Acetylcholine Incorporation by Cholinergic Synaptic Vesicles from Torpedo marmorata

[3H]Acetylcholine efflux and Na+-K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 microM), strophanthidin (500 microM), and parachloromercuribenzene sulfonate (500 microM) each inhibited ouabain-sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 microM), electrical field stimulation (60 V, 60 Hz, 5-ms pulse duration), or the calcium ionophore A23187 (10 micrograms/ml) also inhibited ouabain-sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium-dependent process. Veratridine-induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre-treatment with tetrodotoxin (1 microM). The rate of [3H]acetylcholine efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain-sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+-K+ ATPase.     

Effect of Adenosine, Adenosine Derivatives, and Caffeine on Acetylcholine Release from Brain Synaptosomes: Interaction with Muscarinic Autoregulatory Mechanisms

Synaptosomes, prepared from rat cerebral cortex and hippocampus, were preincubated with [methyl-3H]choline. The effect of adenosine, cyclohexyladenosine, N-ethylcarboxamide adenosine, 2'-deoxyadenosine, and oxotremorine on K+-evoked 3H efflux was investigated. High-voltage electrophoretic separation showed that in the presence of physostigmine, the K+-evoked 3H efflux from hippocampal synaptosomes was 90% [3H]acetylcholine and 10% [3H]choline. Adenosine (30 microM) and oxotremorine (100 microM) both decreased [3H]acetylcholine release from hippocampal synaptosomes. The effect was inversely proportional to the KCl concentration and disappeared at a KCl concentration of 50 mM. Cyclohexyladenosine was approximately 3,000 times more active than adenosine, whereas N-ethylcarboxamide adenosine and 2'-deoxyadenosine were inactive. This indicates that A1 adenosine receptors were involved in the inhibitory effect. Caffeine antagonized the adenosine effect, and at a concentration of 100 microM, it stimulated [3H]acetylcholine efflux. The inhibitory effect of oxotremorine was as great in cortical as in hippocampal synaptosomes. In contrast, adenosine was much less active in cortical than in hippocampal synaptosomes. When inhibitory concentrations of adenosine and oxotremorine were added together into the incubation medium, the effect of adenosine on [3H]acetylcholine release was consistently reduced. An interaction between muscarinic and A1 adenosine presynaptic receptors at a common site modulating acetylcholine release can be assumed.     

Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum

Neuronal varicosities, isolated from the myenteric plexus of guinea pig ileum longitudinal muscle, were incubated with [3H]noradrenaline to label the contents of the noradrenergic secretory vesicles. Exposure of these varicosities to KCl, nicotine, or acetylcholine resulted in the Ca2+ -dependent release of [3H]noradrenaline. Veratridine also evoked a large efflux of [3H] from this preparation, but this release was only partially Ca2+ dependent. The alpha 2-adrenoceptor agonist, clonidine, inhibited the K+-, nicotine-, and acetylcholine-induced release of [3H]noradrenaline. Similarly, exogenously administered (-)noradrenaline was an effective inhibitor of the K+ -evoked release of [3H]noradrenaline. The alpha 2-adrenoceptor antagonist, yohimbine, antagonized the inhibitory actions of both clonidine and (-)noradrenaline on the K+ -evoked release of [3H]noradrenaline from myenteric varicosities. Nicotine, acetylcholine, KCl, and veratridine also released ATP from these guinea pig ileal myenteric varicosities. However, the evoked release of ATP was unaffected by clonidine. These results indicate that myenteric varicosities can take up and release [3H]noradrenaline and that they possess presynaptic alpha 2-adrenoceptors which, when activated, inhibit the release of [3H]noradrenaline. These receptors may play a role in modulating the release of noradrenaline in the myenteric plexus in vivo. In addition, the present results suggest that ATP and [3H]noradrenaline may not be released from the same population of secretory vesicles in neuronal varicosities isolated from guinea pig ileum longitudinal muscle.     

