Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.     

Characteristics of Tyrosine Hydroxylase Activation by K+-Induced Depolarization and/or Forskolin in Rat Striatal Slices

The mechanisms of tyrosine hydroxylase (TH) activation by depolarization or exposure of dopaminergic terminals to cyclic AMP have been compared using rat striatal slices. Tissues were incubated with veratridine or 60 mM K+ (depolarizing conditions), on the one hand, and forskolin or dibutyryl cyclic AMP, on the other. K+-(or veratridine-)induced depolarization triggered an activation of TH (+75%) that persisted in soluble extracts of incubated tissues. This effect disappeared when drugs (EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, Gallopamil) preventing Ca2+- and calmodulin-dependent processes were included in the incubating medium. In contrast, prior in vivo reserpine treatment or in vitro addition of benztropine did not affect the depolarization-induced activation of TH. In vitro studies of soluble TH extracted from depolarized tissues indicated that activation was associated with a marked increase in the enzyme Vmax but with no change in its apparent affinity for the pteridin cofactor 6-methyl-5,6,7,8-tetrahydropterin (6-MPH4) or tyrosine. Furthermore, the activated enzyme from depolarized tissues exhibited the same optimal pH (5.8) as native TH extracted from control striatal slices. In contrast, TH activation resulting from tissue incubation in the presence of forskolin or dibutyryl cyclic AMP was associated with a selective increase in the apparent affinity for 6-MPH4 and a shift in the optimal pH from 5.8 to 7.0-7.2. Clear distinction between the two activating processes was further confirmed by the facts that heparin- and cyclic AMP-dependent phosphorylation stimulated TH activity from K+-exposed (and control) tissues but not that from striatal slices incubated with forskolin (or dibutyryl cyclic AMP). In contrast, the latter enzyme but not that from depolarized tissues could be activated by Ca2+-dependent phosphorylation. These data strongly support the concept that Ca2+- but not cyclic AMP-dependent phosphorylation is responsible for TH activation in depolarized dopaminergic terminals.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Neuronal Activation Regulates the Expression of Opioid Receptors: Possible Role of Glial-Derived Factors and Voltage-Dependent Ion Channels

The previous observation that a continuous chemical depolarization of aggregating rat brain cells with KCl alters the expression of opioid receptors was examined in more detail. In contrast to its significant and converse effect on forebrain and hindbrain cells cultured in serum-containing medium, KCl had only a small and transient effect in serum-free cultures of both types. The basal receptor density in serum-free cultures was similar to the receptor density in KCl-treated serum-containing cultures, but medium conditioned by glial cells restored partially the effect of KCl in serum-free cultures. The effect of KCl in serum-containing forebrain cultures was enhanced by the voltage-dependent calcium channel blocker verapamil, and magnesium and cadmium had a similar, though smaller, effect. The sodium channel activator veratridine had a profound and dose-dependent inhibitory effect on the expression of the receptors in forebrain and hindbrain cultures, and tetrodotoxin blocked the veratridine effect. Information about the selectivity of the effect of neuronal activation on the various opioid receptor subtypes was obtained with the neuroblastoma X glioma hybrid M8 cells that possess only delta type opioid receptors. A Scatchard analysis of [3H]etorphine binding to these cells has shown that depolarization increased the Bmax, but had little, if any, effect on the affinity (KD) of the ligand to the receptors. The significance of depolarization and voltage-dependent sodium and calcium channels on the expression of different opioid receptor subtypes is discussed.     

Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides,calcium, protein kinases and phosphatases and depolarization

Protein phosphorylation was investigated in [³²P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both ₁- and ₂-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the ₂-AR only marginally increased while the stimulation of ₁-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M₂-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated G_i-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of ₁-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

Potassium-induced insulin release and voltage noise measurements in single mouse islets of Langerhans

Summary Insulin release and membrane potential fluctuations in response to increased extracellular potassium [K⁺] _o have been measured in single perifused islets of Langerhans from normal mice. An increase in [K⁺] _o from 5mm to 50mm induced a transient insulin release with a peak at about 1 min. The peak value was [K⁺] _o -dependent but the half-timet _1/2 for the decline was constant at nearly 1 min. 2.5mm cobalt completely inhibited the potassium-induced stimulation of insulin release. The insulin release elicited by 28 and 50mm [K⁺] _o was similar in terms of peak, total release and half-time from maximum release. Stepwise increase in [K⁺] _o from 10 to 28 to 50mm resulted in a normal response to 28mm but no peak of release after the 28 to 50mm increase. The results indicate good correlation between excess voltage noise, thought to reflect calcium channel activity, and insulin release evoked by changing extracellular potassium.     

Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.