Extract of the marine alga Prymnesium patelliferum induces release of acetylcholine from cholinergic nerves in the rat bronchial smooth muscle

An extract of the marine algal flagellate Prymnesium patelliferum enhanced the spontaneous as well as the K⁺ evoked (51 mm K⁺) release of [³H]acetylcholine and endogenous acetylcholine from autonomic cholinergic nerves of rat bronchial smooth muscle. The effects were concentration-dependent and reversible. The enhancement of the K⁺ evoked release by the algal extract was partly dependent on extracellular Ca²⁺ and was significantly suppressed by the organic Ca²⁺ blockers ω-conotoxin GVIA (1 μm ), diltiazem (100 μm ), nifedipine (100 μm ) and flunarizine (100 μm ). The enhancement of the spontaneous release seemed Ca²⁺ independent and not sensitive to the Na⁺ channel blocker tetrodotoxin. Sphingosine (20 μm ), a protein kinase C inhibitor, strongly potentiated the enhancement of spontaneous release of [³H]acetylcholine induced by the algal extract whereas another protein kinase C inhibitor, 1-(5-quinolinesulphonyl)-2-methylpiperazine (H-7) (20 μm ), was without effect. A similar potentiation as seen with sphingosine was observed with procaine (100 μm ) and flunarizine (100 μm ). The results indicate that the enhancement of the K⁺ evoked release of [³H]acetylcholine by the toxic extract of P. patelliferum was partly caused by activation of voltage-dependent Ca²⁺ channels. The increase in the spontaneous release of [³H]acetylcholine and endogenous acetylcholine induced by the algal extract alone may be caused by an ionophore-like property of the algal extract. This effect of the algal extract may be enhanced by compounds that facilitate the interaction of the algal toxin with the plasma membrane such as the lipophilic compounds flunarizine, procaine and sphingosine.     

Visualization of serotonin neurones in the nodose ganglia of the cat. An autoradiographic study

After in vitro incubation of the nodose ganglia (right and left) in tritiated serotonin mixed with norepinephrine, the autoradiographic detection of the amine at the light-microscopic level showed an intense labeling over cell bodies; they have been characterized as gray type neurones at the ultrastructural level. Moreover, a labeling was observed over unmyelinated intraganglionic fibers.The uptake of exogenous serotonin by these neurones was demonstrated to be specific by using different experimental conditions. The same distribution of labeling was observed either after the incubation in ‘high affinity’ concentration (10^?7M), or in saturated concentrations (10^?5M or 10^?4M) of [³H]serotonin. The number of labeled cell bodies decreased after adding fluoxetine (an inhibitor of serotonin uptake) to the tracer.The specificity of the neuronal accumulation of serotonin was confirmed by interganglionic comparisons between the nodose and the superior cervical ganglia incubated under the same conditions. Several hundred cell bodies were labeled in the nodose ganglion incubated in [³H]serotonin (10^?6M) whereas there was no labeling in the superior cervical ganglion. On the other hand, most of the perikarya were labeled in the superior cervical ganglion incubated in [³H] norepinephrine (10^?6M), whereas no significant labeling was found in the nodose ganglion.These results provide some evidence that peripheral sensory neurones are able to take up exogenous serotonin.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

The gadolinium ion: a potent blocker of calcium channels and catecholamine release from cultured chromaffin cells