The effects of cesium chloride on insulin release, ionic fluxes and membrane potential in pancreatic B-cells

Cs+ decreases K+ permeability in nerve and muscle cells. Its effects on the pancreatic B-cell function were studied with mouse islets. In the presence of 3 mM glucose, Cs+ substitution for K+ steadily inhibited 86Rb+ efflux and hyperpolarized the B-cell membrane. Addition of Cs+ to a K+-medium also inhibited 86Rb+ efflux, but depolarized the B-cell membrane. None of these changes altered insulin release. Substitution of Cs+ for K+ in a medium containing 10 mM glucose caused a Ca2+-dependent stimulation of insulin release and 45Ca2+ efflux, produced an initial fall and a secondary rise in 86Rb+ efflux and augmented the electrical activity in B-cells. Reintroduction of K+ to the medium was followed by a marked and transient inhibition of insulin release, that was blocked by ouabain and accompanied by an inhibition of 45Ca2+ and 86Rb+ efflux and by a hyperpolarization of the B-cell membrane. Addition of Cs+ to a K+ medium containing 10 mM glucose stimulated insulin release, 45Ca2+ efflux and 86Rb+ efflux. It also increased the electrical activity in B-cells. In the absence of Ca2+, however, Cs+ addition decreased the rate of 86Rb+ efflux. The effects of Cs+ on the B-cell function may be explained by its ability to decrease K+ permeability of the plasma membrane, by its inability to activate the sodium pump, and by a third unidentified effect likely brought about by the accumulation of intracellular Cs+.     

Effect of osmolality on 86Rb+ uptake and release by Leishmania donovani.

Promastigotes from late-log phase cultures of Leishmania donovani were washed and resuspended in Hanks' Balanced Salt Solution without glucose or phenyl red but with 20 mM (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) (HEPES) (HBSS-, 305 mOsm/kg). They were then added to a solution containing 86Rb such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular 86Rb was measured. Similarly, experiments were performed in which 86Rb was added to the cultures about 18 hr before collection, and the amount of 86Rb released from the washed cells was measured. Under iso-osmotic conditions only about 1.3% of the intracellular 86Rb was released in 900 sec. This increased about 4-fold if the osmolality was reduced from 305-153 mOsm/kg. This is much slower than the very rapid release of alanine in response to hypo-osmotic stress, indicating that alanine release is not via a non-specific pore. Reducing the temperature from 26 degrees C to 3-4 degrees C completely inhibits 86Rb release under iso-osmotic conditions and largely inhibits it under hypo-osmotic conditions. The rate of 86Rb release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either 86Rb uptake or release, but carbonylcyanide P-trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of 86Rb release and, after about a 300 sec exposure, completely inhibited 86Rb uptake. Amiloride partially inhibited 86Rb release, but had no effect on uptake. A decrease in pH from 7.1-5.9 had little effect on 86Rb release under iso-osmotic conditions and slightly increased the rate of release under hypo-osmotic conditions, but it decreased the rate of uptake under both iso-osmotic and hypo-osmotic conditions. Cells taken from 3-day stationary phase cultures released 86Rb more slowly under iso-osmotic conditions than cells from late log phase cultures, but were more responsive to hypo-osmotic stress than were log phase cells. These data appear to rule out an [Na-K-Cl] transporter or a [K-Cl] cotransporter as the means of K+ release, but are consistent with the possibility that a K+/H+ exchanger is present. The possibility that other carrier systems may be present is also discussed.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Stimulation of D-2 Dopamine Receptors Decreases the Evoked In Vitro Release of [3H] Acetylcholine from Rat Neostriatum: Role of K+ and Ca2+

Reportedly, stimulation of D-2 dopamine receptors inhibits the depolarization-induced release of acetylcholine from the neostriatum in a cyclic AMP-independent manner. In the present study, we investigated the role of K+ and Ca2+ in the D-2 receptor-mediated inhibition of evoked [3H]acetylcholine release from rat striatal tissue slices. It is shown that the D-2 receptor-mediated decrease of K+-evoked [3H]acetylcholine release is not influenced by the extracellular Ca2+ concentration. However, increasing extracellular K+, in the presence and absence of Ca2+, markedly attenuates the effect of D-2 stimulation on the K+-evoked [3H]acetylcholine release. Furthermore, it is shown that activation of D-2 receptors in the absence of Ca2+ also inhibits the veratrine-evoked release of [3H]acetylcholine from rat striatum. These results suggest that the D-2 dopamine receptor mediates the decrease of depolarization-induced [3H]acetylcholine release from rat striatum primarily by stimulation of K+ efflux (opening of K+ channels) and inhibition of intracellular Ca2+ mobilization.     

Effect of Potassium on the Release of [3H]Noradrenaline from Rabbit and Human Pulmonary Artery

The release of [3H]noradrenaline ( [3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+-free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic alpha 2-adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+-free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of NA, K-ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+-free solution, the results may indicate involvement of the sodium pump in NA release.     

