, actinomycin D, and mitomycin C indicated that cytotoxicity by transformed lymphocytes depended on protein synthesis but not on short-term RNA or DNA synthesis.     

Preliminary measurements of intracellular calcium in an insect salivary gland using a calcium-sensitive microelectrode

A calcium-sensitive microeletrode was used to measure free intracellular calcium in salivary gland cells of Calliphora during stimulation with 5-hydroxytryptamine (5-HT). The resting level of calcium was approximately 10^?7M or less but increased in a dose-dependent way sometimes to levels in excess of 10^?6M. The onset of the calcium signal was closely related to changes in membrane and transepithelial potential. This calcium response was greatly reduced when the extracellular calcium concentration was reduced from 10^?3 to 10^?4M. This dependence on external calcium is consistent with previous observations that 5-HT acts to increase the permeability of the basal plasma membrane to calcium. These observations indicate that an increase in the intracellular level of calcium is an early event associated with the onset of fluid secretion in this insect salivary gland.     

Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10^?3M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10^?3M or 10^?4M), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2^k) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2^d) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.     

Effects of Organic Nutrients and Hormones on Growth and Development of Tissue Explants from Coconut (Cocos nucifera) and Date (Phoenix dactylifera) Palms Cultured in vitro

The growth response (increase in weight) of cultured explants from seedling date (Phoenix dactylifera L.) and mature coconut (Cocos nucifera L. cv. Malayan Dwarf) palms to source and concentration of organic nitrogen. carbohydrate, auxins, cytokinins and gibberellins was examined. Growth was strongly stimulated by the presence of auxins (10^?7 to 10^?6M), cytokinins (10^?6 to 10^?5M), high concentrations of sucrose (0.2 M), and in the absence of NH₄Cl, by organic sources of reduced nitrogen. Higher concentrations of auxin (2,4-D or NAA at 10^?6 to 10^?5M) which still stimulated growth of Phoenix tissue, proved inhibitory to growth of freshly excised Cocos tissues. Explants from both palms initiated roots when subcultured on a medium with increased levels of auxin (NAA, 2.5 × 10^?6 to 2.5 × 10^?5M) and reduced levels of cytokinin (6-BAP, 5 × 10^?8M). Isolated roots excised from these explants continued growth and produced new laterals when subcultured on media with GA₃ (5 × 10^?7M) and reduced levels of auxin, cytokinin, and either minerals or sucrose.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

Activity of Various Growth Substances in the Avena First Internode Test

The elongation growth of the Avena first internode segments was studied in the presence of one or several of the following growth substances: indoleacetic acid (IAA), 6-fur-furylamino purine (FAP, kinetin), 6-benzylamino purine (BAP), gibberellin A₃ (GA₃) and A₄₊₇ (GA₄₊₇), and abscisic acid (ABA). The cytokinins at concentrations of 10^?7 to 10^?6M stimulated growth with 4 to 6 per cent but this effect was not statistically significant. Concentrations higher than 5 × 10^?6M inhibited growth. FAP and BAP (from 10^?8M to 10^?6M) had no significant interaction with any other growth substance used. The two-factor interactions of IAA × ABA, IAA × GA₃, and GA₃× ABA, as well as the three-factor interaction IAA × ABA × GA₃ were significant. However, the IAA × ABA interaction was significant only when high concentration (10^?6M) of ABA was used. The growth inhibition produced by 10^?7 and 10^?6M ABA was overcome by about equimolar concentrations of IAA. The stimulation of growth by GA₃ and GA₄₊₇ (10^?9 to 10^?7M) was prevented by simultaneous application of ABA, and it was reduced significantly by application of IAA (10^?7 to 10^?8M). GA3 at 10^?8M combined with different concentrations of IAA gave slightly higher elongation than IAA alone but the observed values were significantly lower than expected assuming independent additive action.     

Histamine-induced suppressor factor (HSF): further studies on the nature of the stimulus and the cell which produces it.