Gadolinium (Gd³⁺) is a trivalent ion of the lanthanide series which has a high charge density and a similar ionic radius to Ca²⁺. The effects of gadolinium ions on [³H]noradrenaline output and ⁴⁵Ca fluxes during resting conditions and stimulation were investigated in cultured bovine chromaffin cells. Chromaffin cells isolated from bovine adrenal medullae were plated on collagen-coated dishes (10⁶ cells/dish) in a medium containing 10% fetal calf serum. Seven-day-old cells were used in all studies.Exposure of chromaffin cells to 0.05mM Gd³⁺ produced 80 ± 5% and 81 ± 4% inhibition of the secretory responses to 10^?4M acetylcholine and 56 mM K⁺ respectively. Doubling of the extracellular concentration of Gd³⁺ produced an 87 ± 3% and 100 ± 2% inhibition of the acetylcholine-induced and K⁺-induced release of [ ³H]noradrenaline. Gd³⁺ (0.05 mM) also produced a parallel shift to the right in the dose-response relationship between extracellular Ca²⁺ concentrations and [ ³H]noradrenaline output during acetylcholine stimulation. This observation suggests a competitive antagonism between Ca²⁺ and Gd³⁺ in stimulus-secretion coupling.Gd³⁺ (0.05 mM) was an effective inhibitor (92.3 ± 0.7%) of the uptake of ⁴⁵Ca into chromaffin cells induced by a depolarizing concentration (56 mM) of K⁺. Furthermore, Gd³⁺ also inhibited the increase in both Ca²⁺ ? Ca²⁺ exchange mechanism and [ ³H]noradrenaline output observed in chromaffin cells upon the reintroduction of Ca²⁺ into a Ca²⁺]-free incubation medium.The results, which were obtained using low concentrations of Gd³⁺, indicate that gadolinium is a powerful inhibitor of the Ca²⁺ movements which are required for triggering amine release from chromaffin cells by different secretagogues. Moreover, the results also suggest that Gd³⁺ might be a useful tool for release and electrophysiological studies in other Ca²⁺-dependent systems.     

Development of cholinergic neurones in cultures of rat superior cervical ganglia. Role of calcium and macromolecules

Superior cervical ganglia from 3-day-old rats were grown in vitro for 14 days. Addition of an extract of rat heart to the culture medium led to no change in the activity of tyrosine hydroxylase but a doubling of the activity of choline acetyltransferase in the ganglia.To see whether this effect was due to a change in the concentration of unbound Ca²⁺, the calcium-binding capacity of extracts of adult rat heart was determined in a dialysis-binding study. Both cardiac extracts and serum lowered the levels of free calcium in the medium, but by relatively small amounts. When ganglia were grown in media containing 0, 2.5 and 6 mm Ca²⁺, there was no change in the ratio of choline acetyltransferase activity to tyrosine hydroxylase activity, although the activities of both enzymes increased at lower Ca²⁺ concentrations.When superior cervical ganglia were grown in Rose chambers under cellophane strips, the choline acetyltransferase/tyrosine hydroxylase ratio was higher than when superior cervical ganglia were loose in the medium. When a larger number of ganglia was grown under cellophane this further increased the ratio, while positioning of the ganglia so that substances could diffuse out from under the cellophane decreased the choline acetyltransferase/tyrosine hydroxylase ratio. When ganglia from 3-day-old rats were grown side by side with ganglia from 3-week-old rats, there was an even greater effect on the ratio of the two enzyme activities.We conclude that the ability of cardiac extracts to increase the ratio of choline acetyltransferase activity to tyrposine hydroxylase activity in ganglia is not due to changes in the Ca²⁺ concentration in the medium, but to the presence of a non-dialysable macromolecule. A similar factor also seems to be secreted by the ganglion itself.     

Mechanism of presynaptic actions of adenosine and acetylcholine on noradrenaline release in the guinea-pig heart