Potassium movement during sodium-induced motility initiation in the rat caudal epididymal spermatozoa

The sodium-induced sperm motility initiation of the rat cauda epididymal sperm has been studied in different potassium concentrations. High K+ inhibited motility initiation. At a K+ concentration of 50 mM (concentration found in the rat cauda epididymidis), sperm motility was inhibited by 80%. K+ movement across the sperm membrane has been followed by using 86Rb+ as a marker for K+. When the 86Rb+ preloaded sperm were suspended in a sodium-free medium, there was a little efflux of 86Rb+. However, if they were suspended in a sodium-containing medium, the efflux rate was greatly increased. This increase in 86Rb+ efflux rate was associated with an initiation of sperm motility. Both 86Rb+ efflux and motility initiation were triggered by a K+ ionophore 18-crown-6 (2 X 10(-3)M). However, the ionophore-induced 86Rb+ efflux and motility initiation only occurred in the presence of extracellular Na+. Tetraethylammonium (TEA) chloride, which blocks K+ channels, inhibited motility initiation in a dose-dependent manner. Changes in the membrane potential of sperm have been followed using the fluorescent dye diO-C6-(3) whose fluorescence in sperm suspension changes markedly with changes in sperm membrane potential. During motility initiation, there was a fall in fluorescence of the dye due to increased partition into sperm cells. This observation may indicate a hyperpolarization of the sperm membrane during motility initiation. It was concluded that sperm motility initiation is associated with a complex ionic event. Na+ enters sperm cells in exchange with H+ and K+. This change in the permeability of the sperm membrane to ions is reflected by a change in the sperm membrane potential.     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

Formyl peptide stimulation of superoxide anion release from lung macrophages: sodium and potassium involvement

We examined the role of the monovalent cations Na+ and K+ in the events encompassing the release of O-2 by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP-stimulated O-2 production; and measuring 22Na+, 42K+ and 86Rb+ influx and efflux and intracellular [K+] for control and FMP-stimulated alveolar macrophages. Stimulated O-2 production was relatively insensitive to changes in extracellular K+ or Na+ concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O-2 production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of 22Na+ were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited 22Na+ efflux but had no effect on O-2 release. The influx of 86Rb+ and 42K+ was not altered by the addition of FMP but was virtually abolished in the presence of 10 microM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP-stimulated a prolonged (greater than 20 minutes) increase in 86Rb+ or 42K+ efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+ efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O-2 production under these conditions. It was also observed that there was a loss of intracellular K+ when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+ in the events that lead to FMP-stimulated O-2 release by alveolar macrophages.     

Presynaptic effects of neuropeptide Y on [3H]noradrenaline and [3H]acetylcholine release

The electrically evoked release of radioactivity from mouse vas deferens and rat hypothalamic slices preloaded with [3H]noradrenaline was measured. In addition the release of [3H]acetylcholine from longitudinal muscle strip of guinea-pig ileum was also measured. Neurochemical evidence has been obtained that neuropeptide Y (NPY), although it co-exists and is released with (-)-noradrenaline (NA), it behaves differently as far as its effect on presynaptic modulation of chemical neurotransmission is concerned. It exerts a frequency-dependent presynaptic inhibitory effect on noradrenaline release from mouse vas deferens but has no effect on the electrically evoked release of NA from rat hypothalamus. Unlike NA, NPY does not influence the release of [3H]acetylcholine from the longitudinal muscle strip of guinea-pig ileum and does not potentiate the presynaptic effect of NA. It seems very likely, that the inhibitory effect of NPY is mediated via receptors. Its action is concentration dependent. While exogenous noradrenaline inhibited the release of noradrenaline by 91%, the maximum inhibition reached with NPY was not higher than 60%, indicating that either the intrinsic activity of NPY is lower or much less axon terminals are equipped with NPY receptors. Peptide YY (PYY) also reduced the release of NA from mouse vas deferens.     

