Effects of divalent cations, lanthanum, cation chelators and an ionophore on acetylcholine antinociception.

The antinociceptive effect of intracerebroventricularly administered acetylcholine as measured in the mouse tail-flick test was reduced by intracerebroventricularly injected calcium, magnesium and manganese. Maximum antagonism of acetylcholine-induced antinociception was observed with a 1-hour calcium pretreatment. Significant reduction existed at 2- but not 4-hour pretreatment. Barium and strontium were inactive. The antinociceptive effect of acetylcholine was potentiated by lanthanum and ethylene glycol tetraacetic acid but not by ethylenediamine tetraacetic acid. The ionophore A23187 was shown to increase greatly the antagonistic effect of a low dose of calcium. The ionophore alone did not significantly alter the effect of acetylcholine. Thus, it appears that calcium must penetrate cell membranes to reduce the effect of acetylcholine. In addition to acetylcholine, it was found that the antinociceptive effects of oxotremorine and physostigmine could also be reduced by calcium. These data indicate that alterations in intracellular calcium are involved in cholinergically induced antinociception.     

Effects of some divalent cations on nitrergic relaxations in the mouse corpus cavernosum

Acute effects of some divalent cations (Cd²⁺, Ni²⁺, Co²⁺, Zn²⁺, Mn²⁺ and Sn²⁺) were investigated on neurogenic and endothelium-dependent relaxations in the isolated mouse corpus cavernosum. Neither neurogenic nor endothelium-dependent relaxation was affected by cations at the concentrations used (up to 100 μ M ), except Cd²⁺. Although Cd²⁺ (20 and 40 μ M ) did not cause any significant alteration in the acetylcholine- (ACh) or sodium nitroprusside- (SNP) induced relaxation, it inhibited electrical field stimulation- (EFS) produced relaxation significantly. Zn²⁺ and selenium could not reverse this inhibitory action. Cd²⁺ did block the EFS-evoked guanethidine-sensitive contraction in the presence of N^G-nitro- L -arginine. Elevation of external Ca²⁺ content significantly reduced the inhibitions due to Cd²⁺ on the EFS-induced relaxation and on the EFS-evoked guanethidine-sensitive contraction. In the Ca²⁺-omitted medium, EFS-induced relaxation disappeared, while acetylcholine-elicited relaxation resisted. Verapamil was ineffective on the relaxation produced by EFS or acetylcholine. However, it significantly diminished phenylephrine-induced contractions. These findings suggest that unlike other cations at the concentrations used in the present study, Cd²⁺ may have an effect on an external Ca²⁺-dependent mechanism at the neuronal level, and this effect may be responsible for its acute inhibitory action on the neurogenic relaxation in the mouse corpus cavernosum.     

Selective release of excreted DNA sequences from phytohemagglutinin-stimulated human peripheral blood lymphocytes. Effects of trypsin and divalent cations.

We studied the synthesis of excreted DNA sequences and their release from phytohemagglutinin-stimulated human peripheral blood lymphocytes under conditions permitting optimal cell growth. Cells were labeled by constant exposure to low specific activity [3H]thymidine. Excreted DNA sequences were synthesized during the period of logarithmic cell growth and moved slowly from the high molecular weight chromosomal DNA fraction into the low molecular weight cell DNA fraction (Hirt supernate) from which they could be specifically released by treating the cells briefly with small amounts of various proteases; 1 microgram/ml trypsin for 5 min was optimal. On day 5 of culture, 13.3 +/- 6.9% of the total cellular acid-precipitable [3H]thymidine was released by this treatment. Trypsin-induced release was partially and reversibly inhibited by incubating the cells for 16 h with 5 mM dibutyryl-cyclic AMP. Cells incubated in the absence of divalent cations spontaneously released this Hirt supernatant DNA; after maximal release had occurred under these circumstances, additional trypsin treatment caused no further release of DNA. Trypsin-induced DNA release could be completely and reversibly inhibited by incubating the cells in the presence of 10 mM calcium. Trypsin-released DNA was isolated and analyzed by reassociation kinetics. A major component, representing 54% of the DNA, reassociated with a C0t1/2 of 68 mol.s/liter (the value at which DNA association is 50% complete). The reassociation of this DNA was studied in the presence of an excess of DNA isolated from stimulated lymphocytes on day 3 in culture, and in the presence of an excess of resting lymphocyte DNA. The high molecular weight fraction of day-3 cell DNA contained three times more copies of the trypsin-released DNA major component as compared to resting lymphocyte DNA. Hirt supernatant DNA isolated from day-5 stimulated lymphocytes reassociated in an intermediate component representing 34% of the DNA with a Cot1/2 of mol.s/liter; after cells were treated with trypsin, this component could no longer be identified in the Hirt supernatant fraction, presumably because it had been released into the incubation medium. These data describe a quantitatively reproducible system with which synthesis and release of excreted DNA sequences can be studied.     