Guinea pig lymphocytes are stimulated by histamine to produce a soluble factor with immunosuppressive properties. This factor, termed histamine-induced suppressor factor or HSF, abrogates the production of migration inhibitory factor (MIF) and proliferative response to specific antigen. In the present study we have determined the lymphocyte subpopulation which elaborates HSF, the lymphoid tissue source, the kinetics of its generation in relation to immunization, and the nature of the histamine receptor involved in modification of the release of HSF. HSF activity could be detected in populations of cells from spleen and lymph nodes prior to active immunization of the donor, but not in cells from the donor's blood or thymus. Following immunization with ortho-chloro benzoyl-bovine γ-globulin in complete Freund's adjuvant (CFA), more HSF activity was detected in cells from the donor's spleen and lymph nodes. The peak response was seen 2 weeks postimmunization when significant amounts of HSF also were made by cells from the blood and thymus. Concentrations of T-cell-enriched and B-cell-enriched populations were tested for their ability to make HSF. We found that T-cell-enriched, but not B-cell-enriched populations, made significant amounts of HSF. Cells from the lymph nodes of immunized donors were chromatographed over affinity columns made of insolubilized conjugates of histamine with albumin. The nonretained cells were unable to generate HSF, whereas HSF activity was detected in the cells that were retained by the columns. This finding strongly suggests that the HSF-producing cells have receptors for histamine. Cells from CFA-immune lymph nodes were incubated with H₁ (2-methyl histamine) and H₂ (4-methyl histamine) agonists to determine their relative potency and, therefore, the nature of the histamine receptors on these cells that were modifying HSF release. Although both agonists could induce generation of HSF when high concentrations (10^?3M) were used, only the H₂ agonist stimulated production or release of HSF at lower concentrations (10^?5M). These HSF-producing cells appear to be selectively sensitive to H₂ agonists and likely have a predominance of H₂ receptors. Allergic mediators other than histamine were studied to determine their ability to allow elaboration of HSF-like activity from CFA-immune lymph node cells. Serotonin (10^?3M), slow-reacting substance of anaphylaxis (100 units/ml), eosinophil chemotactic factor (tetrapeptide; 10^?5M), and prostaglandin E₁ (10^?4M) were unable to induce HSF-like activity in lymph node cells from donors immunized with CFA. Furthermore, other agents which raise intracellular levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) such as isoproterenol and cholera toxin, as well as the dibutyryl form of cyclic AMP itself, were also unable to generate HSF-like activity. Thus, histamine is unique among the allergic mediators in stimulating elaboration of the suppressive substance. These findings also suggest that the ability of histamine to stimulate HSF may not reside in the conventional pathway linked to cAMP accumulation, but rather to an as yet undefined pathway of cell activation. A model is presented which further implicates histamine as a modulator of cellular immune reactions.     

Electrical current and cAMP induce lateral branching in the stolon of hydroids

Pulses of cAMP injected ionophoretically or mechanically into the epidermis of the stolon of the hydroid Hydractinia induce lateral branching at the site of stimulation. Up to 72% of the punctured loci developed a bud 6–24 hr after stimulation. Only pulsatile injection in periods of, e.g., 5 min is effective in inducing lateral buds. Controls provided evidence that in the ionophoretic mode the inducing effect derives not only from the cAMP signal but also, in part, from the positive electric current passed through the micropipet during the retention interval: DC (e.g., 8 nA × 1 hr or 20 nA × 2 hr) entering the tissue also evokes a positive response. Additional pulses of cAMP, but not of AMP, enhance the current effect. The threshold dose for a significant amplification has been determined to be 3.6 × 10^?13M (18 pulses à 2 × 10^?14M).     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Inhibitory effects of dibutyryl cyclic AMP and theophylline on the melanosome transformation in the embryonic chick pigmented retina cultured in vitro.