Isolated heart of the guinea-pig was stimulated transmurally (1 Hz), or by excess [K⁺] to evoke release of [³H]noradrenaline. The interactions of tetraethylammonium, acetylcholine and adenosine were investigated on [³H]noradrenaline overflow at different [Ca²⁺] concentrations. One mM tetraethylammonium doubled [³H]noradrenaline overflow, and at 30 mM the overflow was facilitated by about 50-fold. The facilitatory effect of 30 mM tetraethylammonium gradually decreased with reduction in [Ca²⁺] concentration. In 0.1 mM [Ca²⁺] overflow of [³H]noradrenaline was completely blocked. However, addition of 30 mM tetraethylammonium to 0.1 mM [Ca²⁺]-Krebs solution greatly facilitated the overflow. 35 mM K-induced [³H]noradrenaline overflow was facilitated by about 5-fold with 20 mM tetraethylammonium, and the facilitatory effect was blocked by 0.37 μM tetrodotoxin; 75 mM K-induced overflow was facilitated by only 1.5-fold by tetraethylammonium.Electrically induced overflow of [³H]noradrenaline was significantly reduced (30%) by 0.13 μM acetylcholine, and the effect increased up to about 75% with higher concentrations of acetylcholine (1.3 μM). 1.3 μM acetylcholine reduced 35 mM K-induced overflow by 30% and had no effect on 75 mM K-induced overflow. 0.37 μM tetrodotoxin had an almost similar effect on K-induced overflow. Ten-times higher concentrations of acetylcholine were needed to obtain over 70% inhibition of overflow produced by low or high K. The inhibitory effect of acetylcholine on [³H]noradrenaline overflow by 1 Hz was antagonized by increasing concentrations of tetraethylammonium (3 to 30 mM) in 2.5 mM Ca. The antagonism between acetylcholine and tetraethylammonium persisted even when Ca was lowered to 0.02 mM. However, if the overflow was enhanced by other means (high [Ca²⁺] plus phentolamine) to the same extent as with tetraethylammonium and low [Ca²⁺], then acetylcholine blocked overflow by 75%. 3.7 μM adenosine reduced [³H]noradrenaline overflow by about 54%, and this effect was completely prevented by 30 mM tetraethylammonium, either in 2.5 or 0.02 mM Ca.It is proposed that acetylcholine and adenosine interfere with nerve stimulation-evoked release of [³H]noradrenaline, presumably by altering electrical properties (i.e. conduction, resting membrane potential, duration of nerve action potential, etc.) of cardiac sympathetic nerve terminals. This primary action would subsequently lead to a reduction in the availability of [Ca²⁺] for the release process.     

Metabolism of arachidonic acid and prostaglandins in the torpedo electric organ: Modulation by the presynaptic muscarinic acetylcholine receptor

We have found that Torpedo electric organ readily synthesizes prostaglandin E₂ from both exogenous and endogenous arachidonate and that activation of the presynaptic muscarinic acetylcholine receptor increases the rate of prostaglandin E₂ synthesis by inducing the release of tissue arachidonate from its phospholipid pools. The incorporation of radiolabeled arachidonate into tissue phospholipids is slow and Ca²⁺ independent. However, the electric organ slices readily oxidize the externally added, radiolabeled arachidonate via the cyclo-oxygenase pathway, with prostaglandin E₂ being the major product (22 ± 4% of the initial radioactivity). This process is not affected by either Ca²⁺ or mepacrine. Torpedo electric organ slices also synthesize prostaglandin E₂ from endogenous substrates, and release it into the medium. This process, however, is enhanced by Ca²⁺ and inhibited by mepacrine. Activation of the Torpedo muscarinic acetylcholine receptor by the agonist oxotremorine results in a dose-dependent atropine-sensitive increase in the synthesis of prostaglandin E₂ from endogenous tissue substrates and in the concomitant release of arachidonate into the medium. By contrast, oxotremorine has no effect on either the formation of [¹⁴C]prostaglandin E₂ from exogenous arachidonate, the incorporation of radiolabeled arachidonate into tissue phospholipids or its liberation from prelabeled slices.These results suggest that activation of the muscarinic acetylcholine receptor induces lipolysis which results in the liberation of endogenous arachidonate and its subsequent conversion to prostaglandin E₂.     

Uptake of adenosine triphosphate by isolated adrenal chromaffin granules: A carrier-mediated transport

Partly purified chromaffin granules from bovine adrenal medulla were incubated in vitro with [³H]adenosine triphosphate (ATP). After several washes the particles were subjected to density gradient centrifugation. It was demonstrated that chromaffin granules had taken up radioactive material. Thin-layer chromatography revealed that more than 60% of the radioactivity was confined to [³H]ATP. Incubation experiments with a mixture of [³H]ATP and [³²P]ATP indicated that ATP was taken up by chromaffin granules as an intact molecule. The uptake of [³H]ATP was temperature dependent and linear up to 10 min. The rate of uptake depended upon the ATP concentration and levelled off at ATP concentrations above 2 mM. At this ATP concentration the rate was 0.268 nmol ATP/mg protein/min. The uptake of ATP was compared with that of catecholamines. Both processes were activated by Mg²⁺, but inhibited by N-ethylmaleimide and an uncoupler of oxidative phosphorylation. Atractyloside inhibited only the ATP-uptake, whereas reserpine only interfered with that of the catecholamines.It is suggested that this uptake mechanism for ATP is carrier-mediated and that it enables newly formed chromaffin granules to accumulate and to maintain a high nucleotide concentration, which is required for the formation of a storage complex with catecholamines.     

