Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C

1. In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca²⁺ ([Ca²⁺]_i) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca²⁺]_i in thapsigargin-treated cells has allowed a mechanism contributing to this effect to be characterized.
2. Histamine and substance P stimulated [³H]-inositol monophosphate ([³H]-IP₁) accumulation in U373 MG cells. Concentration-response curves of [³H]-IP₁ accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li⁺ yielded best-fit EC₅₀ values of 19.1±1.5 μM for histamine and 5.7±1.3 nM for substance P.
3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 μM histamine resulted in a transient 597±50 nM increase in [Ca²⁺]_i. The best-fit EC₅₀ for histamine was 4.6±2.2 μM. The initial, transient, histamine response was often followed by further small transient increases in [Ca²⁺]_i.
4. Treatment of U373 MG cells with 5 μM thapsigargin, followed by the readdition of 1.8 mM Ca²⁺ to the perfusion buffer, resulted in a steady-state level of [Ca²⁺]_i 97±5 nM above pretreated levels (measured 400 s after readdition of Ca²⁺). Perfusion of histamine (100 μM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca²⁺]_i. This effect of histamine was normally reversible upon washout. The best-fit EC₅₀ for the histamine response was 0.8±0.2 μM. Substance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca²⁺]_i.
5. Neither 100 μM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn²⁺ in U373 MG cells pretreated with 5 μM thapsigargin, indicating that the depressant effect on steady-state raised [Ca²⁺]_i was probably not due to a block of Ca²⁺ entry.
6. The depressant effect of histamine on [Ca²⁺]_i was blocked by 1 μM mepyramine, and was partially reduced by pre-incubation with 1 μM staurosporine (61±7% reduction) and with Ro 31-8220 (24±10% and 50±6% reduction by 1 and 10 μM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine.
7. Neither 1 μM staurosporine nor 10 μM KN-62 inhibited the binding of [³H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17±1% and 55±2% by 1 and 10 μM Ro 31-8220, respectively. However, [³H]-IP₁ accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 μM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 μM, similarly potentiated the response to 100 μM histamine in 3 out of 4 experiments. KN-62 (10 μM) did not stimulate histamine-induced [³H]-IP₁ accumulation.
8. In HEPES buffer to which no Ca²⁺ had been added, histamine stimulated a transient 451±107 nM increase in [Ca²⁺]_i. Pretreatment with 1 μM and 10 μM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca²⁺]_i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca²⁺]_i. H-89 did not alter the histamine response.
9. The effect of histamine in stimulating Ca²⁺ extrusion was not confined to U373 MG cells, since 100 μM histamine also caused a rapid decrease in steady-state levels of [Ca²⁺]_i in thapsigargin-treated human HeLa cells.
10. The results indicate that agonists which increase [Ca²⁺]_i via activation of phosphoinositide metabolism can also stimulate a homeostatic mechanism which acts to reduce [Ca²⁺]_i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca²⁺-extrusion pump.

     

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca²⁺([Ca²⁺]_i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca²⁺-channel blocker.
2. Midazolam (30 and 100 μM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 μM NA.
3. NA (10 μM) produced a phasic, followed by a tonic increase in both [Ca²⁺]_i and force. Midazolam (10–100 μM) did not modify the resting [Ca²⁺]_i, but attenuated the NA-induced phasic and tonic increases in [Ca²⁺]_i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 μM) attenuated the NA-induced tonic, but not phasic, increases in [Ca²⁺]_i and force.
4. In Ca²⁺-free solution containing 2 mM EGTA, NA (10 μM) transiently increased [Ca²⁺]_i and force. Midazolam (10–100 μM), but not nicardipine (0.3 μM), attenuated this NA-induced increase in [Ca²⁺]_i and force, in a concentration-dependent manner. However, midazolam (10 and 30 μM), had no effect on the increases in [Ca²⁺]_i and force induced by 10 mM caffeine.
5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca²⁺- storage sites, NA slowly increased [Ca²⁺]_i and force. Nicardipine (0.3 μM) did not modify the resting [Ca²⁺]_i but partly attenuated the NA-induced increases in [Ca²⁺]_i and force. In the presence of nicardipine, midazolam (100 μM) lowered the resting [Ca²⁺]_i and further attenuated the remaining NA-induced increases in [Ca²⁺]_i and force.
6. The [Ca²⁺]_i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca²⁺ (0.16–2.6 mM) in Ca²⁺-free solution containing 100 mM K⁺. NA (10 μM) shifted the [Ca²⁺]_i-force relationship to the left and enhanced the maximum Ca²⁺-induced force. Under these conditions, whether in the presence or absence of 10 μM NA, midazolam (10 and 30 μM) attenuated the increases in [Ca²⁺]_i and force induced by Ca²⁺ without changing the [Ca²⁺]_i-force relationship.
7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca²⁺ mobilization. Midazolam attenuates NA-induced Ca²⁺ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca²⁺ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

