Calcium/Calmodulin-Dependent Protein Kinase II Mediates Hippocampal Glutamatergic Plasticity During Benzodiazepine Withdrawal

Benzodiazepine withdrawal anxiety is associated with potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) currents in hippocampal CA1 pyramidal neurons attributable to increased synaptic incorporation of GluA1-containing AMPARs. The contribution of calcium/calmodulin-dependent protein kinase II (CaMKII) to enhanced glutamatergic synaptic strength during withdrawal from 1-week oral flurazepam (FZP) administration was further examined in hippocampal slices. As earlier reported, AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, along with increased single-channel conductance in neurons from 2-day rats, estimated by non-stationary noise analysis. Input–output curve slope was increased without a change in paired-pulse facilitation, suggesting increased AMPAR postsynaptic efficacy rather than altered glutamate release. The increased mEPSC amplitude and AMPAR conductance were related to CaMKII activity, as intracellular inclusion of CaMKIINtide or autocamtide-2-related inhibitory peptide, but not scrambled peptide, prevented both AMPAR amplitude and conductance changes. mEPSC inhibition by 1-naphthyl acetyl spermine and the negative shift in rectification index at both withdrawal time points were consistent with functional incorporation of GluA2-lacking AMPARs. GluA1 but not GluA2 or GluA3 levels were increased in immunoblots of postsynaptic density (PSD)-enriched subcellular fractions of CA1 minislices from 1-day FZP-withdrawn rats, when mEPSC amplitude, but not conductance, was increased. Both GluA1 expression levels and CaMKIIα-mediated GluA1 Ser⁸³¹ phosphorylation were increased in PSD-subfractions from 2-day FZP-withdrawn rats. As phospho-Thr²⁸⁶CaMKIIα was unchanged, CaMKIIα may be activated through an alternative signaling pathway. Synaptic insertion and subsequent CaMKIIα-mediated Ser⁸³¹ phosphorylation of GluA1 homomers contribute to benzodiazepine withdrawal-induced AMPAR potentiation and may represent an important hippocampal pathway mediating both drug-induced and activity-dependent plasticity.     

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 two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells

1. In A7r5 cells loaded with the Ca²⁺ indicator fura-2, we examined the effect of a Ca²⁺ channel blocker SK&F 96365 on increases in intracellular free Ca²⁺ concentrations ([Ca²⁺]i) and Mn²⁺ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed.
2. Higher concentrations (⩾10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca²⁺]i: removal of extracellular Ca²⁺ abolished only the latter. A blocker of L-type voltage-operated Ca²⁺ channel (VOC) nifedipine at 1 μM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 μM).
3. Lower [ET-1] (⩽1 nM) evoked only a sustained elevation in [Ca²⁺]i which depends on extracellular Ca²⁺. The elevation was partly sensitive to nifedipine but not SK&F 96365.
4. In the presence of 1 μM nifedipine, higher [ET-1] increased the rate of Mn²⁺ quenching but lower [ET-1] had little effect.
5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of −60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca²⁺ in the bath solution with Mn²⁺. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365.
6. In a bath solution containing only Ca²⁺ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn²⁺, whereas that at higher [ET-1] was partly sensitive to SK&F 96365.
7. These results indicate that in addition to VOC, ET-1 activates two types of Ca²⁺-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn²⁺.

     

Spider Polyamine Toxin

In the early 1980s, a new type of polyamine toxins was found in the venom of several orb-web spiders. Joro spider toxins (JSTXs) in the venom of Nephila clavata and Nephila spider toxins (NSTXs) derived from Nephila maculata blocked postsynaptic glutamate receptors in the invertebrate and vertebrate nervous system. Subsequently, chemical characterization and synthesis of JSTXs and NSTXs were carried out. These toxins and a synthetic analog, 1-naphthylacetylated spermine (Naspm), effectively suppressed glutamate channel responses of AMPA/KA type in mammalian central neurons. By use of recombinant subunit receptors expressed in Xenopus oocytes, JSTX was found to cause subunit specific block of the Ca²⁺-permeable AMPA receptors. This specific nature of JSTX was utilized to identify Ca²⁺-permeable AMPA receptors in various neurons and glial cells. The JSTXs strongly suppressed excitatory postsynaptic currents (EPSCs) in the hippocampal CA1 neurons after transient brain ischemia. The results indicate that JSTXs are effective at blocking abnormal EPSCs that may induce Ca²⁺ accumulation leading to delayed neuronal death after transient ischemic insult. Recent evidence shows that Ca²⁺-permeable AMPA receptors are involved in a variety of nervous diseases including amyotrophic lateral sclerosis (ALS) and allodynia. The JSTXs are potentially useful to understand pathogenesis of these diseases.     

