α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Enhancement of extractable ethylene at light/dark transition in primary leaves of paclobutrazol-treated Phaseolus vulgaris seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol], a triazole growth retardant, increased the 1-aminocyclopropane-1-carboxylic acid (ACC) level and resulted in reduced ethylene production, estimated as ethylene release in a closed system or by vacuum-extraction, in the primary leaves of Phaseolus vulgaris L. cv. Juliska seedlings exposed to light. At the light/dark transition, a definite enhancement of the endogenous ethylene level was observed by vacuum-extraction of primary leaves of treated plants and the ethylene deficiency of retardant-treated leaves ceased. The concentration of ACC after the light/dark transition followed the pattern for ethylene, and the increase in ACC content was paralleled by a decrease in malonyl-ACC.
It is concluded that the internal level of ethylene is not necessarily lower in the primary leaves of paclobutrazol-treated bean plants, but under special environmental conditions in vivo it may reach that of the control.     

The structural features of effective antagonists of the luteinizing hormone releasing hormone

Summary The structure-activity data of 6 years on 395 analogs of the luteinizing hormone releasing hormone (LHRH) have been studied to determine effective substituents for the ten positions for maximal antiovulatory activity and minimal histamine release. The numbers of substituents studied in the ten positions are as follows: (41)¹-(12)²-(12)³-(5)⁴-(47)⁵-(52)⁶-(16)⁷-(18)⁸-(4)⁹-(8)¹⁰. In position 1, DNal and DQal were effective with the former being more frequently the better substituent. DpClPhe was uniquely effective in position 2. Positions 3 and 4 are very sensitive to change. D3Pal in position 3 and Ser in position 4 of LHRH were in the best antagonists. PicLys and cPzACAla were the most successful residues in position 5 with cPzACAla being the better substituent. Position 6 was the most flexible and many substituents were effective; particularly DPicLys. Leu⁷ was most often present in the best antagonists. In position 8, Arg was effective for both antiovulatory activity and histamine release; ILys was effective for potency and lesser histamine release. Pro⁹ of LHRH was retained. DAlaNH₂ ¹⁰ was in the best antagonists.Abbreviations AABLys N -(4-acetylaminobenzoyl)lysine - AALys N -anisinoyl-lysine - AAPhe 3-(4-acetylaminophenyl)lysine - Abu 2-aminobutyric acid - ACLys N -(6-aminocaproyl)lysine - ACyh 1-aminocyclohexanecarboxylic acid - ACyp 1-aminocyclopentanecarboxylic acid - Aile alloisoleucine - AnGlu 4-(4-methoxy-phenylcarbamoyl)-2-aminobutyric acid - 2ANic 2-aminonicotinic acid - 6ANic 6-aminonicotinic acid - APic 6-aminopicolinic acid - APh 4-aminobenzoic acid - APhe 4-aminophynylalanine - APz 3-amino-2-pyrazinecarboxylic acid - Aze azetidine-2-carboxylic acid - Bim 5-benzimidazolecarboxylic acid - BzLys N -benzoyllysine - Cit citrulline - Cl₂Phe 3-(3,4-dichlorphenyl)alanine - cPzACAla cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alnine - cPmACAla cis-3-[4-(4-pyrimidylcarbonyl)aminocyclohexyl]alanine - Dbf 3-(2-dibenzofuranyl)alanine - DMGLys N -(N,N-dimethylglycyl)lysine - Dpo N -(4,6-dimethyl-2-pyrimidyl)-ornithine - F₂Ala 3,3-difluoroalanine - hNal 4-(2-naphthyl)-2-aminobutyric acid - HOBLys N -(4-hydroxybenzoyl)lysine - hpClPhe 4-(4-chlorophenyl)-2-amino-butyric acid - Hse homoserine, 2-amino-4-hydroxybutanoic acid - ICapLys N -(6-isopropylaminocaproyl)lysine - ILys N -isopropyllysine - Ind indoline-2-carboxylic acid - INicLys N -isonicotinoyllysine - IOrn N -isopropylornithine - Me₃Arg N^G,N^G,N^G-trimethylarginine - Me₂Lys N ,N -dimethyllysine - MNal 3-[(6-methyl)-2-naphtyl]alanine - MNicLys N -(6-methylpicolinoyl)lysine - MPicLys N -(6-methylpicolinoyl)lysine - MOB 4-methoxybenzoyl - MpClPhe N-methyl-3-(4-chlorphenyl)lysine - MPZGlu glutamic acid,-4-methylpiperazine - Nal 3-(2-naphthyl)alanine - Nap 2-naphthoic acid - NicLys N -nicotinoyllysine - NO₂B 4-nitrobenzoyl - NO₂Phe 3-(4-nitrophenyl)alanine - oClPhe 3-(2-chlorphenyl)alanine - Opt O-phenyl-tyrosine - Pal 3-(3-pyridyl)alanine - 2Pal 3-(2-pyridyl)alanine - 2PALys N -(3-pyridylacetyl)lysine - pCapLys N -(6-picolinoylaminocaproyl)lysine - pClPhe 3-(4-chlorophenyl)alanine - pFPhe 3-(4-fluorophenyl)-alanine - Pic picolinic acid - PicLys N -picolinoyllysine - Pip piperidine-2-car-boxylic acid - PmcLys N -(4-pyrimidylcarbonyl)lysine - Ptf 3-(4-trifluromethyl phenyl)alanine - Pz pyrazinecarboxylic acid - PzAla 3-pyrazinylalanine - PzAPhe 3-(4-pyrazinylcarbonylaminophenyl)alanine - Qal 3-(3-quinolyl)alanine - Qnd-Lys N -quinaldoyllysine - Qui 3-quinolinecarboxylic acid - Qux 2-quinoxalinecarboxylic acid - Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid - TinGly 2-thienylglycine - tNACAla trans-3-(4-nicotinoylaminocyclohexyl)-alanine - tPACAla trans-3-(4-picolinoylaminocyclohexyl)alanine     