Spontaneous and evoked release of acetylcholine and a cholinergic false transmitter from brain slices: comparison to true and false transmitter in subcellular stores

Rat cerebral cortex slices were incubated with [¹⁴Ccholine and [³Hhomocholine to allow synthesis of [¹⁴Cacetylcholine and [³Hacetylhomocholine; release of the acetylated compounds was tested in the presence of eserine and hemicholinium-3. Both [¹⁴Cacetylcholine and [³Hacetylhomocholine were released spontaneously by a Ca²⁺-independent mechanism; exposure of the tissue to a high K⁺ medium resulted in a Ca²⁺-dependent increase in [¹⁴Cacetylcholine release (30-fold) and in [³Hacetylhomocholine release (5 to 7-fold). Thus, spontaneous and K⁺-evoked transmitter release could be distinguished on the basis of their molar ratios of true to false transmitters and these ratios were compared to the molar ratios of the transmitters in subcellular fractions prepared from the incubated tissue. The molar ratio of acetylcholine to acetylhomocholine released from the tissue spontaneously differed from the ratio in subcellular fractions containing occluded transmitters. The molar ratio of acetylcholine to acetylhomocholine released by K⁺ differed from the ratio in a fraction (H) containing occluded transmitters but was similar to the ratio in the fraction (D) containing monodisperse synaptic vesicles and the fraction (0) containing the majority of a soluble cytoplasmic marker. Comparison of the transmitter contents of subcellular fractions from unstimulated and K⁺-stimulated tissue showed that the three fractions (D, H and O) lost equal proportions of both transmitters as a result of K⁺ stimulation.It is concluded that acetylhomocholine may be released from brain slices both spontaneously and in response to stimulation via mechanism similar to those that release acetylcholine, but that there must be some differences in specificity of the acetylcholine storage and/or release processes. The results also indicate that spontaneous transmitter release originates from extravesicular stores; the results are consistent with a vesicular site of origin of evoked transmitter release but do not distinguish between nerve terminal vesicles and cytoplasm as the immediate source of evoked transmitter release.     

Acetylcholine synthesis from recaptured choline by a sympathetic ganglion

1. The recapture and re-use of choline formed by the hydrolysis of released acetylcholine (ACh) was studied in the superior cervical ganglion of the cat using radioactive tracer techniques. The ganglion's ACh store was labelled by perfusion, during preganglionic nerve stimulation, with Krebs solution containing [(3)H]choline.2. Preganglionic stimulation (5 Hz for 20 min) of ganglia containing [(3)H]ACh released similar amounts of radioactivity when perfusion was with neostigmine-choline-Krebs or with hemicholinium-Krebs. This indicated that neostigmine does not increase transmitter release.3. The amount of radioactivity collected from stimulated ganglia during perfusion with choline-Krebs was 39% of the amount of radioactivity collected during perfusion with medium containing neostigmine or hemi-cholinium. This difference in release was almost (85%) accounted for at the end of the experiment by extra radioactive ACh in the ganglia perfused with choline-Krebs. It is concluded that during preganglionic nerve stimulation approximately 50-60% of endogenously produced choline is recaptured for ACh synthesis; thus, during activity preganglionic nerve terminals appear selectively to accumulate choline.4. However, chronically decentralized ganglia accumulated as much choline as did acutely decentralized ganglia, and this was interpreted as indicating that at rest preganglionic nerve terminals do not selectively accumulate choline.5. Increased exogenous choline concentration increased the amount of radioactivity collected during nerve stimulation in the absence, but not the presence, of an anticholinesterase agent. The spontaneous efflux of radioactivity was little affected by changes in external choline levels. It is concluded that exogenous choline and choline made available from released transmitter compete for uptake into nerve terminals.     

