Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium-induced activation of tyrosine hydroxylase. Calcium (12.5–200 μ M ) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 m M ) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μ M ) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μ M ) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM ) decreased cell-free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.     

Effects of Pentobarbitone on 45Ca2+ Transport by Rat Brain Mitochondria

The effects of pentobarbitone on the transport of 45Ca2+ by rat brain mitochondria were studied, using the Ruthenium Red-EGTA quench technique. In the presence of succinate and inorganic phosphate, mitochondria rapidly accumulate 45Ca2+. Pentobarbitone (0.1-1.0 mM) stimulates the initial rate of Ca2+ transport. In contrast, pentobarbitone (1 mM) did not affect the NaCl (50 mM)-induced efflux of 45Ca2+ from mitochondria. Dibucaine (60 micro M), a clinically used local anaesthetic, inhibits both 45Ca2+ uptake an efflux. The results suggest that barbiturate stimulation of mitochondrial Ca2+ uptake may, in combination with effects on other Ca2+ sequestering processes, contribute to the inhibitor of transmitter release observed at a number of synapses.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Calcium Binding Sites of Synaptosomes from Insect Nervous System as Probed by Trivalent Terbium Ions

Terbium binding to synaptosomes from the central nervous system of the locust was studied by fluorescence spectroscopy and electron microscopy. The protein-sensitized fluorescence of terbium was used to characterize the calcium binding sites of synaptosomes. As judged by electron microscopy and x-ray analysis, terbium ions produced electron-dense patches in regular arrays on the outer surface of synaptosomal membranes and induced marked aggregation of synaptic vesicles in isolated terminals.     

Studies on the Interaction of Ca2+ Ions with Some Fractions of the Neurospecific S-100 Protein

Abstract: Fractions of neurospecific S-100 protein were purified from bovine brain and their physicochemical properties were studied. Conformational changes caused by the binding of calcium to S-100 protein fractions were detected by means of differential and fluorescence spectroscopy. Fractions demonstrating opposite shifts of their spectra also differ in the distribution in double-phase system. The number of calcium-binding centers and their association constants were determined by means of equilibrium dialysis and gel filtration. The nature of the differences in the interaction of various S-100 protein fractions with calcium is discussed.     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

From compartmental analysis of radioisotope elutin measurements, fluxes of Ca²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to complete nutrient solutions containing a range of calcium concentrations ([Ca₀]) from 2 eq l^-1 to 20 meq l^-1, increasing in 10-fold steps for Ca²⁺. Except for the calcium counter-ion (usually NO ₃ ^- , sometimes Cl^- at the highest [Ca₀]), the composition of the nutrient solution was other-wise the same at all calcium concentrations. Compartmental analysis indicated that the cytoplasm had a high content of exchangeable Ca²⁺ but, in the light of evidence from animal studies, ionic activity of calcium in the cytoplasm was assumed to be no greater than 0.002 eq ml^-1. With the Ussing-Teorell flux equation as the criterion, it was concluded that at all values of [Ca₀] tested, Ca²⁺ entered the cytoplasm passively and was actively pumped back into the external solution. Entry of calcium to the vacuole from the cytoplasm was active in all cases. The conclusions regarding the character of ion transport across the plasmalemma were the same as when the whole calcium content of the cytoplasm was taken to contribute to the ionic activity. However, the electrochemical activity gradient was very much steeper than formerly estimated. Calcium was transported to the stele in proportion to the calcium content of the cytoplasm and moved in the xylem almost exclusively in the basipetal direction.     

Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Effects of acid irrigation and liming on two clones of Norway spruce

Summary The effect of acid irrigation on the growth of rooted cuttings ofPicea abies (L.) Karst, was investigated in a pot experiment lasting 3 years. It involved two clones of Norway spruce, H 253 Bogstad I and H 254 Bogstad II. Irrigation water of pH 5.4, 4.0, 3.0 and 2.5 was used. Liming was included in the experiment.After the experimental period, the plants of all treatments were growing reasonably well. However, those plants irrigated at pH 2.5 were slightly discoloured. The plant mortality was only 3% throughout the experiment, and was not connected to acid irrigation. The limiting growth factor was N. All other nutrient elements measured in the plants were close to optimal concentration. Plants irrigated at pH 2.5, and to some extent at pH 3, contained excessively high concentrations of Al, t-S and SO₄. The total amount of Ca, Fe and Mn taken up by the plants decreased with increasing soil acidity. The increased growth of clone H 254 relative to H 253, produces a corresponding impression on soil characteristics. Soil acidity is governed by acid irrigation and CaCO₃ application, but the clonal effects are also of importance. Norway spruce appears to be tolerant to Al concentrations as high as 50 mmol/kg in the needles.     

Existence and stability of periodic travelling wave solutions to Nagumo's nerve equation

Summary Nagumo's nerve conduction equation has a one-parameter family of spatially periodic travelling wave solutions. First, we prove the existence of these solutions by using a topological method. (There are some exceptional cases in which this method cannot be applied in showing the existence.) A periodic travelling wave solution corresponds to a closed orbit of a third-order dynamical system. The Poincaré index of the closed orbit is determined as a direct consequence of the proof of the existence. Second, we prove that the periodic travelling wave solution is unstable if the Poincaré index of the corresponding closed orbit is + 1. By using this result, together with the result of the author's previous paper, it is concluded that the slow periodic travelling wave solutions are always unstable. Third, we consider the stability of the fast periodic travelling wave solutions. We denote by L(c) the spatial period of the travelling wave solution with the propagation speed c. It is shown that the fast solution is unstable if its period is close to L_min, the minimum of L(c).