Tyrosine Hydroxylase in "Leaky" Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP-Dependent Systems

Abstract: The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca²⁺, the other on cyclic AMP. The Ca²⁺-dependent system is half-maximally activated by 1-2 μ M Ca²⁺ and 0.5 m M ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 m M ) does not inhibit significantly Ca²⁺-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 μ M cyclic AMP and about 0.3 m M ATP. Ca²⁺-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10⁻⁸ M Ca²⁺ plus 2 × 10⁻⁵ M cyclic AMP or of 10⁻⁵ M Ca²⁺, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca²⁺-dependent system, whereas the other site(s) are phosphorylated by both the Ca²⁺- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.     

ACCUMULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE IN BRAIN SLICES: INTERACTION OF LOCAL ANAESTHETICS AND DEPOLARIZING AGENTS

Abstract— Membrane depolarizing agents such as veratridine, ouabain and high concentrations of potassium ions elicit a remarkable accumulation of cyclic AMP in brain slices incubated in vitro , and this accumulation, but not that elicited by biogenic amines, is prevented by a membrane stabilizer, cocaine. The effect of various local anaesthetics (compounds which are known to stabilize the membrane of peripheral sensory nerves) on the accumulation of cyclic AMP elicited by depolarizing agents in incubated slices of guinea pig brain has now been examined. At optimal concentrations the anaesthetics inhibited by more than 95 per cent the accumulation of cyclic AMP elicited with veratridine, ouabain, and high concentrations of potassium ions. The order of the inhibitory potency vs. veratridine was: dibucaine (ED₅₀= 9.5 ± 10⁻⁶ M) > tetracaine > cocaine (ED₅₀= 1·3 ± 10⁻⁴ M) > lidocaine > procaine (ED₅₀= 1.7 ± 10⁻³M). This order is consistent with the order of their local anaesthetic potency, but is not consonant with the order of the relative toxicity of these agents when used as spinal anaesthetics.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

MORPHOLOGICAL CHANGES OF CULTURED MYOCARDIAL CELLS DUE TO CHANGE IN EXTRACELLULAR CALCIUM ION CONCENTRATION

Increase in the extracellular Ca²⁺ concentration from low (≤ 10⁻⁷ M) to normal (10⁻³ M) caused morphological changes of cultured myocardial cells obtained from fetal mouse heart. The extracellular Na⁺ and K⁺ concentrations of the normal medium (10⁻³ M Ca²⁺) did not significantly affect the genesis of these morphological changes. Like Ca²⁺, Ba²⁺ and Sr²⁺, but not Mg²⁺, Co²⁺ or Ni²⁺, could induce morphological changes. Increase in the extracellular Ca²⁺ concentration from 10⁻⁸ M to 10⁻³M also caused excess uptake of ⁴⁵Ca²⁺ by cultured myocardial cells. B–16CW 1 cells, which did not show these morphological changes, did not take up excess ⁴⁵Ca²⁺ on this treatment. Treatments, such as addition of verapamil or incubation at pH 6.3, which reduced the genesis of morphological changes, reduced the rate of ⁴⁵Ca²⁺ uptake by myocardial cells. These facts show that the morphological changes of myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal are due to excess uptake of Ca²⁺ by the myocardial cells.
The morphological changes of cultured myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal were reversed on further incubation of the cells in medium with or without Ca²⁺.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

SAXITOXIN TETRODOTOXIN AND THE METABOLISM AND CATION FLUXES IN ISOLATED CEREBRAL TISSUES

Abstract— Saxitoxin and tetrodotoxin at low concentrations (10⁻⁷-10⁻⁸ M) exerted similar inhibitory effects on the increase in lactate production and the redistrjbution of Na⁺ and K⁺ that normally accompany electrical stimulation of rat cerebral cortical slices. In contrast, the toxins exerted dissimilar effects on the production of lactate in response to low concentrations of Ca²⁺ in the medium. Inhibition by tetrodotoxin occurred at a higher concentration of Ca²⁺ and was significantly greater than that produced by saxitoxin at concentrations of Ca²⁺ below 0.75 mM. These differences were not related to differential effects on the redistribution of Na⁺ and K⁺ under such conditions. The toxins had different effects on Ca²⁺ influx. Tetrodotoxin, but not saxitoxin, inhibited the influx of Ca²⁺ in the absence of electrical stimulation. The influx of Ca²⁺ increased when electrical pulses were applied and tetrodotoxin inhibited this increase, whereas saxitoxin potentiated influx of Ca²⁺ during stimulation. Our results suggest that metabolic responses to conditions that increase excitability are not governed solely by changes in the distribution of Na⁺ and K⁺. The differential effects of the toxins on Ca²⁺ fluxes suggest that one site of Ca²⁺ entry during electrical stimulation may be functionally independent of Na⁺ entry.     