Inhibition by acetylcholine of the stimulation-evoked release of [3H]acetylcholine from the guinea-pig myenteric plexus

The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum was incubated with [³H]choline and then superfused with Tyrode's solution. Exposure to [³H]choline resulted in the formation of [³H]acetylcholine which was released upon electrical field stimulation. The effects of exogenous acetylcholine, physostigmine and scopolamine on the stimulation-evoked release of [³H]acetylcholine were studied.In the absence of a cholinesterase inhibitor exogenous acetylcholine (10^?5 M) only slightly inhibited (by 26%) the evoked release of [³H]acetylcholine. If the cholinesterase activity of the preparation was reduced by about 50% in the presence of 10^?7M physostigmine, exogenous acetylcholine caused a concentration-dependent depression of the release evoked at 1 Hz. At a concentration of 10^?5 M acetylcholine the release was reduced by 76%. Scopolamine (10^?9 M) shifted the concentration-response curve for the inhibitory action of acetylcholine in a parallel manner to the right. From the dose ratio a pA₂ value of 9.8 for scopolamine against the release-inhibitory effect of acetylcholine was calculated. Physostigmine also inhibited the stimulation-evoked release of [³H]acetylcholine in a concentration-dependent manner, the maximal effect measured being an 85% reduction by 10^?5 M physostigmine. In the absence of a cholinesterase inhibitor scopolamine enhanced the evoked release of [³H]acetylcholine. The facilitatory effect was more marked at a stimulation frequency of 3 Hz (2-fold increase) than at 1 Hz (1.4-fold increase).It is concluded that extracellular acetylcholine decreases the stimulation-evoked release of neuronal acetylcholine. This inhibition is specifically mediated by a stimulation of presynaptic muscarinic receptors. The increase by scopolamine of the evoked release of [³H]acetylcholine suggests that previously liberated acetylcholine may trigger the negative feedback mechanism of acetylcholine release even if the cholinesterase activity is not inhibited, and that the presynaptic muscarinic receptors of the myenteric plexus have a physiological role in regulating the release of acetylcholine.     

An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction

The hyperpolarization produced by the application of 10^?5 M d-tubocurarine to the end-plate of mouse diaphragm-phrenic nerve preparation pretreated with an irreversible anticholinesterase was studied. This hyperpolarization has been attributed to a non-quantal release of acetylcholine by the nerve terminals. The usual hyperpolarization effect of about 10 mV was mimicked by adding 10^?7 M acetylcholine to the bath which contained 15mM Ca²⁺ to block the non-quantal release. The hyperpolarization effect was maximal (9.2 ± 0.8mV) in saline solution with the normal concentrations of Ca²⁺ (2mM) and K⁺ (5mM). Reducing [Ca²⁺] in the bath decreased the hyperpolarization effect. In Ca-free solution with 4.10^?4 M ethleneglycoltetra-acetate the hyperpolarization effect was3.8 ± 0.6mV. An increase of [Ca²⁺] also reduced the hyperpolarization effect and it was absent in 15mM of Ca²⁺. When the external concentration of Ca²⁺ was kept constant (2mM), both decreasing and increasing K⁺ concentration from 5 mM diminished the hyperpolarization effect. However, the decrease at elevated K⁺ concentrations was due to a reduction of membrane resistance and when the hyperpolarization effect was corrected for the change in size of the miniature endplate potential, it was unchanged in solutions with increased [K⁺] up to 13 mM. No traces of non-quantal release were observed during repetitive stimulation of the phrenic nerve in a medium containing 0.3 mM Ca²⁺. The hyperpolarization effect was not found in denervated muscles that lacked signs of neuromuscular transmission. The hyperpolarization effect was blocked by botulinum toxin while some evoked release of ACh was present. The time required for the block by 1 μg/ml toxin was shorter than for 0.25 μg/ml. The application of α-bungarotoxin produced a hyperpolarization of the membrane in the junctional region. The addition of d-tubocurarine failed to produce the hyperpolarization effect. The hyperpolarization effect was also absent when muscles were incubated with the desensitization potentiating drug, SKF-525A. The hyperpolarization effect was unchanged by replacement of Na⁺ with guanidine and was absent following the replacement of Na⁺ with arginine.The data suggest the channel responsible for the hyperpolarization effect is the same as that giving the endplate potential. The results are in agreement with the hypothesis that the hyperpolarization effect reflects a sustained non-quantal release of acetylcholine from the nerve terminal into the synaptic cleft.     