Effect of arachidonic acid on [3H]d-aspartate outflow in the rat hippocampus

The aim of this study was to investigate the effect of arachidonic acid on [³H]d-aspartate outflow in rat hippocampus synaptosomes and slices. Arachidonic acid 1) increased basal outflow of [³H]d-aspartate in both synaptosomes and slices, and 2) increased K⁺-evoked overflow in slices but not in synaptosomes. The latter effect was dependent (at least in part) on arachidonic acid metabolism, most likely mediated by lipo-oxygenase metabolites and free radical production. It was prevented by nordihydroguaiaretic acid but not by indomethacin, and was significantly reduced by free radical scavengers (superoxide-desmutase and catalase). This effect was dependent upon stimulation since it could not be observed after a continuous perfusion of arachidonic acid in the absence of stimulation. Furthermore, it was long-lasting since a 30 min perfusion of arachidonic acid was sufficient to exert a significant effect on a stimulation following termination of the application.     

Taurine in the developing cat: Uptake and release in different brain areas

Taurine is an important modulator of neuronal activity in the immature brain. In kittens, taurine deficiency causes serious dysfunction in the cerebellar and cerebral visual cortex. The processes of taurine transport in vitro were now studied for the first time in different brain areas in developing and adult cats. The uptake of taurine consisted initially of two saturable components, high- and low-affinity, in synaptosomal preparations from the developing cerebral cortex and cerebellum, but the high-affinity uptake component completely disappeared during maturation. The release of both endogenous and preloaded labeled taurine from brain slices measured in a superfusion system was severalfold stimulated with a slow onset by depolarizing K⁺ (50 mM) concentrations. K⁺ stimulation released markedly more taurine from the cerebral cortex, cerebellum and brain stem in kittens than in adult cats. The responses were largest in the cerebellum. Both uptake and release of taurine are thus highly efficient in the brain of kittens and may be of significance in view of the vulnerability of cats to taurine deficiency.     