The activation of neuronal nitric-oxide synthase by various divalent cations

Nitric-oxide synthase (NOS; EC 1.14.13.39) catalyzes the oxidation of L-arginine to nitric oxide (NO(.)) and L-citrulline via the intermediate N(omega)-hydroxy-L-arginine. Of the three distinct isoforms of NOS that have been characterized, the constitutive neuronal NOS (NOS I) generates NO(.) associated with long-term potentiation (LTP) and early brain development. All of the NOS isoforms contain an N-terminal oxidase and a C-terminal reductase domain connected by a Ca(2+)/calmodulin binding region. To activate NOS I, Ca(2+) has to bind to calmodulin, allowing electron transport through both domains. Calcium ions are tightly regulated in cells. However, a number of other metal ions that bind and activate calmodulin may also activate NOS I. One such metal ion may be Pb(2+), which is associated with neurobehavioral and psychological alterations, including the inhibition of LTP. The effect of various divalent cations on NOS I activity was tested, and the results presented herein demonstrate that Pb(2+) and Sr(2+) can activate NOS I to a level similar to that found for Ca(2+). Finally, there is a synergy between Pb(2+) and Ca(2+) resulting in maximal activation of NOS I using minimal concentrations of both metal ions.     

Age-related differences in the effects of some muscarinic agents on acetylcholine release from rat neostriatal slices

The purpose of this study was to investigate whether the muscarinic modulation of neostriatal acetylcholine release changes with senescence. Neostriatal slices from Fischer 344 rats aged 3, 10 and 28 months were prepared and incubated in Krebs-Ringer bicarbonate buffer oxygenated with 95% O2/5% CO2. Acetylcholine release from slices of each age group was monitored in the presence or absence of muscarinic agents, and the release in the presence of the drug was compared to the release from slices of age-matched controls in the absence of drug. The muscarinic agonist, oxotremorine, and two muscarinic antagonists, atropine and pirenzepine, were tested for their effects on acetylcholine release. Pirenzepine is selective in its interaction with the M1 muscarinic receptor subtype; atropine and oxotremorine are nonselective in their actions. Of the three drugs tested, pirenzepine displayed a significant age-related difference in its effects on acetylcholine release. Whereas the effects of pirenzepine (50 microM) on acetylcholine release modulation in slices from the 3-month rats were negligible, the M1-selective antagonist increased the release of acetylcholine from slices of 10- and 28-month rats by another 42 and 192% (P less than .05), respectively. Atropine (1 microM) was also tested, and an increase in acetylcholine release by another 64, 104 and 218% (all P less than .05) was observed in slices from the 3-, 10- and 28-month rats, respectively. In the presence of oxotremorine (50 microM), acetylcholine release decreased in slices from the 3-month rats by 35% (P less than .1), but changed by only 7 and 15% in the 10- and 28-month slices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular calcium-dependent and -independent effects of methylmercury on spontaneous and potassium-evoked release of acetylcholine at the neuromuscular junction