mTORC1 Inhibition in the Nucleus Accumbens ‘Protects' Against the Expression of Drug Seeking and ‘Relapse' and Is Associated with Reductions in GluA1 AMPAR and CAMKIIα Levels

The mechanistic target of rapamycin complex 1 (mTORC1) is necessary for synaptic plasticity, as it is critically involved in the translation of synaptic transmission-related proteins, such as Ca²⁺/Calmodulin-dependent kinase II alpha (CAMKIIα) and AMPA receptor subunits (GluAs). Although recent studies have implicated mTORC1 signaling in drug-motivated behavior, the ineffectiveness of rapamycin, an mTORC1 inhibitor, in suppressing cocaine self-administration has raised questions regarding the specific role of mTORC1 in drug-related behaviors. Here, we examined mTORC1''s role in three drug-related behaviors: cocaine taking, withdrawal, and reinstatement of cocaine seeking, by measuring indices of mTORC1 activity and assessing the effect of intra-cerebroventricular rapamycin on these behaviors in rats. We found that withdrawal from cocaine self-administration increased indices of mTORC1 activity in the nucleus accumbens (NAC). Intra-cerebroventricular rapamycin attenuated progressive ratio (PR) break points and reduced phospho-p70 ribosomal S6 kinase, GluA1 AMPAR, and CAMKIIα levels in the NAC shell (NACsh) and core (NACc). In a subsequent study, we treated rats with intra-NACsh infusions of rapamycin (2.5 μg/side/day for 5 days) during cocaine self-administration and then tracked the expression of addiction-relevant behaviors through to withdrawal and extinction. Rapamycin reduced drug seeking in signaled non-drug-available periods, PR responding, and cue-induced reinstatement, with these effects linked to reduced mTORC1 activity, total CAMKIIα, and GluA1 AMPAR levels in the NACsh. Together, these data highlight a role for mTORC1 in the neural processes that control the expression and maintenance of drug reward, including protracted relapse vulnerability. These effects appear to involve a role for mTORC1 in the regulation of GluA1 AMPARs and CAMKIIα in the NACsh.     

Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia

1. The effect of mGlu receptor agonists on intracellular calcium (Ca²⁺) in rat cortical neurons and glial cells was studied. The responses evoked consisted of two phases; an initial transient response followed by a sustained plateau. In both cell types the order of potency of group I mGlu receptor agonists was DHPG>1S,3R ACPD>3-HPG.
2. The selective mGlu5 agonist CHPG elicited responses in both cell types as did S4C3-HPG which is thought to be an mGlu5 agonist at high concentrations. S4-CPG had no effect on intracellular Ca²⁺ levels nor did it inhibit the action of 1S,3R ACPD. These results suggest that the responses in both cell types are mediated by mGlu5 receptors.
3. In the absence of extracellular Ca²⁺ ions, 1S,3R ACPD (100 μM) induced only a transient Ca²⁺ response which decayed to baseline with a time constant of approximately 20 s in both cell types. Subsequent readdition of Ca²⁺ (2 mM) to the external solution in the continued presence of 1S,3R ACPD induced a sustained Ca²⁺ plateau.
4. The sustained Ca²⁺ plateau could be blocked by a number of inorganic cations, with an order of potency of Zn²⁺⩾La³⁺>Cd²⁺⩾Co²⁺>Ni²⁺>Mg²⁺. Similar concentrations of Zn²⁺ had little effect on Ca²⁺-influx evoked by 25 mM K⁺.
5. It is concluded that the Ca²⁺-entry pathway activated by mGlu5 receptors resembles store-operated Ca²⁺-entry pathways that have been described in other cell types.