When the retinal pigment epithelial cells of chick embryo are cultured in monolayer conditions, the pigment granules are lost from the cytoplasm. The first structural change in depigmentation is the transformation of pigment granules into the degradative organelles designated as the dense body and melanosome complex. The cells are grown in medium containing DBcAMP of various doses from 10^?5 to 10^?2M. Cell proliferation is retarded by treatment with DBcAMP (10^?3M). The transformation of pigment granules is almost completely prevented in all 1-day cultured cells. In 5-day cultured cells continuously treated with more than 10^?4M, the transformation is not only prevented, but the synthesis of pigment granules is stimulated. A similar result is obtained by the administration of 10^?3M theophylline. 5′-AMP does not prevent the transformation of pigment granules but seems to stimulate the synthesis of pigment granules. On the other hand, cGMP is ineffective both on prevention of transformation and on synthesis of pigment granules. The mechanisms of the transformation of pigment granules are discussed.     

Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiatio from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22°–24°C when the injury was located 800 μm from the soma. Elevating the temperature to 32°C or moving the lesion site to 400 or 1500 μm from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 × 10^?5 M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10^?5 M) did. The calcium channel blocker lanthanum (10^?6–10^?4 M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10^?3–10^?5 M), and the calcium ionophore A23187 (10^?5 M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH₄Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10^?5 M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10^?5 M) had no effect. Inhibition of protein synthesis with anisomycin (2 × 10^?4 to 2 × 10^?6 M) did not significantly suppress sprouting 24 h after axotomy. Both d and l isomers of glutamate (300 μM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.     

INFLUENCES OF COLCHICINE, VINBLASTINE AND CYTOCHALASIN ON THE RELEASE OF 5-HYDROXYTRYPTAMINE FROM RAT BRAIN SYNAPTOSOMES

Rat brain synaptosomes preincubated with [³H]5-HT. were further incubated and the release of [³H]5-HT from the preparation was studied. The spontaneous release consisted of an initial rapid phase followed by slower release. Incubation with 60 mM-KCl increased the release while 60 mw-NaCl did not affect it. The effect of KG was abolished when NaCl was omitted from the medium. The potassium-induced release was Ca²⁺ -dependent while that induced by tyramine (10^?5-10^?4M) and the spontaneous release did not depend on Ca²⁺. Vinblastine (10^?5–2.5 X 10^?4 M) caused an increase in the spontaneous release and an decrease in the potassium-induced release, while it completely inhibited the release by tyramine at 2.5 X 10^?4 M. Colchicine (5 X 10^?5–10^?3M) and cytochalasin D (10^?5, 10^?4 M) failed to produce any change in the release. Cytochalasin B (10^?5, 10^?4M) increased the spontaneous release and decreased the potassium-induced release but it did not affect the release by tyramine. Colchicine (10^?3 M). vinblastine (10^?4 M) and cytochalasin B (10^?4 M) did not affect significantly Na⁺.K⁺-. Mg²- and Ca²⁺ -ATPase activities. These results suggest that none of microtubules. microfilaments and contractile protein participates in the mechanism of [³H]5-HT release from synaptosomes, in vitro.     

Electrophysiological studies of fetal mouse olfactory bulb explants during development of synaptic functions in culture

Explants of 18- or 19-day fetal mouse olfactory bulb have been maintained in culture for periods up to 5 weeks. Compound action potentials can be evoked in the bulb explants by 1 day in vitro, and by 3–4 days, synaptically mediated slow wave discharges can be demonstrated in bicuculline (10^?5 M). The capability of the bulb explants to generate these slow-wave discharges has also been revealed by the introduction of picrotoxin (10^?5 M), d-tubocurarine (10^?4 M) and chloride-free medium, but not of strychnine (up to 3 × 10^?5 M). The data indicate early functional development of inhibitory, as well as excitatory, synaptic systems. In addition, the selective and reversible depression of these slow wave potentials by GABA (1–5 × 10^?4 M), but not by glycine (up to 3 × 10^?3 M), indicates a GABA-ergic component in the inhibitory network. Single unit extracellular recordings have been obtained from the presumptive mitral cells which, in culture, are spontaneously active even as early as 1–2 days after explantation. Correlative Bodian silver-impregnations demonstrate the presence of neurons in these explants which resemble typical mitral cells. Studies of mitral cells using paired stimuli suggest the development in vitro of an inhibitory system analogous to that known to suppress the excitability of their in situ counterparts following orthodromic or antidromic activation. These data, as well as the pharmacological sensitivities of the mitral cells in culture to GABA (5 × 10^?4 M) and bicuculline (10^?5 M), indicate that granule-to-mitral synapses may develop characteristic functions in olfactory bulb explants.     