Acute bath administration of methylmercury (MeHg) to the murine neuromuscular junction causes an initial surge in the frequency of occurrence of miniature end-plate potentials (MEPPs), followed by a complete suppression of asynchronous spontaneous release. The goals of the present study were to determine: whether the MeHg-induced in MEPP frequency was dependent upon extracellular Ca++, whether MeHg produced this effect by actions within the motor nerve terminal and whether the secondary suppression of release was due to transmitter depletion. Conventional intracellular microelectrode recording measurements of MEPPs were made from myofibers of the isolated hemidiaphragm of the rat. Increasing the bath concentration of Ca++ from 1 to 2 or 4 mM decreased the time period required by 100 microM MeHg to produce a peak increase of spontaneous release from 52 to 39 to 28 min, respectively. Further increasing bath Ca++ to 8 mM actually increased this period back to 49 min. Increasing [Ca++]o had no consistent effect on the magnitude of the MeHg-induced increase in MEPP frequency. After depolarization of the nerve terminal with elevated extracellular K+ (15 mM) the time to peak increases in MEPP frequency was shortened from approximately 40 min to 1 to 2 min. The time required for MeHg to cause complete cessation of MEPPs was also shortened. In experiments conducted in K+-depolarized preparations to which no Ca++ was added, MeHg still increased MEPP frequency, although not as rapidly, or to the same extent as in solutions containing Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of divalent cations and sulfhydryl reagents on the p-aminohippurate (PAH) transporter of renal basal-lateral membranes

A specific system of transport for p-aminohippurate (PAH) is demonstrated in rabbit renal basal-lateral membrane vesicles. The PAH uptake into an intravesicular space is inhibited by probenecid in concentrations above 0.2 mM. The transport is saturable and is also temperature-dependent with an optimum between 37 and 45 degrees C. Divalent cations are able to enhance the uptake 2- to 3-fold. The stimulatory effect of the divalent cations diminishes in the following order: Mg++ = Mn++, Ba++, Ca++ and Sr++. Maximum stimulation occurs between 2.5 and 5 mM Mg++. The divalent cation stimulatory effect is not the result of changes in the size of the vesicles, in the degree of vesiculation, in the net charge of the membrane or of a transient potential difference across the membrane. Several inhibitors, more inhibitory than probenecid, were found. These are: lithium diiodosalicylate; 4-acetamido-4'-isothiocyano 2,2'-disulfonic acid stilbene; the mercurials, mersalyl acid, p-chloromercuriphenyl sulfonate and Hg++; and 5,5'-dithiobis(nitrobenzoate). Among these, mersalyl acid is the most potent inhibitor for PAH uptake. Its inhibitory effect is probably a combination of its reactivity toward sulfhydryl groups and its anionic character. The results with sulfhydryl reagents indicate that the PAH transport system contains sulfhydryl groups which are essential for the uptake activity. These sulfhydryl groups are probably buried in a hydrophobic region within the lipoprotein matrix of the basal-lateral membrane.     

Effect of zinc and other cations on the release of the eosinophil cationic protein

The eosinophil cationic protein, ECP, is a unique eosinophil granule constituent, which is released extracellularly after exposure of the eosinophils to a non-phagocytosable surface such as complement-coated Sephadex beads. The ECP is released to some extent even in the absence of Ca2+ and Mg2+, though both these cations augment the release reaction tested alone, and an optimal release is observed only in the presence of 2 mmol/l Ca2+ and 2 mmol/l Mg2+ in the medium. Zn2+ at concentrations from 0.25-4.0 mmol/l inhibited the release of ECP in a dose-dependent fashion, with or without Ca2+ and Mg2+ in the medium. Mn2+ had dual effects, stimulating the ECP release in the absence of Mg2+ and Ca2+, and inhibiting the release in the presence of these cations. Li1+ caused minor inhibition of ECP release, but only in the absence of Ca2+ and Mg2+. The inhibitory effect of Zn2+ was immediate and reversible after washing of the cells, suggesting that the inhibition is due to interaction with the plasma membrane functions.     

Effects of divalent cations, cation chelators and an ionophore on morphine analgesia and tolerance.

