Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2

Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV _max while the apparentK _m was slightly increased. In the presence of 4mm CaCl₂ inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl₂ polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl₂ reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Biology of Subanguina picridis,a Potential Biological Control Agent of Russian Knapweed

The knapweed nematode, Subanguina picridis, forms galls on the leaves, stems, and root collar of Russian knapweed, Acroptilon repens. After being revived from a dormant, cryptobiotic state, second-stage juveniles required at least 1 month in a free-living state before becoming infective. Galls were induced on relatively slow-growing host plants that retained their apical meristems at or near the soil surface for 2-5 weeks. Galls developed extensive areas of nutritive tissue. The nematode was introduced from the Soviet Union and released in Canada for the biological control of Russian knapweed.     

Characterization of a Ca-dependent maxi K channel in the apical membrane of a cultured renal epithelium (JTC-12.P3)

Summary A Ca and potential-dependent K channel of large unit conductance was detected in the apical membrane of JTC-12.P3 cells, a continuous epithelial cell line of renal origin. The open probability of the channel is dependent on membrane potential and cytoplasmic-free Ca concentration. At cell-free configuration of the membrane patch, the open probability shows a bell-shaped behavior as function of membrane potential, which decreases at larger depolarization. With increasing Ca concentration, the width of the bell-shaped curve increases and the maximum shifts into the hyperpolarizing direction. For the first time the kinetics of this channel was analyzed under cell-attached conditions. In this case the kinetics could sufficiently be described by a simple open-closed behavior. The channel has an extremely small open probability at resting potential, which increases exponentially with depolarization. The low probability induces an uncertainty about the actual number of channels in the membrane patch. The number of channels is estimated by kinetic analysis. It is discussed that this K channel is essential for the repolarization of the membrane potential during electrogenic sodium-solute cotransport across the apical membrane.     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: II. Modulation by γ-Aminobutyric Acid and Serotonin

In the accompanying paper we reported that 3,4-dihydroxyphenylethylamine (dopamine) induced light-adaptive retinomotor movements in teleost photoreceptors and that this effect was mediated by D2 dopamine receptors located on the photoreceptors themselves. In this study, we investigated the effects on cone retinomotor movement of three agents that have been reported by others to modulate retinal dopamine release: gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT, serotonin), and melatonin. We report here that the GABA antagonists bicuculline and picrotoxin induced light-adaptive cone contraction in dark-adapted green sunfish retinas cultured in constant darkness; thus they mimic the effect of light or exogenously applied dopamine. Since their effects were blocked by either the D2 dopamine antagonist sulpiride or by Co2+, it seems likely that these agents act by enhancing retinal dopamine release. The GABA agonist muscimol produced effects opposite to those of GABA antagonists. Muscimol inhibited light-induced cone contraction in previously dark-adapted retinas and induced dark-adaptive cone elongation in light-adapted retinas. These results suggest that in green sunfish retinas, as has been reported for other retinas, GABA inhibits dopamine release. 5-HT induced light-adaptive cone contraction in dark-adapted retinas; thus 5-HT also mimics the effect of light or exogenously applied dopamine. The effect of 5-HT was blocked by sulpiride, Co2+, or the 5-HT antagonist mianserin. These results suggest that 5-HT induces cone contraction by stimulating dopamine release. Melatonin neither inhibited dopamine-induced cone contraction in retinas cultured in the dark nor induced cone elongation in retinas cultured in the light. Our results suggest that both GABA and 5-HT (but not melatonin) affect cone retinomotor movements in green sunfish by modulating dopamine release: GABA by inhibiting and 5-HT by stimulating dopamine release. We report in the companion paper that dopamine induced contraction in isolated cone fragments. Together these observations strongly suggest that dopamine serves as the final extracellular messenger directly inducing light-adaptive cone retinomotor movement, and that GABA and 5-HT affect these movements by modulating dopamine release.     

γ-Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

The specific binding of [N-methyl-3H]flunitrazepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+-dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzodiazepine receptors in mammals. Thus binding is enhanced by gamma-aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5-4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photoaffinity-labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.