Control of luminescence in phengodid beetles

Luminescence control in four rare species of phengodid beetles was investigated. Behavioural evidence suggests that Phrixothrix, Stenophrixothrix, and Diplocladon light organs are neurally controlled whereas those of Phengodes are not. Light organs on isolated body segments of Phrixothrix and Stenophrixothrix responded to electrical stimulation by luminescing. These two species exhibited a post-anoxic pseudoflash response similar to that of lampyrid beetles whose light organs are neurally controlled. Application of noradrenaline (5 × 10^?5 M) or amphetamine (5 × 10^?3 M) to Phrixothrix light organs produced luminescence. Brief electrical stimulation during eserine treatment (5 × 10^?4 M) produced fluctuating, long-lasting luminescence, but only if the segmental ganglia were left intact. The light organs of Phengodes did not respond significantly to drugs or to electrical stimulation, indicating again that this genus does not have neural control of light organs.     

Free calcium changes associated with hormone action in amphibian oocytes

Ca²⁺-sensitive electrodes and the photoproteins obelin and aequorin were used with the oocytes of the anuran Xenopus laevis and the urodeles Ambystoma mexicanum and Pleurodeles waltlii in order to detect any changes in internal free Ca²⁺ which might result from progesterone or agonist stimulation. A dramatic Ca²⁺ surge was recorded: from 0.7 × 10^?6M in the unstimulated oocyte to 7 × 10^?6M after stimulation but before germinal vesicle breakdown (GVBD). Ca²⁺ efflux was also measured, but it accounted for less than 0.2% of the internal Ca²⁺ transient; this efflux did not take place in the absence of external calcium. The Ca²⁺ surge and maturation in response to progesterone, p-hydroxymethylenesulfonate (PHMPS), or Mn²⁺ occurred normally even when divalent cations were absent from the external medium. In contrast, external divalent cations were necessary for the induction of meiosis and a Ca²⁺ transient by the K⁺ ionophore valinomycin. HCO₃^? also triggers meiosis and causes Ca²⁺ release, but the release occurs with quite different kinetics. Incompletely grown or seasonally dormant oocytes as well as 10 mM theophilline- or procaine-treated oocytes neither release Ca²⁺ nor respond to the hormone. We conclude that intracellular released Ca²⁺ is likely to be the major “second messenger” following hormone stimulation in amphibian oocytes as in starfish.     

Development of an immunological response and changes in the activity of an ectogalactosyltransferase

Ectoglycosyltransferases have been postulated to play a role in intercellular recognition and association. Since considerable interaction among immunocompetent cells is required for the generation of the immune response, it was of interest to examine the involvement of these enzymes in this reaction. Thymic and splenic lymphocytes prepared from normal mice were capable of transferring galactose from uridine diphosphogalactose to endogenous acceptors. The transfer required the presence of Mn²⁺; the optimal concentration was 0.04 M. The K_M values for UDP-galactose were calculated to be 10^?5M for thymic lymphocytes and 0.7 × 10^?5M for splenic lymphocytes. After immunizing the mice with DNP₃-ovalbumin, thymic lymphocytes, but not splenic lymphocytes, showed a significant increase in their ability to catalyze the transfer of galactose as compared with those from nonimmunized mice. Whether the increase in activity can be related to some intercellular reaction of the immune response remains to be determined.     

Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA: II. Formation of multiple promoter-like complexes at non-promoter sites

The interactions between Escherichia coli RNA polymerase holoenzyme and a 3800 base-pair restriction fragment of bacteriophage T7 DNA (Mbo-IC) have been examined by electron microscopy. In addition to exhibiting weak, non-specific interactions (K_a ～ 10⁴ M^?1), RNA polymerase is able to form up to 15 to 20 relatively stable complexes with this template (K_a est 10⁹ M^?1). Only one of these complexes is formed at the T7 promoter E, that maps at 92.2 ± 1% on the conventionalgenome. The remaining complexes seem to be situated non-randomly on this fragment and possibly involve interactions with specific DNA sequences. The association kinetics of formation have been examined and give rise to a second-order rate constant of ～ 10⁵ M^?1s^?1. Formation of these complexes is markedly reduced at low temperatures. Under standard binding conditions (50 mM-NaCl) the dissociation rate of these complexes is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; ~\; 30\; </mtext><mtext>min</mtext>}}$), but increases rapidly with increasing salt concentration and at reduced temperatures. It is unaffected by the presence of heparin up to 5 μg/ml. Thus it appears that E. coli RNA polymerase can form complexes with promoter-like properties at many different sites on T7 DNA.     

An inhibitory effect of arachidonic acid on subsequent responses of canine cerebral arteries to serotonin and other agonists

Arachidonic acid (AA) at 10^?4M and 10^?3M produced a phasic contraction in isolated canine basilar arteries that peaked rapidly and then slowly declined. This contraction was evidently due to the conversion of AA to prostanoids because it was blocked by cyclooxygenase inhibitors and because 11, 14, 17 eicosatrienoic acid (10^?3M), which is not a cyclooxygenase substrate, failed to produce a contraction. When the artery was exposed to 10^?3M AA for 20 min and washed, subsequent contractile responses to 10^?6M serotonin (5-HT) were only 10% of control. Contractions produced by prostaglandin E₂ (10^?5M), uridine triphosphate (10^?4M) and potassium (5.5×10^?4M) were inhibited to a lesser degree than 5-HT, the response to potassium being the least affected (66% of control). This damaging effect of 10^?3M AA did not occur if the artery was washed at peak contraction nor with 10^?4M AA. Autooxidation products were evidently not responsible for the damage because prior oxygenation (90 min) of 10^?4M AA had no such effect. Pretreatment with superoxide dismutase or ascorbate did not prevent the inhibition, suggesting that free radical reactions were not involved. Pretreatment with indomethacin (3×10^?4M) or meclofenamate (10^?4M) also failed to prevent the inhibitory phenomenon. Saponin, a detergent, produced similar inhibitory effects but 11, 14, 17 eicosatrienoic acid or oleate (10^?3M) did not. The arteries partially recovered from the inhibition with time. In conclusion, AA produced contraction in basilar arteries by inducing prostaglandin synthesis but can produce secondarily by an unidentified mechanism an inhibition of the contractile responses evoked by various agonists that is both time and concentration dependent.     

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes,coenzyme analogues,substrates, and inhibitors

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M^?1 cm^?1, 407 nm (3500 M^?1 cm^?1), 505 nm (300 M^?1 cm^?1), 570 nm (?130 M^?1 cm^?1), and 680 nm (?80 M^?1 cm^?1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry.The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH > 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal ion which acts as a Lewis acid in both substrate coordination and catalysis.     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-ahphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine. and glutathione have been investigated, with the goal of understanding the relationship between RS^?-Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto ammo acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]_o), pH, and the structure of the redox partners. With excess cysteine present at 25°C, pH 5 0, I = 0 2 M (NaOAc), decay of 1:1 cys-S^?-Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)²⁺ (6 1 × 10³M^?1sec^?1), Cu(Me₅dien)²⁺ (2.7 × 10³M^?1 sec^?1), Cu(en)₂²⁺ (2.1 × 10³M^?1 sec^?1), and Cu(dien)₂²⁺ (4.7 × 10³ M^?1 sec ^?1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results Moderate reactivity decreases in the cysteine-Cu(dien)²⁺, Cu(Me₅dien)²⁺ reactions with increasing [H⁺] (pH 4–6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS^?-Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)²⁺ and Cu(Me₅dien)²⁺. This behavior suggests the mtermediacy of di-μ-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)²⁺ and Cu(Me₅dien)²⁺ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me₆tren)²⁺ (k(25°C) 7.5 × 10⁷ M^?2 sec^?1, I = 0.2 M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR^?-forming pathway. Formation of a coordinated disulfide in the rate-determining step is purposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me₆(tren)Cu(I) product.