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kainic acid evoked release of D-[3H]aspartate from rat striatum in vitro: characterization and pharmacological modulation

A presynaptic stimulatory action of kainic acid (KA) on the release of glutamate from corticostriatal neurons is thought to contribute to the toxic effect of KA on cell bodies of neurons in the striatum. To characterize the action of KA on the presynaptic amino acid release, its effect was evaluated on the spontaneous efflux of D-[3H]aspartate (D-[3H]Asp), a marker for glutamatergic neurons, from slices of rat striatum in superfusion experiments. In the concentration range 0.5-10.0 mM, KA significantly increased the spontaneous efflux of D-[3H]Asp. Under similar conditions potassium (K+, 25 mM), veratridine, D-aspartic acid (D-Asp), and N-methyl-D-L-aspartic acid (NMDLA) also induced the efflux of the radiolabelled amino acid. The stimulatory effect of KA, like that of K+, was partly calcium dependent. The action of veratridine, D-Asp, and NMDA was not calcium dependent. Tetrodotoxin (TTX) blocked the action of veratridine on D-[3H]Asp efflux but did not affect the action of KA. In a sodium-free perfusion medium the action of KA was greatly reduced. Dihydrokainic acid produced an effect on D-[3H]Asp efflux comparable in magnitude with that produced by KA. The latter, at a dose of 5 mM, also stimulated the efflux of D-[3H]Asp from the cortex, hippocampus and the septum but its effect on these regions was weaker than its striatal effect. The action of several agents, which previously have been found to depress transmitter release in other systems and (or) to modify the neurotoxic action of KA in vivo, was evaluated on the KA-evoked D-[3H]Asp efflux from striatal slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depression of potassium-evoked striatal acetylcholine release by delta-receptor activation: inhibition by cholinoactive agents

To examine the role of delta-opioid receptors in the modulation of striatal acetylcholine (ACh) release, the action of D-Pen2,L-Pen5-enkephalin, a selective delta-opioid receptor agonist, was tested on [3H]ACh release from slices of the rat caudate-putamen. Slices, incubated with [3H]choline, were superfused with a physiological buffer and stimulated twice by exposure to a high potassium (K+) concentration. In the absence of a cholinesterase inhibitor, 1 microM D-Pen2,L-Pen5-enkephalin produced a 46 and 35% decrease in the release of [3H]ACh evoked by 15 and 25 mM K+, respectively. The depressant action of the enkephalin analogue was concentration dependent, with a maximal effect on K+-evoked [3H]ACh release occurring at 1.0 microM, and was completely blocked in the presence of the delta-opioid receptor selective antagonist, ICI 174864 (1 microM). In the presence of the cholinesterase inhibitors physostigmine (10 microM) and neostigmine (10 microM), or the muscarinic receptor agonist oxotremorine (10 microM), D-Pen2,L-Pen5-enkephalin did not depress the K+-evoked release of [3H]ACh. Atropine (1 microM) blocked the inhibitory effect of physostigmine on the depressant action of D-Pen2,L-Pen5-enkephalin. The results of this study indicate that delta-opioid receptor activation is associated with an inhibition of striatal ACh release, but this opioid-cholinergic interaction is not apparent under conditions of presynaptic muscarinic receptor activation.     

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na+ on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na+ hyperpolarized the B-cell membrane, inhibited 86Rb+ and 45Ca2+ efflux, but did not affect insulin release. In contrast, Li+ substitution for Na+ depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca2+ efflux and insulin release. Na+ replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca2+ efflux and insulin release. In the absence of Ca2+, 45Ca2+ and 86Rb+ efflux were inhibited and insulin release was unaffected by choline substitution for Na+. Na+ replacement by Li+ in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca2+ efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca2+, Li+ still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na+ plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.     

Differential Sensitivity to Blockade by 4-Aminopyridine of Presynaptic Receptors Regulating [3H]Acetylcholine Release from Rat Hippocampus

The inhibitory effect of an adenosine analogue, R-N6-phenylisopropyl adenosine (R-PIA), of the cholinergic agonist carbachol, and of morphine on 3H efflux from [3H]choline-labeled field-stimulated rat hippocampal slices was compared with that produced by two inhibitors of N- and L-type Ca2+ channels, omega-conotoxin (CgTx; conotoxin GVIA) and cadmium chloride. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release, with a maximal effect (an almost threefold increase) at 100 microM. 4-AP (100 microM) did not affect the actions of CgTx, cadmium chloride, and R-PIA but almost abolished the effect of carbachol and morphine. The present results indicate that presynaptic muscarinic and opiate receptors reduce acetylcholine release by a mechanism that is somewhat different from that used by adenosine A1 receptors. Furthermore, the results indicate that presynaptic A1 receptors on hippocampal cholinergic neurons do not primarily regulate 4-AP-dependent potassium channels, but that they might act directly on a Ca2+ conductance.     