     

[Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity

1. Cytosolic calcium concentration ([Ca²⁺]_i) by indo 1 microspectrofluorimetry in freshly isolated cells and isometric contraction of isolated rings were measured in response to muscarinic cholinoceptor stimulation in rat tracheal smooth muscle.
2. In isolated myocytes, acetylcholine (ACh, 0.031 μM) caused a rapid and graded increase in [Ca²⁺]_i up to a net amplitude of 492±26 nM (n=19) which gradually declined. The EC₅₀ for ACh was 0.13 μM. This first [Ca²⁺]_i peak was followed, when the ACh concentration increased, in approximately 5060% of the cells, by successive peaks of decreased amplitude ([Ca²⁺]_i oscillations) superimposed on the plateau phase. Whereas the percentage of cells exhibiting [Ca²⁺]_i oscillations remained consistent, the frequency of these oscillations increased to up to 10 min⁻¹ with an ACh concentration of 100 μM.
3. Removal of extracellular calcium (in the presence of EGTA, 0.4 mM) or addition of the voltage-dependent Ca²⁺-channel blocker verapamil (10 μM) did not alter the first [Ca²⁺]_i peak, the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca²⁺-ATPase, thapsigargin (1 μM), completely abolished the [Ca²⁺]_i response. Thapsigargin (1 μM) also blocked the caffeine (5 mM)-induced transient rise in [Ca²⁺]_i.
4. Atropine (a non-selective muscarinic cholinoceptor antagonist) and 4-diphenyl acetoxy N-methyl piperidine (4-DAMP, a selective M₃ antagonist) inhibited the [Ca²⁺]_i response to muscarinic cholinoceptor activation with an IC₅₀ of 13 and 20 nM, respectively. Pirenzepine (a selective M₁ antagonist) also totally inhibited the [Ca²⁺]_i response to ACh but with a higher IC₅₀ of 2 μM. Methoctramine (a selective M₂ antagonist) up to a concentration of 10 μM caused only a 40% inhibition. The effect of muscarinic antagonists on cumulative concentration-response curves (CCRC) for carbachol was assessed at the following concentrations: atropine and 4-DAMP at 3, 10 and 30 nM; pirenzepine 0.3, 1 and 3 μM, and methoctramine at 1, 3 and 10 μM. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA₂ values were 9.2, 8.8, 6.7 and 6.3, respectively.
5. In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca²⁺]_i oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca²⁺]_i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca²⁺]_i and the contractile responses are primarily dependent on activation of the M₃ receptor subtype.

     

ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia

1. ATP (10–100 μM), but not glutamate (100  μM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 μM) nor glutamate (100 μM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 μM), which is metabolized in the cytosol to BAPTA (an intracellular Ca²⁺ chelator), completely inhibited the plasminogen release evoked by ATP (100 μM). The Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 induced plasminogen release in a concentration-dependent manner (0.3 μM to 10 μM).
2. ATP induced a transient increase in the intracellular calcium concentration ([Ca²⁺]_i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 μM) had no effect on [Ca²⁺]_i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μM) stimulated an increase in [Ca²⁺]_i similar to that of the first application (21 out of 21 cells).
3. The ATP-evoked increase in [Ca²⁺]_i was totally dependent on extracellular Ca²⁺, 2-Methylthio ATP was active (7 out of 7 cells), but α,β-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca²⁺]_i. Suramin (100 μM) was shown not to inhibit the ATP-evoked increase in [Ca²⁺]_i (20 out of 20 cells). 2′- and 3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), a selective agonist of P2X₇ receptors, evoked a long-lasting increase in [Ca²⁺]_i even at 1 μM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′-triphosphate-2′, 3′-dialdehyde (oxidized ATP, 100 μM), a selective antagonist of P2X₇ receptors, blocked the increase in [Ca²⁺]_i induced by ATP (10 and 100 μM).
4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca²⁺]_i via the ionotropic P2X₇ receptor which stimulates the release of plasminogen from the microglia.

