Activation of central muscarinic receptors inhibit Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

Effects of Hg and CH3Hg on Ca fluxes in rat brain microsomes

A permanent increase in cytosolic Ca²⁺ levels seems to be associated with various pathological situations which may result in cell death. Hg²⁺ and CH₃Hg⁺ are potent neurotoxic agents, but the precise molecular mechanism(s) underlying their effects are not sufficiently understood. In the present study we investigated the potential role of Ca²⁺-ATPase located in the endoplasmic reticulum as a molecular target for mercury. Hg²⁺ and CH₃Hg⁺ inhibited Ca²⁺-ATPase and Ca²⁺ uptake by brain microsomes with similar potencies. However, the inhibitory potency of Hg²⁺ was higher than that of CH3Hg+, probably reflecting differences in the affinity for the sulfhydryl groups of these compounds. Passive or unidirectional Ca²⁺ efflux (measured in the absences of Ca²⁺-ATPase ligands) was increased significantly by CH₃Hg⁺ and Hg²⁺. Again, the potency of Hg²⁺ was higher than that of CH₃Hg⁺. Blockers of Ca²⁺ channels (ruthenium red, procaine, heparin) did not affect the increase in passive Ca t+ efflux induced by mercury compounds, possibly indicating that Ca²⁺ release occurs through Ca²⁺-ATPase. Addition of physiological concentrations of glutathione (GSH) simultaneously with mercury abolished the inhibitory effects of both forms of Hg on Ca Z+-transport. However, if the enzyme was first inhibited with Hg²⁺ or CH₃Hg⁺ and subsequently treated with GSH, the reversal of inhibition was about 50%, suggesting that part of the cysteinyl residues involved in the inhibitory actions of mercury in Ca t+-transport bind to mercury with an extremely high affinity.     

A high affinity calcium-stimulated ATPase in synaptic plasma membranes detectable in the presence and absence of exogenous magnesium

A Ca2+-stimulated ATPase activity detectable in the presence of submicromolar free Ca2+ was characterized in synaptic plasma membrane preparations. In the absence of exogenous magnesium, Ca2+-stimulated ATPase showed a K0.5 Ca2+ of 0.1 microM, Vmax of 125 nmol Pi/mg per min and a Hill number of 1.2. The addition of 1 mM MgCl2 increased basal ATPase activity by about 10-fold. After this activity had been subtracted, apparent values for Ca2+-stimulated ATPase were 0.033 microM (K0.5 Ca2+), 172 nmol Pi/mg per min (Vmax) with a Hill number of 4. These activities were non-significantly affected by ouabain, sodium azide, theophylline and sodium fluoride. The results obtained indicated that high affinity Ca2+-stimulated ATPase activity could be in synaptic plasma membrane-enriched fraction detected both in the presence and absence of exogenous magnesium. Furthermore, the data indicated that magnesium was required for calcium transport.     

Cholesterol oxidation reduces Ca + Mg-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes

These experiments examined effects of cholesterol oxidation on Ca²⁺+Mg²⁺-ATPase activity, Na⁺+K⁺-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 fromBrevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a¹t₁₂ of 1.19±0.09 min and a second pool with a²t₁₂ of 38.30±4.16 min. Activity of Ca²⁺+Mg²⁺-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca²⁺+Mg²⁺-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca²⁺+Mg²⁺-ATPase activity was not observed. Activity of Na⁺+K⁺-ATPase was not affected by different levels of cholesterol oxidation (5%–40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relatiobship observed between SPM interdigitation and Ca²⁺+Mg²⁺-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Studies on the relationship between Ca efflux from mitochondria and the release of amino acid neurotransmitters

It is known that transmitter release depends upon the entry of calcium ions into the synaptic terminal. Mitochondria have been suggested to play a key role on the regulation of intracellular Ca²⁺ concentration, thereby influencing transmitter liberation and synaptic transmission. Here we report the stimulatory effect of DNP and FCCP, uncouplers of oxidative phosphorylation, and quinidine, known to induce muscle contractures and increase Ca²⁺ efflux from muscle mitochondria, on both [¹⁴C]glutamic acid and [³H]GABA unstimulated release from synaptosomes and loss of⁴⁵Ca²⁺ from preloaded whole brain mitochondria. Results showed that the spontaneous efflux of non-putative neurotransmitters, leucine and α-aminoisobutyric acid, was less affected or not stimulated at all, under similar experimental conditions. Data suggest that calcium ions released from mitochondria are able to trigger neurotransmitter release.     

Differential effect of a dihydropyridine derivative to Ca entry pathways in neuronal preparations

Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca²⁺ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca²⁺-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheochromocytoma cells, while the high K^+- stimulated Ca²⁺ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K⁺-stimulated Ca²⁺ influx and transmitter releases. It was then suggested that the voltage-dependent Ca²⁺ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

Immunologic localization and kinetic characterization of a Na/Ca exchanger in neuronal and non-neuronal cells

The plasma membrane Na⁺/Ca²⁺ exchanger is believed to play a role in the regulation of Ca²⁺ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na⁺/Ca²⁺ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The V_max was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted 90% of the exchanger activity in the primary neurons and 70 and 50% of the activity in NCB-20 and CV-1 cells respectively. Thus the expression of the 36-kDa protein appears to be closely associated with the Na⁺/Ca²⁺ exchanger in neuronal cells and, possibly to a lesser extent, in non-neuronal cells.     