Effects of forskolin and analogues on nicotinic receptor-mediated sodium flux,voltage-dependent calcium flux,and voltage-dependent rubidium efflux in pheochromocytoma PC12 cells

1. Forskolin, a naturally occurring diterpene that activates adenylate cyclase, HL706, a water-soluble derivative of forskolin (6 beta-[(piperidino)acetoxy]-7-desacetylforskolin) that is less potent than forskolin in activating adenylate cyclase, and 1,9-dideoxyforskolin, an analogue that does not activate adenylate cyclase, were examined for effects on the nicotinic receptor-mediated 22Na+ flux, a high potassium-induced 45Ca2+ flux through L-type calcium channels, and a high potassium-induced 86Rb+ efflux through a calcium-dependent potassium channels in PC12 cells. 2. Forskolin and analogues at 30 microM completely blocked carbamylcholine-elicited flux of 22Na+ through the nicotinic receptor-gated channel. 1,9-Dideoxyforskolin had an IC50 value of 1.6 microM with forskolin and HL706 being two- to three fold less potent. 3. Forskolin and its analogues appear to be noncompetitive blockers of the neuronal nicotinic receptor-channel complex in PC12 cells, but unlike many noncompetitive blockers, did not markedly enhance desensitization. Instead, forskolin, but not HL706 or 1,9-dideoxyforskolin, slightly antagonized the desensitization evoked by high concentrations of carbamylcholine. N-Ethylcarboxamidoadenosine, an adenosine analogue that elevates cyclic AMP and 8-bromo-cyclic AMP had no effect on desensitization. 4. Forskolin, HL706, and 1,9-dideoxyforskolin in the presence of carbamylcholine inhibited the binding of a noncompetitive blocker, [3H]perhydrohistrionicotoxin, to the muscle-type nicotinic receptor-channel complex in Torpedo electroplax membranes with IC50 values of 20 microM. Forskolin had no effect on [3H]perhydrohistrionicotoxin binding in the absence of carbamylcholine, while HL706 and 1,9-dideoxyforskolin still inhibited binding in the absence of carbamylcholine. 5. Forskolin, but not HL706 or 1,9-dideoxyforskolin had a slight inhibitory effect on the binding of [125I]alpha-bungarotoxin to acetylcholine recognition sites in Torpedo membranes. 1,9-Dideoxyforskolin at 30 microM, but not forskolin or HL706, markedly inhibited depolarization-evoked 45Ca+ flux and 86Rb+ efflux in PC12 cells, suggesting that 1,9-dideoxyforskolin has nonspecific inhibitory effects on a variety of ion channels.     

Furosemide and Ca2+ affect 86Rb+ efflux from pancreatic beta-cells by different mechanisms

The interaction between furosemide, calcium and D-glucose on the 86Rb+ efflux from beta-cell-rich mouse pancreatic islets was investigated in a perifusion system with high temporal resolution. Raising the glucose concentration from 4 to 20 mM induced an initial decrease in 86Rb+ efflux, which was followed by a steep increase and then a secondary decrease. Removal of extracellular calcium increased the 86Rb+ efflux at 4 mM D-glucose but reduced it at 20 mM. The initial biphasic changes in 86Rb+ efflux induced by 20 mM D-glucose were inhibited by calcium deficiency. Furosemide (100 microM) reduced the 86Rb+ efflux rate both at 4 and 20 mM D-glucose and the magnitudes appeared to be similar at either glucose concentration. Furosemide (100 microM) reduced the glucose-induced (10 mM) 45Ca+ uptake but did not affect the basal (3 mM D-glucose) 45Ca+ uptake. However, the ability of furosemide (100 microM) to reduce the 86Rb+ efflux at a high glucose concentration (20 mM) was independent of extracellular calcium. The inhibitory effects of furosemide and calcium deficiency on the 86Rb+ efflux rate appeared to be additive. It is concluded that the effect of furosemide on 86Rb+ efflux is not secondary to reduced calcium uptake and that the effects of furosemide and calcium deficiency are mediated by different mechanisms. The effect of furosemide is compatible with inhibition of loop diuretic-sensitive co-transport of Na+, K+ and Cl- and the effect of calcium deficiency with reduced activity of calcium-regulated potassium channels.