The analgesic effect of morphine was antagonized in mice by intracerebroventricular injection of Ca++, Mg++ and Mn++ and was potentiated by ethylene glycol tetraacetic acid but was not altered by Sr++, Ba++, Ni++, Hg++, Cd++ or ethylenediamine tetraacetic acid. The antagonistic effect of Ca++ was not altered by pretreatment with pargyline or 6-hydroxydopamine indicating that altered release of catecholamines or serotonin was not involved in this action of Ca++. Induction of morphine tolerance by pellet implantation also did not alter the antagonistic effect of Ca++. The antagonistic effects of Ca++ and naloxone were additive in both nontolerant and tolerant animals and the apparent affinity of naloxone for its receptors, as estimated by in vivo pA2 determinations, was not altered by Ca++. However, the ionophore X537A was found to increase greatly the narcotic antagonist effect of a low dose of Ca++ although the ionophore alone did not alter the effects of morphine. This indicates that Ca"++ must penetrate cell membranes in order to reduce the analgesic effects of morphine. These findings indicate the importance of Ca++ localization in the actions of narcotic agonists and antagonists.     

Inhibitory effects of the antiparkinsonian drugs memantine and amantadine on N-methyl-D-aspartate-evoked acetylcholine release in the rabbit caudate nucleus in vitro.

Slices of the rabbit caudate nucleus were incubated with [3H]choline or [3H]dopamine and then superfused continuously with Mg(++)-free medium. Stimulation with N-methyl-D-aspartate (NMDA), alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA), L-glutamate and kainic acid (in that rank order of potencies) caused a concentration-dependent increase in [3H]ACh efflux, which was abolished in the presence of Mg++. This kind of release was Ca(++)-dependent and tetrodotoxin-sensitive. In contrast, NMDA was hardly effective in stimulating [3H]ACh release from hippocampal or cortical slices, as well as [3H]dopamine release from slices of rabbit caudate nucleus. Hence, the presence of cell bodies of stimulated neurons seems to be a prerequisite for the induction of release via NMDA receptors. Dizocilpine [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate] at nanomolar concentrations, as well as memantine and amantadine at low micromolar concentrations, inhibited the L-glutamate- and NMDA-evoked [3H]ACh release in a concentration-dependent, noncompetitive and use-dependent manner. Also (+/-)-2-amino-5-phosphopentanoic acid at micromolar concentrations depressed the L-glutamate- and NMDA-induced release, acting, however, in a competitive manner. It is concluded that, by antagonizing NMDA receptor-mediated ACh release, memantine and amantadine may act as functional "anticholinergics" when administered clinically to treat Parkinson's disease.     

The fundamental roles of monovalent and divalent cations with sulfates on molybdenite flotation in the absence of flotation reagents

Due to regional shortage of freshwater, the use of saline/seawater for Cu–Mo sulfide ore flotation has received considerable attention. However, the effects of various salts, especially the cations present in seawater, on molybdenite flotation and the mechanisms involved remain unclear due to the complexity of the solutions applied. In this work, the influence of some common cations (i.e., Na⁺, K⁺, Ca²⁺ and Mg²⁺) with sulfate (SO₄²⁻) anions on molybdenite flotability was investigated in the absence of flotation reagents (i.e., frothers and collectors) at pH 10. Flotation results indicated a greater depression of molybdenite recovery with increased sulfate salt concentration. The underlying mechanisms responsible for the deleterious effects in the presence of Na⁺ and K⁺ can be attributed to the increased repulsive forces between molybdenite particles and bubbles owing to increased molybdenite oxidation to produce e.g., MoO₄²⁻ and HMoO₄⁻. However, the increased depression observed in the presence of Mg²⁺ and Ca²⁺ is likely due to the adsorption of precipitated Mg(OH)₂ and CaMoO₄, respectively, onto molybdenite surfaces. These clearly show the different depressing mechanisms due to monovalent and divalent sulfates on molybdenite flotation in the absence of flotation reagents, to reveal the influence of these sulfate salts on its natural flotability.

Schematic of molybdenite oxidation and flotation in the presence of various cations. (a) Oxidised molybdenite edge, in the presence of (b) Na⁺, (c) K⁺, (d) Ca²⁺, and (e) Mg²⁺.     