     

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg

1. The sea urchin egg homogenate is an ideal model to characterize Ca²⁺-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP₃- and ryanodine-sensitive Ca²⁺-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist.
2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca²⁺-releasing agent, NAADP, compared to inositol trisphosphate (InsP₃) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors.
3. NAADP-induced Ca²⁺-release was blocked by L-type Ca²⁺-channel blockers and by Bay K 8644, while InsP₃- and cyclic ADPR-induced Ca²⁺-release were insensitive to these agents. L-type Ca²⁺-channel blockers did not displace [³²P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion.
4. Similarly, a number of K⁺-channel antagonists blocked NAADP-induced Ca²⁺-release selectively over InsP₃- and cyclic ADPR-induced Ca²⁺-release. Radioligand studies showed that these agents were not competitive antagonists.
5. As has been shown for InsP₃ and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca²⁺-release mechanisms.
6. The presence of K⁺ was not essential for NAADP-induced Ca²⁺-release, since substitution of K⁺ with other monovalent cations in the experimental media did not significantly alter Ca²⁺ release by NAADP. On the contrary, cyclic ADPR and InsP₃-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K⁺. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca²⁺-release. While the alkaline condition permitted InsP₃ and cyclic ADPR-induced Ca²⁺-release, the acidic condition completely hampered both Ca²⁺-release mechanisms.
7. The present results characterize pharmacologically and biochemically the novel Ca²⁺-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

     

Ca2+ mobilization in the aortic endothelium in streptozotocin-induced diabetic and cholesterol-fed mice

1. Experiments were performed to compare Ca²⁺ mobilization in the aortic endothelium in streptozotocin (STZ)-induced diabetic and cholesterol-fed mice with that in age-matched controls.
2. The intracellular free Ca²⁺ ([Ca²⁺]_i) in the fura PE-3 loaded endothelium of aortic rings was dose-dependently increased by cumulative administration of acetylcholine (ACh). ACh caused a transient rise in [Ca²⁺]_i in Ca²⁺-free medium. The ACh-induced increase in [Ca²⁺]_i in normal or Ca²⁺-free medium was significantly weaker in both STZ-induced diabetic and cholesterol-fed mice.
3. The weaker [Ca²⁺]_i response in Ca²⁺-containing medium in STZ-induced diabetic and cholesterol-fed mice was normalized by chronic administration of cholestyramine.
4. The increased low density lipoprotein (LDL) levels seen in both STZ-induced diabetic and cholesterol-fed mice were normalized by the same chronic administration of cholestyramine (300 mg kg⁻¹, p.o. daily for 10 weeks). Chronic administration of cholestyramine had no effect on the plasma glucose level.
5. Lysophosphatidylcholine (LPC) decreased the [Ca²⁺]_i responses to ACh in the aortic endothelium from normal mice.
6. These results suggest that ACh increases both Ca²⁺ influx and Ca²⁺ release from storage in the aortic endothelium. The weaker [Ca²⁺]_i influx seen in the endothelium of aortae from both STZ-induced diabetic and cholesterol-fed mice was improved by the chronic administration of cholestyramine, and we suggest that this improvement is due, at least in part, to a lowering of the plasma LDL level. It is further suggested that LPC may have an important influence over Ca²⁺ mobilization in the endothelium.

     

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.

     

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 Val158Met polymorphism of the COMT gene is associated with increased pain sensitivity in morphine-treated patients undergoing a painful procedure after cardiac surgery

Aims

The catechol-O-methyltransferase (COMT) Val158Met polymorphism affected pain sensitivity of healthy volunteers upon application of experimental pain stimuli. The relevance of these findings in morphine-treated postoperative cardiac patients undergoing painful healthcare procedures is unknown; therefore, the aim of this study was to investigate whether the COMT Val158Met polymorphism increases pain sensitivity in morphine-treated patients undergoing an unavoidable painful routine procedure after cardiac surgery.