In vitro TRH release from hypothalamus slices varies during the diurnal cycle

We have previously described a daily rhythm in thyrotropin releasing hormone (TRH) and TRH mRNA in the rat hypothalamus. To determine whether TRH release fluctuates in a diurnal manner, we have measured basal and potassium stimulated release from hypothalamic slices, and compared it to release from olfactory bulb slices, during the diurnal cycle. Basal TRH release was higher at 7:00 h than at any other time (1:00, 13:00 or 19:00 h) in either hypothalamus or olfactory bulb. The ratio of stimulated over basal release was higher in the hypothalamus at 19:00 h, when TRH content was highest. Potassium stimulated TRH release from olfactory bulb was not different from basal release at any time. TRH release fluctuations were not due to a rhythm of extracellular inactivation: the activity of pyroglutamyl aminopeptidase II, an ectoenzyme responsible for TRH inactivation, was constant throughout the cycle. Our data demonstrate that diurnal variations of TRH release occur in vitro and that the enhanced responsiveness to potassium stimulation in hypothalamus is correlated with increased levels of peptide.     

Changes in dormancy of Sisymbrium officinaleseeds do not depend on changes in respiratory activity

Effects of dark incubation at different temperatures were studied on dormancy and respiratory activity of seeds of Sisymbrium officinale (L.) Scop. Because germination of this species absolutely depends on the simultaneous action of light and nitrate, changes in dormancy could be studied in darkness without the interference of early germination events. Upon the start of incubation rates of O₂ uptake and CO₂ release rose. This was followed by a gradual decrease until stable levels of O₂ uptake and CO₂ release were achieved. Seeds kept for prolonged periods at 24^°C, showed neither a change in germination capacity nor in rates of O₂ uptake and CO₂ release. Respiratory quotients were 0.55–0.7. The initial rise in O₂ uptake correlated with the rate of water uptake and with breaking of primary dormancy. However, the subsequent decline in O₂ uptake was not generally linked to induction of secondary dormancy. An increased O₂ uptake was not required during breaking of secondary dormancy. It is concluded that changes in dormancy are not generally related to changes in respiratory activity. However, germination strongly depends on respiration. The increase in O₂ uptake started well before radicle protrusion. A far red irradiation only reversed this increase when it was given before germination escaped from its red light antagonising action. The contribution of different respiratory pathways was followed during prolonged incubation at 24^°C in darkness. KCN at 1.5 mM was needed to inhibit the cytochrome pathway (CP) and benzohydroxamic acid (BHAM) at 30 mM to inhibit the alternative pathway (AP). These concentrations did not exert any side effects. Electron flow was predominantly via the CP, maximally 10% was via the AP. Flow through the CP declined during the first 6 days and residual respiration remained constant. Therefore, the contribution of residual respiration became relatively more important with prolonged incubation. KCN at concentrations that almost completely inhibited flow through the CP, did not dramatically reduce germination. BHAM already inhibited germination at concentrations that do not inhibit oxygen uptake.     

Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture and after axotomy: The role of cholinergic differentiation factor/leukemia inhibitory factor

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum Against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy. 1994 John Wiley & Sons, Inc.     

Life history and reproductive potential of the agarophyte Gelidium robustum in California

The reproductive potential of the tetrasporangial phase of Gelidium robustum was studied for 16 months at two sites off Santa Barbara, California. In all samples tetrasporangial thalli were always more abundant than gametangial ones. Tetratrasporangial sori were present throughout the duration of the study but relative fecundity was highest [300–400 sori g^–1 (w. wt)] in spring/summer samples of consecutive years, as a result of increasing numbers both of tetrasporangial branchlets per plant and of sori per branchlet. On the other hand, laboratory experiments showed that tetraspore release per sorus was highest (150–250 spores sorus^–1 d^–1) in winter. Inferring from these field and laboratory data plants released up to ± 34 000 tetraspores g^–1 (w. wt) d^–1 in the spring/summer of the second study year. Tetraspore germination, under defined culture conditions, also showed a marked seasonality increasing sharply from less than 10% in winter up to almost 60% in spring/summer, thus coinciding with the period of maximal spore output per plant. These results suggest that although relatively high numbers of tetraspores may be released by G. robustum plants all year round these might not always have the potential to germinate and recruit.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.