Autoradiographic localization of monoamine uptake in the central nervous system of a marine mollusc (Mactra stultorum L., pelecypoda)

Uptake of [³H]serotonin and [³H]dopamine has been investigated in the central nervous system of the marine mussel, Mactra stultorum L. by means of light and electron microscopic autoradiography. It was established that among the axon profiles of the neuropil of the ganglia, axons containing dense-core vesicles with 700–1500Ådiameter play the primary role in the uptake of both serotonin and dopamine. Although the nerve cell bodies in the cortical layer were not extensively labelled, glial cells and processes in this layer took up large amounts of the labelled amines.This autoradiographic study shows that transmitter re-uptake is a possible means of transmitter inactivation in the central nervous system of marine mussels and that glial elements might also participate in the uptake and inactivation of transmitter.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Potassium-induced secretion of melanocyte-stimulating hormone from isolated pars intermedia cells signals participation of voltage-dependent calcium channels in stimulus-secretion coupling

The recent discovery of action potentials with a Ca component in pars intermedia cells raised the question whether voltage-dependent Ca²⁺ influx participates in stimulus-secretion coupling in these cells. To test this, a study was made of the effects of a depolarizing concentration of K⁺ on melanocyte-stimulating hormone (MSH) output from isolated pars intermedia cells of rats and mice obtained by tissue disaggregation and maintained in culture for periods ranging from 2 h to 10 days. The cells were placed in a column which was perfused thereby permitting continuous monitoring of MSH output. The cells secreted MSH promptly upon exposure to 50 mM K⁺ and this effect was abolished when Ca²⁺ was omitted or when Co²⁺ was added to block Ca channels. It is concluded that activation of voltage-dependent Ca channels in pars intermedia cells allows influx of Ca ions that provides an adequate stimulus for MSH secretion. Furthermore, inhibitory effects of Ca²⁺ lack and of Co²⁺ addition on basal MSH output suggest the operation of such a Ca-dependent mechanism during spontaneous secretory activity. Secretory responses to high [K⁺], although intense, were brief and waned rapidly during continuous exposure to K⁺. Experiments showing that Ca²⁺ fails to elicit secretion when introduced with a delay of several minutes after exposure to high [K⁺] indicate that the transience of the response is due to inactivation of voltage-dependent Ca channels. The brevity of the response of the isolated pars intermedia cells to high [K⁺] may explain, at least in part, why stimulant effects of excess K⁺ have not been seen previously, or in the present experiments in intact neurointermediate lobes of rats. When the much smaller, and seemingly more favorable, neurointermediate lobes of mice were used, K⁺ elicited a Ca-dependent secretion of MSH comparable with that seen in the isolated cells. The demonstration that pars intermedia cells respond directly to a depolarizing stimulus by secreting MSH encourages the view that electrical activity in these cells is involved in the regulation of secretion.     

K+-stimulated release of labeled γ-aminobutyrate,glycine and taurine in slices of several regions of rat central nervous system

The effect of depolarization by high K⁺ concentration (68.5 mm) on the release of [³H] γ-aminobutyrate (GABA), [¹⁴C]glycine and [³⁵S]taurine was studied in superfused slices of rat cerebellum, striatum, hypothalamus, colliculi, cerebral cortex and ventral and dorsal halves of spinal cord. The release of [³H]GABA was notably stimulated by K⁺-depolarization in all regions studied, particularly in the cerebral cortex and the hypothalamus. The Ca²⁺-dependence of this phenomenon was studied in the cortex and ventral spinal cord; in both regions the K⁺-stimulated release was abolished when Ca²⁺ was omitted from the superfusing medium. The release of [¹⁴C]glycine was also stimulated in all regions, except the cerebellum, but to a lesser extent than that of GABA. This stimulation was Ca²⁺-dependent in the ventral spinal cord but not in the cerebral cortex. The release of [³⁵S]taurine was not affected by K⁺-depolarization in any of the regions studied.These results are consistent with a widely distributed neurotransmitter role for GABA. The Ca²⁺-dependence of glycine release in the spinal cord is in agreement with a role of this amino acid as a transmitter in this region. The finding that [³⁵S]taurine release was not stimulated by K⁺-depolarization in any of the regions studied, under experimental conditions identical to those resulting in an enhancement of [³H]GABA and [¹⁴C]glycine release, argues against a neurotransmitter role of this amino acid in brain and spinal cord.     