Histamine H1 receptors in UC-11MG astrocytes and their regulation of cytoplasmic Ca

Experiments were carried out on UC-11MG human astrocytoma cells, a continuous cell line that expresses a broad range of the biochemical and electrophysiological properties found in well-differentiated astrocytes. Because of a number of recent reports that astrocytes may express receptors for a variety of neuro-active substances, we measured the effects of 12 different neurotransmitters on intracellular free Ca²⁺ (Ca_i²⁺) in UC-11MG cells. Of these neurotransmitters only histamine was found to have a significant effect. Further characterization of the nature of the histamine response showed that UC-11MG cells express mepyramine-sensitive H₁ receptors the activation of which causes both mobilization of Ca²⁺ from intracellular stores and entry of Ca²⁺ from the extracellular solution. No evidence was found for the presence of H₂ receptors. The Ca_i²⁺ response was maximal at 300 μM histamine and was attenuated by increasing cell density. We suggest that this neurotransmitter may play a role in astrocytic function in the human CNS.     

Adenosine A1 receptors inhibit Ca channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A₁ receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca²⁺]_i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [³H]ACh, but not the release of [¹⁴C]GABA, was potentiated when adenosine A₁ receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca²⁺]_i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A₁ receptors. Our results show that adenosine A₁ receptors inhibit Ca²⁺ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A₁ receptors.     

Repetitive application of caffeine sensitizes caffeine-induced Ca release in bullfrog sympathetic ganglion neurons

Cytosolic free calcium concentration ([Ca(2+)](i)) was recorded from cultured bullfrog sympathetic ganglion cells loaded with the Ca(2+)-indicator Fura-2 or Fura-6F. Repetitive application of caffeine at a low concentration, which either failed to produce any [Ca(2+)](i) elevation or induced a small gradual increase in [Ca(2+)](i) at first challenge, produced a drastic increase in the amplitude of Ca(2+) release (caffeine response). The caffeine response eventually reached peak amplitude and then remained constant even if caffeine application were continued. This augmentation was maintained for up to 2 h, and was achieved not only by repetitive application but also by a long exposure of caffeine. However, this augmentation was neither achieved by repetitive administration of high K(+)-solution, nor caused by inhibition of phosphodiesterase by caffeine. The repetitive or sustained application of caffeine is suggested to increase the caffeine sensitivity of the calcium release channel to calcium, thus causing the potentiation of the caffeine response.     

Homeostatic compensation maintains Ca2+ signaling functions in Purkinje neurons in the leaner mutant mouse

Several human neurological disorders have been associated with mutations in the gene coding for the alpha1 subunit of the P/Q type voltage-gated calcium channel (alpha1A/Ca(v)2.1). Mutations in this gene also occur in a number of neurologically affected mouse strains, including leaner (tg(la)/tg(la)). Because the P-type calcium current is very prominent in cerebellar Purkinje neurons, these cells from mice with alpha1 subunit mutations make excellent models for the investigation of the functional consequences of native mutations in a voltage-gated calcium channel of mammalian central nervous system. In this review, we describe the impact of altered channel function on cellular calcium homeostasis and signaling. Remarkably, calcium buffering functions of the endoplasmic reticulum and calcium-binding proteins appear to be regulated in order to compensate for altered calcium influx through the mutant channels. Although this compensation may serve to maintain calcium signaling functions, such as calcium-induced calcium release, it remains uncertain whether such compensation alleviates or contributes to the behavioral phenotype.     

Calcium transport in and out of brain nerve endings in vitro—the role of synaptosomal plasma membrane Ca-ATPase in Ca-extrusion

The effects of cellular cations and ATP on calcium transport in and out of the nerve endings (synaptosomes) of mice brain were studied. The synaptosomes accumulated⁴⁵Ca time-dependently in the absence of ATP or other additions for at least 10 min. When ATP was present, the overall⁴⁵Ca accumulation was decreased and was maximal at about 4 min, after which it started to decline. Studies on the effects of cations with or without ATP at 4 min revealed selective activities for different cations. Mg²⁺ inhibited⁴⁵Ca accumulation in the absence of ATP but increased⁴⁵Ca accumulation when ATP was present. Similarly, ATP increased⁴⁵Ca accumulation only when Mg²⁺ was present. Na⁺, on the other hand, inhibited⁴⁵Ca accumulation both in the presence and absence of ATP and/or Mg²⁺. K⁺ increased⁴⁵Ca accumulation in the presence of ATP with or without Mg²⁺; however, K⁺-stimulation was not noted in the presence of 100 mM Na⁺, and in fact, K⁺ became inhibitory. The ATP-stimulated⁴⁵Ca accumulation in the presence of Mg²⁺ peaked within 4–6 min and then declined, suggesting release of⁴⁵Ca. Compatible with this notion, in⁴⁵Ca-loaded synaptosomes, ATP evoked⁴⁵Ca release which was accompanied by the appearance of P_i in the medium. Although ATP-activated⁴⁵Ca-release can occur in the presence of Mg²⁺, Mg²⁺ is not required and, infact, is inhibitory. Rapid release of⁴⁵Ca was also noted when⁴⁵Ca-loaded synaptosomes were incubated in the presence of Na⁺ without ATP. It is concluded that Mg²⁺, Na⁺,⁺K and ATP each has a specific role in regulating Ca²⁺ permeability of the plasma membrane, calcium binding and calcium extrusion.     