     

The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase

1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca²⁺]_i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃), [Ca²⁺]_i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
2. Opioid receptor binding was assessed with [³H]-diprenorphine ([³H]-DPN) as a radiolabel. Ins(1,4,5)P₃ and cyclic AMP were measured by specific radioreceptor assays. [Ca²⁺]_i was measured fluorimetrically with Fura-2.
3. Scatchard analysis of [³H]-DPN binding revealed that the B_max varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [³H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pK_i values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na⁺ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pK_i value of 6.76±0.04.
4. Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC₅₀=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml⁻¹ for 24 h)-sensitive and naloxone-reversible manner (K_i=1.7 nM). Morphine (1 μM) and [D-Ala², MePhe⁴, gly(ol)⁵]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen², D-Pen⁵], enkephalin (DPDPE, 1 μM) did not.
5. Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P₃ formation, with a pEC₅₀ of 7.95±0.15 (n=5). PTX (100 ng ml⁻¹ for 24 h) abolished, whilst Ni²⁺ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P₃ response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P₃ formation. Fentanyl (1 μM) also caused an increase in [Ca²⁺]_i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca²⁺]_i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca²⁺]_i.
6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca²⁺]_i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca²⁺ ([Ca²⁺]_i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca²⁺ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca²⁺ and an influx of extracellular Ca²⁺, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA₂ activation.
2. In RAW 264.7 cells UTP (100 μM) and thapsigargin (1  μM) caused 2 and 1.2 fold increases, respectively, in [³H]-AA release. The release of [³H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca²⁺-free buffer, BAPTA (30 μM)-containing buffer or in the presence of the cPLA₂ inhibitor MAFP (50 μM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml⁻¹) or 4-bromophenacyl bromide (100 μM). By contrast, aristolochic acid (an inhibitor of sPLA₂) had no effect on UTP and thapsigargin responses.
3. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 μM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca²⁺]_i rise.
4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM–3 μM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 μM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release.
5. Short-term treatment with PMA (1 μM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca²⁺]_i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA.
6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 μM), Ro 31-8220 (10 μM), Go 6976 (1 μM) and the down-regulation of PKC.
7. Following treatment of cells with SK&F 96365 (30 μM), thapsigargin-, but not UTP-, induced Ca²⁺ influx, and the accompanying AA release, were down-regulated.
8. Neither PD 98059 (100 μM), MEK a inhibitor, nor genistein (100 μM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin.
9. We conclude that UTP-induced cPLA₂ activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca²⁺, which is essential for the activation of cPLA₂ by UTP and thapsigargin. The [Ca²⁺]_i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca²⁺]_i was also shown to be involved in AA release.

     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

1. To characterize increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) associated with discharge of action potentials, membrane potential and [Ca²⁺]_i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques.
2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca²⁺]_i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca²⁺]_i transients with an almost constant amplitude.
3. Nicardipine (1 μM) and La³⁺ (10 μM), blockers of voltage-dependent Ca²⁺ channels (VDCCs), abolished both the action potential discharge and the [Ca²⁺]_i transient.
4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca²⁺-activated K⁺ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca²⁺]_i transients.
5. Thapsigargin (1 μM), an inhibitor of SR Ca²⁺-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca²⁺]_i transients without affecting action potentials. In addition, in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca²⁺ current but exerted a similar effect on its concomitant rise in [Ca²⁺]_i to that on the action potential-triggered [Ca²⁺]_i transient.
6. Similar action potential-triggered [Ca²⁺]_i transients were induced by brief exposures to high-K⁺ solution. They were not decreased, but rather increased, after depletion of intracellular Ca²⁺ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open-lock of Ca²⁺-induced Ca²⁺ release (CICR)-related channels.
7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca²⁺]_i. Intracellular Ca²⁺ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca²⁺]_i transients by replenishing cytosolic Ca²⁺. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca²⁺]_i transient.

     

The ability of a new hypoglycaemic agent,A-4166, compared to sulphonylureas,to increase cytosolic Ca2+ in pancreatic β-cells under metabolic inhibition