EFFECTS OF CADMIUM TOXICITY ON GROWTH AND ELEMENTAL COMPOSITION OF MARINE PHYTOPLANKTON

Some classes of marine phytoplankton are believed to be more tolerant of high concentrations of trace metals than others, but the results of experimental tests of this hypothesis are ambiguous. Eleven species of phytoplankton representing five classes were grown in Aquil medium containing Cd concentrations between 10⁻⁸ and 10⁻⁵ M ([Cd²⁺]= 10^−9.85 to 10^−6.84 M), and growth rates and intracellular concentrations of Cd, C, N, and S were measured. The mean Cd²⁺ concentration (pCd⁵⁰) that reduced the growth rate of each species to 50% of its maximum varied by 2.5 orders of magnitude, from 10^−6.23 for Emiliania huxleyi to 10^−8.79 for Synechococcus sp. Taxonomic trends in Cd resistance were not apparent in these data. Cadmium quotas (mol Cd·L⁻¹ cell volume) were lowest in species of Bacillariophyceae (ANOVA, P < 0.001), suggesting that they might regulate Cd transport differently than other taxa. Cellular S:C molar ratios increased in four of seven phytoplankton grown at high pCd (7.37–6.84) compared to low Cd ion concentrations (no added Cd), a result of increases in S·L⁻¹ cell volume. Nitrogen:carbon molar ratios were also higher in Cd-exposed phytoplankton, as changes in N and S were highly correlated ( r = 0.98, P < 0.0001). In two species that were examined, S:C ratios increased as a linear function of increasing Cd concentration. The results demonstrate large variability in Cd resistance among phytoplankton that is primarily a function of interspecific differences in Cd detoxification.     

Attraction of zebrafish, Brachydanio rerio, to alanine and its suppression by copper

Preference responses of zebrafish to 10⁻³, 10⁻⁴ and 10⁻⁵M alanine (Ala) were concentration- dependent. Behavioural responses to copper (Cu) and Cu + Ala mixtures were also assessed. Zebrafish avoided 100 and 10 μg Cu l⁻¹, but not 1 μg l⁻¹. Mixtures of 10⁻³ m Ala+ 100 μg Cu l⁻¹ and 10 ⁴ M Ala + 10 μg Cu 1⁻¹ were avoided as intensely as was Cu alone. Responses to 10⁻³ M Ala + 10 or 1 μg Cu l⁻¹ and 10 ⁴ M Ala +1 μg Cu l⁻¹ did not differ statistically from controls (no detectable preference or avoidance). These results demonstrate, firstly, that a concentration of a pollutant avoided by itself (10 μg Cu l⁻¹) may not be avoided when encountered with an attractant chemical stimulus (Ala) and may suppress the preference for an attractant stimulus, and secondly, that a concentration of a pollutant not avoided by itself and not considered deleterious (1 μg Cu l⁻¹) suppresses attraction to Ala (an important constituent of prey odours for many fishes).     

Effect of angiotensin II on transepithelial potential in the isolated perfused flounder (Platichthys flesus) gill

Angiotensin II (Asp¹, Val⁵) perfused through isolated flounder gills inhibited the transepithelial potential by up to 25 per cent at a concentration of 10⁻⁹M. There was no effect on gill haemodynamics and the subsequent response to 10⁻⁵ M adrenaline was normal.     