A high affinity Ca-ATPase in enriched nerve-ending plasma membranes

A preparation of enriched synaptosomal plasma membrane (SPM) from mouse brain was found to contain a Mg²⁺-independent high affinity Ca²⁺-activated ATPase. The preparation readily accumulated Ca²⁺ at micromolar concentration from the medium in the presence or absence of Mg²⁺ and released previously accumulated calcium upon the addition of ATP. It is concluded that SPM Ca²⁺-ATPase may have a functional role in removal of cytosol Ca²⁺.     

Ca-independent activity of Ca/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells

We investigated the involvement of Ca²⁺-independent activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the α isoform of CaM kinase II (Nb2a/α cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca²⁺-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca²⁺-independent activity. The autophosphorylation and appearance of Ca²⁺-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (αT286A kinase) or Asp (αT286D kinase) was examined. αT286A kinase was not converted to a Ca²⁺-independent form, and αT286D kinase had Ca²⁺-independent activity significantly as an autophosphorylated kinase. Cells expressing αT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing αT286D kinase had much longer neurites than Nb2a/α cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca²⁺-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.     

Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved

Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.     

Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells

Autoinhibition is a common mechanism observed in neurons to regulate neurotransmission. Released neurotransmitter interacts with presynaptic autoreceptors to inhibit subsequent release. The requisite elements for autoinhibition are present in chromaffin cells: secretory granules contain millimolar levels of ATP which is coreleased with catecholamines upon stimulation and the cells express purinergic receptors. We were interested to determine whether autoinhibition produced by ATP binding to purinergic receptors plays an important role in catecholamine release from chromaffin cells. In these studies, short depolarizations were used to elicit transmitter release measured by membrane capacitance. We find that stimulation of chromaffin cells results in the release of endogenous ATP which may suppress Ca(2+) channel currents and secretion. In the presence of a maximal concentration of ATP, both the amount of secretion and the maximal rate of release are about half that observed in the absence of ATP. ATP-mediated inhibition of secretion was blocked by Reactive Blue-2 suggesting the involvement of P(2Y) purinergic receptors. Prepulses to positive potentials that relieve the Ca(2+) channel block largely relieve the inhibition of secretion. Furthermore, when secretion is plotted as a function of Ca(2+) influx there is no apparent change in the relationship between control cells and those stimulated in the presence of ATP and prepulses. These results suggest that ATP diminishes secretion by inhibiting Ca(2+) influx into the cells. Our results indicate that feedback inhibition by ATP, mediated primarily by Ca(2+) channels, may be an important regulator of catecholamine release in chromaffin cells.     

Prostaglandins block a Ca-dependent slow spike afterhyperpolarization independent of effects on Ca influx in visceral afferent neurons

The blockade of a slow Ca2+-activated K+-dependent afterhyperpolarization (AHPs) in rabbit visceral sensory neurons by the prostaglandins, PGE1 and PGD2, was investigated to determine whether the blockade was indirectly due to a reduction in Ca2+ influx. The prostaglandins (PGs) could block the AHPs in the absence of any change in Ca2+-dependent spikes elicited in the presence of tetrodotoxin and tetraethylammonium bromide. A PG-induced decrease in Ca2+-dependent spike width observed in some neurons was temporally dissociated from the PG-induced block of the AHPs. In addition, a slow afterhyperpolarization produced by the application of the Ca2+ ionophore, A23187, was blocked by the PGs. It is concluded that a reduction in Ca2+ influx is not responsible for the PG-induced blockade of the AHPs.     

Does intracellular release of Ca participate in the afterhyperpolarization of a sympathetic neurone?

The afterhyperpolarization (AHP) of an action potential in the bullfrog sympathetic ganglion cell was highly sensitive to anions (a factor affecting Ca²⁺ release¹⁶) filled in a recording electrode; it was slower for citrate ion than for Cl⁻. The AHP recorded with a ‘KCl-electrode’ was suppressed drastically by D-600 (Ca²⁺-antagonist⁶) and prolonged significantly by caffeine (promoting Ca²⁺ release^4,9), while the AHP recorded with a ‘K₃-citrate-electrode’ was affected only slightly by these agents. Thus, these results suggest that Ca²⁺ entry during an action potential is the main origin of Ca²⁺ for the AHP recorded with a ‘KCl-electrode’, and favour the idea that the intracellular release of Ca²⁺ by an action potential as well as the Ca²⁺ influx participates in the mechanism of the AHP recorded with a ‘K₃-citrate-electrode’.