1. N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new non-sulphonylurea oral hypoglycaemic agent which stimulates insulin release by increasing cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in β-cells.
2. We studied comparative effects of A-4166 and sulphonylureas on [Ca²⁺]_i, measured by dual-wavelength fura-2 microfluorometry, in single rat pancreatic β-cells under normal conditions and conditions where glucose metabolism was inhibited.
3. A glucokinase inhibitor, mannoheptulose (10 mM), a mitochondrial respiratory inhibitor, KCN (100 μM), and uncouplers, dinitrophenol (DNP, 50 μM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.3 μM), were used to abolish glucose-induced increases in [Ca²⁺]_i in a reversible manner.
4. Under control conditions, A-4166 was one order more potent than tolbutamide in increasing [Ca²⁺]_i, and maximal responses were evoked by 30 μM A-4166 and 300 μM tolbutamide. These equipotent concentrations were employed for the comparative study where glucose metabolism was inhibited.
5. In the presence of mannoheptulose, [Ca²⁺]_i responses to tolbutamide, but not those to A-4166, were attenuated in a reversible manner.
6. KCN, DNP and FCCP inhibited [Ca²⁺]_i responses to tolbutamide to a much greater extent than those to A-4166. Responses to tolbutamide even at 3.3 times the equipotent concentration (1000 μM) were also markedly attenuated by these inhibitors. Responses evoked by another sulphonylurea, gliclazide, were inhibited by DNP to a larger extent than A-4166-induced responses.
7. The results indicate that A-4166 acts more effectively than sulphonylureas to increase [Ca²⁺]_i in β-cells during metabolic inhibition.

     

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca²⁺]_i, in a population of rat striatal astrocytes loaded with the fluorescent Ca²⁺ probe Fura2, by means of fluorescence spectrometry.
2. ATP triggered a fast and transient elevation of [Ca²⁺]_i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5′-O-(2-thiodiphosphate) (ADPβS), as well as uridine-5′-triphosphate (UTP) resembled that of ATP, while α,β-methylene-ATP (α,β-meATP) and β,γ-methylene-ATP (β,γ-meATP) were totally ineffective.
3. Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response.
4. Extracellular Ca²⁺ did not contribute to the observed [Ca²⁺]_i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni²⁺ (1 mM) or Mn²⁺ (1 mM) did not modify the nucleotide responses.
5. Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide-evoked [Ca²⁺]_i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved.
6. Both application of the G-protein blocker guanosine-5′-O-(2-thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml⁻¹) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 μM), significantly reduced the ATP-evoked [Ca²⁺]_i rise.
7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca²⁺]_i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

     

Effects of κ-opioid receptor stimulation in the heart and the involvement of protein kinase C

1. The role of protein kinase C (PKC) in mediating the action of κ-receptor stimulation on intracellular Ca²⁺ and cyclic AMP production was determined by studying the effects of trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte.
2. U50,488H at 2.5–40 μM decreased both the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the κ-agonist, that were blocked by a selective κ-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ-receptor stimulation.
3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca²⁺]_i transient induced by caffeine, that depletes the SR of Ca²⁺, was used as an indicator of Ca²⁺ content in the SR. The caffeine-induced [Ca²⁺]_i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine-induced [Ca²⁺]_i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca²⁺ from the SR. When the increase in IP₃ production in response to κ-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ-receptor stimulation does not involve IP₃. The observations suggest that PKC may act directly at the SR.
4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ-receptor stimulation on Ca²⁺ in the SR and cyclic AMP production, both of which play an essential role in Ca²⁺ homeostasis in the heart.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells

1. The effects of exogenous adenosine 5′-triphosphate (ATP) and α,β-methylene ATP (α,βmeATP) on C6BU-1 cells transfected with P2X₂ and P2X₃ subtypes, separately or together (P2X₂₊₃), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods.
2. Untransfected C6BU-1 cells showed no intracellular Ca²⁺ ([Ca²⁺]_i) increase in response to depolarizing stimulation with high K⁺ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca²⁺ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca²⁺ influx through the P2X subtypes.
3. In C6BU-1 cells transfected with the P2X₂ subtype, ATP (more than 10 μM) but not α,βmeATP (up to 100 μM) evoked a rise in [Ca²⁺]_i.
4. In the cells transfected with the P2X₃ subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 μM of α,βmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca²⁺]_i rise and the current response was seen only with the P2X₃ subtype. In addition, the agonist-induced rise in [Ca²⁺]_i was observed only after the first application because of desensitization of this subtype.
5. In C6BU-1 cells co-transfected with P2X₂ and P2X₃, ATP at 1 μM evoked a [Ca²⁺]_i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X₂ and P2X₃, when compared to transfection with the P2X₃ subtype alone. The desensitization of the P2X₂₊₃ was apparently slower than that of the P2X₃ subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca²⁺]_i rise.
6. These results suggest that the P2X₂ and P2X₃ subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca²⁺ dynamics than that by homogenously expressed P2X₂ or P2X₃

     

Relationships between structure and vascular activity in a series of benzylisoquinolines