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release

Electrophysiological measurements of cell capacitance (C _m) and biochemical assays of [³H] serotonin ([³H]5-hydroxytryptamine or [³H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced C _m changes (ΔC _m), indicative of exocytosis, with the same voltage and Ca²⁺ dependency as the inward Ca²⁺ currents (I _Ca). Ba²⁺ was able to substitute for Ca²⁺ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca²⁺-versus Ba²⁺-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [³H]5-HT to study hormone release from cell populations. [³H]5-HT was released in a depolarization-, Ca²⁺- and time-dependent manner. Ba²⁺ also substituted for Ca²⁺ in depolarization-induced [³H]5-HT release. Thapsigargin, used to deplete Ca²⁺ stores, had no effects on Ca²⁺-triggered C _m increases, but Ca²⁺-triggered [³H]5-HT release was abolished. Ba²⁺-triggered [³H]5-HT release, however, was only slightly affected by Ca²⁺ store depletion. Ba²⁺ was found to act directly as a secretagogue of [³H]5-HT in intact cells, but not in C _m measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action. Received: 18 October 1995/Received after revision and accepted: 9 January 1996     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

心肌缺血再灌注损伤时细胞核膜钙泵功能的改变

目的:研究心肌缺血再灌注损伤时心肌细胞核膜钙泵(Ca²⁺-ATPase)功能的改变。方法:采用大鼠心肌缺血再灌注模型,密度梯度离心分离纯化细胞核,定磷法测定Ca-ATPase活性,[⁴⁵Ca²⁺]直接测定核钙摄取。结果:缺血再灌注损伤动物血浆MDA和FFA显著高于正常大鼠(P<0.01),再灌注心肌细胞核膜Ca-ATPase在[Ca²⁺]i较低时,酶活力低于正常细胞,而在[Ca²⁺]i较高浓度50μmol/L时酶活力高于正常细胞。对[⁴⁵Ca²⁺]摄取的变化与Ca-ATPase活性变化相似。结论:心肌缺血再灌注损伤时核膜Ca²⁺-ATPase功能发生了改变。     

Incorporation of [3H]2-deoxyglucose into glycogen in nervous tissues

Mice were injected with [³H]2-deoxyglucose and after 1 h high molecular weight glycogen was extracted from brain, liver and muscle tissues. 1–2% of the total radioactivity in each tissue was recovered in the glycogen fraction. Isolated buccal ganglia of the pond snail,Planorbis, and isolated abdominal ganglia of the horse leechHaemopis, were exposedin vitro to [³H]2-deoxyglucose for 1 h. 1–10% of the total radioactivity in these tissues was located in the high molecular weight glycogen fraction. Treatment of the extracted labelled glycogen fractions with amyloglucosidase caused release of the label in a manner consistent with the breakdown of labelled glycogen. Ganglia of snail and leech were exposed to [³H]2-deoxyglucose, fixed in glutaraldehyde and osmium tetroxide solutions, and prepared for autoradiography using aqueous histological processing. Light and electron microscope autoradiography showed that over 90% of the label was positively associated with glycogen particles (α- and β-particles). Certain previously published reports on the incorporation of 2-deoxyglucose into glycogen are discussed in relation to these findings.It is concluded that [³H]2-deoxyglucose is partially incorporated into glycogen in nervous tissue; the labelled 2-deoxyglycogen withstands aqueous histological processing and can be visualized directly by autoradiography.