Protein Synthesis in a Cell-free System from Rat Brain Sensitive to ACTH-like Peptides

Abstract: Protein synthesis was measured using a cell-free system obtained from subcortical rat brain tissue. The concentrations of Mg²⁺ and K⁺ and the amount of tissue, during both the preparation and the final assay, were critical to the incorporation of amino acids as expressed per milligram protein. Even under optimal conditions mainly elongation of growing peptide chains was measured. Behaviorally active fragments of ACTH modulated the activity of the system in a biphasic manner; i.e., at a low concentration (10⁻⁸ M) of ACTH a stimulation of between 10 and 70% was found; a high concentration (10⁻⁴ M) was inhibitory (50 to 70%). Structure-activity studies revealed that the stimulatory effect was confined to the N-terminus of the peptide (1–24), whereas the C-terminal sequence was responsible for the inhibition. The stimulation by ACTH_1–24 was dependent on Ca²⁺ and Mg²⁺. Cyclic AMP (10⁻⁵ M) stimulated the amino acid incorporation too. When a similar cell-free extract was prepared from brain tissue of hypophysectomized rats, the lower in vivo protein synthesis in these animals was preserved in the present cell-free system. The data are discussed in terms of a possible direct intracellular effect of ACTH on brain protein synthesis.     

In vitro carbon dioxide excretion from erythrocytes of two species of Antarctic fishes and its inhibition by catecholamines

This study was designed to investigate whether the blood of Pagothenia borchgrevinki , exhibits a Haldane effect, and whether activation of a Na⁺/H⁺ antiporter increases transport of intracellular protons and Bohr protons out of the erythrocytes resulting in inhibition of CO₂ excretion in both P. borchgrevinki , and Dissostichus mawsoni. When carbon dioxide dissociation curves were determined from blood samples pooled from three fish under oxygenated and deoxygenated conditions a Haldane effect was observed. Using an in vitro , CO₂ excretion assay, the rate of HCO₃⁻ dehydration was determined on blood and plasma equilibrated under an N₂atmosphere then rapidly oxygenated with air in the presence of 10⁻⁵ M noradrenaline or acetazolamide (10⁰⁴M). Whole blood and plasma from P. borchgrevinki , and D. mawsoni , were equilibrated with 0·5% CO₂ in air and assayed in the presence of 10⁻⁵ M noradrenaline. Erythrocyte CO₂ excretion rates were depressed significantly by noradrenaline in both species. The whole blood HCO₃⁻ dehydration rate was depressed significantly following rapid oxygenation in the presence of acetazolamide indicating that the pathway of CO₂ excretion included activation of intracellular carbonic anhydrase and an adrenergic receptor.     

The rooting ability of shoots raised 'in vitro' from the apple rootstock A2 in juvenile and in adult growth phase

The apple rootstock A2 can be readily propagated in vitro both in the juvenile and in the adult growth phase. Shoots were produced by meristem tip culture from the apple rootstock A2 in different growth phases. The influence of growth phases and different concentrations of PG and IBA was investigated as to rooting percentage, survival percentage, number of roots per rooted shoot, root length, shoot length and formation of callus. IBA at 15 μ M without PG gave a significantly lower rooting percentage than 5 and 10 μ M IBA. PG together with IBA stimulated rooting, the optimum concentrations of PG being, however, not the same for the different growth phases. For the adult growth phase, 10⁻⁴ M PG promoted rooting, whereas 10⁻³ M PG markedly inhibited rooting. In the juvenile growth phases, both 10⁻⁴ and 10⁻³ M PG stimulated rooting. PG at 10⁻⁴ M also increased the number of roots. The longest roots were obtained at 10⁻³ M PG and 5 μ M IBA. PG at 10⁻³ M reduced callus formation at all IBA concentrations used. Neither shoot length nor root length influenced the survival percentage.     

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L₆ myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L₆ myoblasts was stimulated more than three fold by 5 × 10⁻⁵ M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L₆ cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L₆ cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f₂b. Therefore, the data show that differentiation in L₆ cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923

Abstract Growth of Bacillus sp. DSM 2923 on nicotinic acid in mineral medium was dependent on the concentration of sodium molybdate added. Addition of increasing amounts of tungstate to the medium resulted in an inhibition of growth on nicotinic acid or 6-hydroxynicotinic acid as sole source of carbon and energy. Chlorate-resistant mutants were isolated which were not able to degrade nicotinic acid and 6-hydroxynicotinic acid nor to reduce nitrate. Additionally, enzyme activities of nicotinic acid dehydrogenase and 6-hydroxynicotinic acid dehydrogenase increased with increasing concentrations of molybdate (10⁻⁸ to 10⁻⁶ M) added to the medium, and decreased with increasing amounts of tungstate (10⁻⁶ to 10⁻⁵ M) in the medium.     