1. In the present work, the properties of 3-methyl isoquinoline, 3,4-dihydropapaverine, tetrahydropapaverine and tetrahydropapaveroline were compared with those of papaverine and laudanosine. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KC1, and a determination of the affinity of the compounds for α₁-adrenoceptors and calcium channel binding sites, with [^#H]-prazosin, [^#H]-nitrendipine and [^#H]-(+)-cis-diltiazem binding to rat cerebral cortical membranes. The effects of papaverine derivatives on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined.
2. The three papaverine derivatives show greater affinity than papaverine for the [^#H]-prazosin binding site. They are therefore more selective as inhibitors of [^#H]-prazosin binding as opposed to [^#H]-(+)-cis-diltiazem, while papaverine appears to have approximately equal affinity for both. [^#H]-nitrendipine binding was not affected by either papaverine or papaverine derivatives in concentrations up to 100 μM. 3-Methylisoquinoline had no effect on any of the binding sites assayed.
3. Contractions evoked by noradrenaline (1 μM) in rat aorta were inhibited in a concentration-dependent manner by 3,4-dihydropapaverine, tetrahydropapaverine and with a lower potency, by tetrahydropapaveroline. In Ca²⁺-free solution, tetrahydropapaverine and to a lesser extent, tetrahydropapaveroline, inhibited the noradrenaline (1 μM) evoked contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (10 mM). This suggests that these alkaloids do not act at the intracellular level, unlike papaverine which inhibits the contractile response to caffeine and noradrenaline.
4. Inositol phosphates formation induced by noradrenaline (1 μM) in rat aorta was inhibited by tetrahydropapaverine (100 μM) and tetrahydropapaveroline (300 μM), thus suggesting that α_1D-adrenoceptors are coupled to phosphoinositide metabolism in rat aorta.
5. Unlike papaverine, which has a significant effect on all the PDE isoforms, the three alkaloids assayed did not have an inhibitory effect on the different forms of PDE isolated from bovine aorta.
6. These results provide evidence that papaverine derivatives with a partially or totally reduced isoquinoline ring have a greater affinity for α₁-adrenoceptors and a lower affinity for benzothiazepine sites in the Ca²⁺-channel than papaverine. This structural feature also implies a loss of the inhibitory activity on PDE isoforms. The planarity of the isoquinoline ring (papaverine) impairs the interaction with the α₁-adrenoceptor site and facilitates it with the Ca²⁺-channels and PDEs, whereas the more flexible tetrahydroisoquinoline ring increases the binding to α₁-adrenoceptors.

     

Effects of bradykinin on signal transduction,cell proliferation,and cytokine,prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B₂-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells.
2. The aims of the present studies were to demonstrate the specific binding of [³H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca²⁺-mobilization ([Ca²⁺]_i), cell proliferation (via [³H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E₂ (PGE₂).
3. Specific [³H]-BK binding comprised 83±2% of the total binding, and was of high affinity (K_d=1.66±0.52 nM, n=5), saturable (B_max=640±154 fmol g⁻¹ wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]BK; icatibant): K_i=0.17±0.07 nM; BK: K_i=1.0±0.11 nM; [Tyr⁸]-BK: K_i=12.9±2.3 nM; [des-Arg⁹]-BK: K_i>9,200 nM (all n=3–5)).
4. BK potently stimulated PI turnover (EC₅₀=2.3±0.3 nM; n=7) and [Ca²⁺]_i mobilization (EC₅₀=8–20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM–10 μM Hoe-140, a selective B₂-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC₅₀=3.0±1.6 μM). BK-induced [Ca²⁺]_i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
5. BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [³H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1α (IL-1α; 10 ng ml⁻¹) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 μg ml⁻¹) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE₂ from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
7. In conclusion, these data indicate that the CEPI cells express high-affinity [³H]-BK binding sites representing B₂-subtype BK receptors coupled to PI turnover and [Ca²⁺]_i mobilization which appear to stimulate [³H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE₂, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

     