Presynaptic interactions between acetylcholine and adrenergic antagonists on norepinephrine release

A previously unknown interaction between acetylcholine (ACh) and adrenergic mechanisms is described, which increases the likelihood of a physiological association between the two divisions of the autonomic nervous system. In particular, ACh had a novel effect to disengage the neuronal mechanism that is purported to regulate the release of norepinephrine from sympathetic nerves. Brief exposure to ACh (1.4 X 10(-7) M-1.4 X 10(-5) M) inhibited the stimulation-evoked release of [3H]norepinephrine from guinea pig atria and ureter and rabbit aorta but higher concentrations (8.8 X 10(-5) or 1.4 X 10(-4) M) or prolonged exposure to moderate concentrations either had no visible effect or enhanced release. ACh blocked the ability of yohimbine, the presynaptic alpha receptor antagonist, to enhance the liberation of 3H-transmitter during field stimulation at 2 and 5 Hz, and it did so in all three of the test tissues. This effect was not attributable to a direct competition between ACh and yohimbine for presynaptic alpha sites and ACh did not act like yohimbine to increase transmitter release. The antagonistic effect of ACh bore no relation to the direct effect of ACh on adrenergic neurotransmitter release and occurred regardless of whether ACh itself inhibited, enhanced or did not affect transmitter liberation. Atropine blocked the effect of ACh on 3H-transmitter efflux and restored the capacity of yohimbine to enhance transmitter release. Inhibition of neurotransmitter release by norepinephrine was partially antagonized by ACh and this antagonism was also countered by atropine. Enhancement of norepinephrine release by phenoxybenzamine was also blunted by ACh. These findings cannot be incorporated into a model of neurotransmitter regulation that interprets the enhancement of norepinephrine release by adrenergic antagonists as the result of interruption of an on-going negative feedback system. The action of yohimbine appears linked to activation of presynaptic sites and not simply to their passive occupancy. A working model is offered to account for the interaction between ACh and adrenergic antagonists.     

Effect of divalent cations and pH on intrinsic factor-mediated attachment of vitamin B12 to intestinal microvillous membranes

Calcium, but not other divalent cations, is required for optimal uptake of intrinsic factor-bound (57)Co-labeled cyanocobalamin (IFB(12)) by microvillous membranes isolated from hamster ileal-absorptive cells. Chelation of divalent cations by disodium ethylenediaminetetraacetate (EDTA) promptly removes IFB(12) previously attached to microvillous membranes. High concentrations of CaCl(2) or MgCl(2) also markedly inhibit membrane uptake of IFB(12) and rapidly remove previously attached IFB(12). Similarly, reduction of pH to below 5.4 prevents membrane attachment of IFB(12) and removes virtually all IFB(12) already bound to microvillous membranes. The effects of calcium depletion, increased salt concentrations, and acidification on membrane uptake of IFB(12) were completely reversible. These findings are consistent with the concept that the formation of calcium salt bridges is essential for attachment of IFB(12) to the ileal-absorptive surface.     

Adeno-associated virus vectors show variable dependence on divalent cations for thermostability: implications for purification and handling

Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations. Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance. Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile.     

Interactions of acetylcholine and epinephrine on the dynamics of insulin release in vitro.

An in vitro system for perifusion of rat pancreatic islets has been utilized to define the effects of epinephrine on acetylcholine-induced insulin release over varying concentrations of the two agents. Perifusion of islets with epinephrine before challenge with acetycholine produced marked enhancement of both phases of cholinergically induced insulin release; enhancement of the first phase being more marked with increase in acetylcholine concentration and the converse being observed with the second phase. Perifusion of islets with epinephrine during stimulation with acetylcholine produced inhibition of insulin release, an effect dependent upon the concentration of epinephrine and of acetylcholine. There was an order of difference in the acetycholine concentration needed to overcome significant epinephrine-mediated inhibition of the first phase of insulin release (5 X 10(-4) mug/ml) and that needed to overcome inhibition of the second phase (5 X 10(-3) mug/ml). Comparison of the effects of various concentrations of epinephrine on glucose- and acetyl-choline-induced insulin release revealed that epinephrine was a less potent inhibitor of the first phase of acetylcholine-induced insulin release than of the first phase of glucose-induced insulin release. These data provide some insight into the potential interactions between cholinergic and adrenergic autonomic systems in modifying insulin release.