Putative role of ethylene in Datura metel microspore embryogenesis

The role of ethylene in microspore embryogenesis was analyzed by studying the effect of cobalt ions, silver ions, methionine and Ethrel on the androgenic response of in vitro cultured anthers of Datura metel L. Cobalt and silver ions, provided in the form of cobalt chloride and silver nitrate, respectively, decreased the average number of embryos/plantlets developed per anther, at all the concentrations tested. In contrast, methionine (10⁻⁵–10⁻³ M ) and Ethrel (10⁻⁶ and 10⁻⁵ M ) stimulated embryo formation. The optimal enhancement was recorded at 10⁻⁵ M of both methionine and Ethrel.     

Stimulatory effectiveness of amino acids on the olfactory response in an algivorous marine teleost, the rabbitfish Siganus fuscescens Houttuyn

The detection of threshold concentrations and relative stimulatory effectiveness (RSE) for 19 amino acids were studied by olfactory bulbar electroencephalogram (EEG) in the algivorous rabbitfish. The threshold concentrations for 19 amino acids ranged from 10⁻¹⁰ to 10⁻⁵ M. l -Alanine was the most effective amino acid, and the threshold concentration was estimated to be from 10⁻¹⁰ to 10⁻⁹M. These results were compared with the olfactory response in herbivorous and carnivorous fishes previously reported. The comparison of RSE for rabbitfish with those for other fishes showed correlation coefficients ranging from r = 0.97 to r = 0.50, but high similarities did not correspond with difference in feeding habits for herbivores and carnivores. The role of amino acids in fish chemosensory behaviour and the olfactory response of the rabbitfish was compared with the gustatory response reported previously. It was found that l -serine was the most potent stimulant in both chemoreceptors, l -proline and l -glutaminic acid were the most potent gustatory stimulants, and l -alanine, l -glutamine, l -arginine and l -lysine were the most potent olfactory stimulants.     

Effects of phenolic acids on ammonia oxidation by terrestrial autotrophic nitrifying microorganisms

Abstract It has been hypothesized that vegetation in certain ecosystems inhibits nitrification in soil by producing phenolic compounds that inhibit oxidation of ammonia by nitrifying microorganisms. This hypothesis is based largely on a report that very low concentrations (10⁻⁶ M–10⁻⁸ M) of several phenolic acids (notably ferulic acid) completely inhibited NO₂⁻ production in an aqueous suspension of soil treated with (NH₄)₂SO₄ and a nutrient solution suitable for growth of Nitrosomonas and other autotrophic nitrifying microorganisms. To evaluate this hypothesis, we determined the effects of three ohenolic acids (ferulic acid, caffeic acid, and p -coumaric on nitrite production by representatives of three genera of terrestrial autotrophic nitrifying microorganisms ( Nitrosospira, Nitrosomonas , or Nitrosolubos ) grown on a defined medium containing NH₄⁺. We found that nitrite production by the Nitrososspira was not inhibited by ferulic acid, caffeic acid, or p -coumaric acid at concentrations of 10⁻⁶ or 10⁻⁵ M and was only slightly inhibited when these acids were at a concentration of 10⁻⁴ M. We also found that ferulic acid did not markedly inhibit nitrite production by the three genera of nitrifying microorganisms studied, even when its concentration was as high as 10⁻³ M. These observations invalidate the hypothesis tested because the phenolic acids studied did not significantly retard ammonia oxidation by autotrophic microorganisms even when their concentration in cultures of these microorganisms greatly exceeded their concentrations in soils.     

In vitro effects of zinc on lymphoid cells of the carp, Cyprinus carpio L.

Cells from the spleen, anterior kidney, middle kidney, thymus and peripheral blood of common carp were tested in culture at 22°C. Unlike mammals, zinc was not found to be a mitogen but to have a suppressive effect. This was usually detectable at very low concentrations (10⁻⁷M), although the degree of suppression varied depending on the organ investigated. Direct toxic effects of the metal were only observed at high concentrations (0·5 × 10⁻³ m or greater). The use of carp serum supplement rather than foetal calf serum was important in demonstrating these effects.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.