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 μM–3 mM) produced concentration-dependent, well-sustained contraction. Its −logEC₅₀ was 3.74±0.05 (mean±s.e.mean) and its maximal effect was equivalent to 97.5±4.2% of the response to acetylcholine (ACh, 1 mM).
2. Vanadate (200 μM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 μM), zileuton (10 μM), a mixture of atropine, mepyramine and phentolamine (each at 1 μM), or by mast cell degranulation with compound 48/80.
3. Vanadate (200 μM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 μM) or to a Ca²⁺-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 μM) in Ca²⁺-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca²⁺-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca²⁺ stores. In such tissues cyclopiazonic acid (CPA; 10 μM) prevented Ca²⁺-induced recovery of vanadate-induced contraction.
4. Tissue incubation in K⁺-rich (80 mM) PSS, K⁺-free PSS, or PSS containing ouabain (10 μM) did not alter vanadate (200 μM)-induced contraction. Ouabain (10 μM) abolished the K⁺-induced relaxation of human bronchus bathed in K⁺-free PSS. This action was not shared by vanadate (200 μM). The tissue content of Na⁺ was increased and the tissue content of K⁺ was decreased by ouabain (10 μM). In contrast, vanadate (200 μM) did not alter the tissue content of these ions. Tissue incubation in a Na⁺-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 μM).
5. Vanadate (200 μM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 μM), staurosporine (1 μM) and calphostin C (1 μM). Genistein (100 μM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate.
6. Vanadate (0.1–3 mM) and ACh (1 μM–3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca²⁺-free medium either alone or in combination with ryanodine (10 μM).
7. In human cultured tracheal smooth muscle cells, histamine (100 μM) and vanadate (200 μM) each produced a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).
8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 μM) in human bronchus was associated with cellular depolarization.
9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca²⁺ from an intracellular store. The Ca²⁺ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca²⁺]_i that are close to basal.

     

Inhibition of [3H]-U69593 binding and the cardiac effects of U50,488H by calcium channel blockers in the rat heart

1. The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 ((5a,7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)-benzeneacetamide), a specific κ-opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (4 nM) in a dose-dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine.
2. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of κ-receptor stimulation by a specific κ-receptor agonist, U50,488H (trans-(±-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80–800 nmol dose-dependently induced arrhythmias, which were completely abolished by a selective κ-receptor antagonist, nor-BNI (nor-binaltorphimine,17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-binorphinan-3,4′,14, 14′-tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose-dependent manner. The ED₅₀ values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca²⁺ influx in the cardiac muscle.
3. Measurement of [Ca²⁺]_i in the absence of free extracellular Ca²⁺ by a spectrofluorometric method, with fura-2 as Ca²⁺ indicator, showed that U50,488H 5×10⁻⁵ M slowly increased [Ca²⁺]_i in single ventricular myocytes and this effect was abolished by pretreatment with nor-BNI (5 μM), or ryanodine (5 μM). Verapamil 1 and 10 μM abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 μM had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine-induced Ca²⁺ transient.
4. The observations suggest that CCBs may inhibit the actions of κ-receptor stimulation at the level of the κ-receptor.

     

Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

1. Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca²⁺]_i) entry through nicotinic ACh receptors (AChRs) by use of Ca²⁺-aequorin luminescence.
2. ACh (0.1–3 mM, 20 μl) elicited biphasic elevation of [Ca²⁺]_i (fast and slow Ca²⁺ mobilization) in muscle cells. The peak amplitude of the slow Ca²⁺ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mM) of applied ACh.
3. A pulse of nicotinic agonists, (−)-nicotine (10 mM) and 1,1-dimethyl-4-phenyl-piperazinium (10 mM), but not a muscarinic agonist pilocarpine (10 mM), also elicited a biphasic Ca²⁺ signal.
4. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 μg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca²⁺ component caused by ACh application was observed.
5. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca²⁺ signal reflecting Ca²⁺ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

     

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[³H]-arginine to L-[³H]-citrulline.
2. Elevation of extracellular K⁺ to 20, 35 or 60 mM concentration-dependently augmented L-[³H]-citrulline production. The response to 35 mM KCl was abolished by N^G-nitro-L-arginine (100 μM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl₂ concentration up to 10 mM also prevented the K⁺ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity.
3. However, the effect of high K⁺ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine.
4. The 35 mM K⁺ response was insensitive to ω-conotoxin GVIA (1 μM) and nifedipine (100 μM), but could be prevented in part by ω-agatoxin IVA (0.1 and 1 μM). The inhibition caused by 0.1 μM ω-agatoxin IVA (∼30%) was enhanced by adding ω-conotoxin GVIA (1 μM) or nifedipine (100 μM). Further inhibition (up to above 70%) could be observed when the three Ca²⁺ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 μM) the K⁺-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μM ω-conotoxin GVIA plus 100 μM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX.
5. It was concluded that elevation of extracellular [K⁺] causes NO synthase activation by external Ca⁺ entering cells mainly through channels of the P/Q-type. Other Ca²⁺ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.