Methods

One hundred and seventeen postoperative cardiac patients in the intensive care unit were genotyped for the COMT Val158Met polymorphism. All patients were treated with continuous morphine infusions for pain at rest, and received a bolus of morphine (2.5 or 7.5 mg) before a painful procedure (turning and/or chest drain removal) on the first postoperative day. Numerical rating scale (NRS) scores were evaluated at the following four time points: at baseline (at rest), and before, during and after the painful procedure.

Results

Overall mean NRS scores were significantly higher in patients carrying the Met-variant allele. During the painful procedure, the mean NRS score was significantly higher for Met/Met patients compared with Val/Met and Val/Val patients (mean NRS 3.4 ± 2.8, 2.7 ± 2.4 and 1.7 ± 1.7, respectively; P = 0.04). In Met/Met patients, the increase in NRS scores during the painful procedure compared with the baseline NRS score was clinically relevant (ΔNRS ≥ 1.3) and statistically significant and appeared to be independent of sex and the morphine bolus dose.

Conclusions

Our results show that the COMT Val158Met polymorphism contributes to variability in pain sensitivity after cardiac surgery of morphine-treated patients in the intensive care unit, because Met-allele carriers were more sensitive to overall pain and procedure-related pain.     

Enhanced isoflurane suppression of excitatory synaptic transmission in the aged rat hippocampus

1. The effects of the volatile anaesthetic, isoflurane, were investigated on evoked dendritic field excitatory postsynaptic potentials (f.e.p.s.p.) and antidromic and orthodromic population spikes recorded extracellularly in the CA1 cell layer region in the in vitro hippocampal slice taken from young mature (2–3 months) and old (24–27 months) Fisher 344 rats.
2. Isoflurane depressed the f.e.p.s.ps and the orthodromically-evoked population spikes in both old and young hippocampi. However, the magnitude of the anaesthetic-induced depression was greater in slices taken from old rats compared to those taken from young rats during the application of different isoflurane concentrations (0.5–5%).
3. In the presence of the GABA_A antagonist, bicuculline methiodide (15 μM), isoflurane suppressed the f.e.p.s.ps to the same extent as was observed in the absence of the GABA_A antagonist.
4. Orthodromically evoked population spikes were suppressed by isoflurane in a manner quantitatively similar to the suppression of the f.e.p.s.ps. However, antidromic population spikes and presynaptic volleys evoked in young and old slices were resistant to anaesthetic action. In addition, paired pulse facilitation ratio of the evoked dendritic f.e.p.s.ps was not affected in both young and old slices during the application of isoflurane.
5. When slices were exposed to low Ca²⁺/high Mg²⁺ solution, isoflurane (1 and 3%) depressed the f.e.p.s.ps in aged slices to the same extent as in young slices.
6. The augmented anaesthetic depression of f.e.p.s.ps in old compared to young hippocampi in the absence and presence of bicuculline, and the lack of anaesthetic effects on antidromic population spikes and presynaptic volleys in old and young slices, suggest that the increased sensitivity of anaesthetic actions in old hippocampi is due to age-induced attenuation of synaptic excitation rather than potentiation of synaptic inhibition. Furthermore, elimination of the increased sensitivity of old slices to anaesthetic actions when the slices were perfused with low Ca²⁺/high Mg²⁺ medium, which presumably would decrease intracellular [Ca²⁺], suggests that the enhanced anaesthetic effects in aged neurones might be related to increased intraneuronal [Ca²⁺] in the synaptic terminal.

     

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

1. Two types of Ca²⁺ channel α₁-subunits were co-expressed in Xenopus oocytes with the Ca²⁺ channel α₂- and β₁-subunits. The Ba²⁺ current through the α_1Cα₂β and the α_1Bα₂β channels had electrophysiological and pharmacological properties of L- and N-type Ca²⁺ channels, respectively.
2. Amlodipine had a strong blocking action on both the L-type and N-type Ca²⁺ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca²⁺ channel was comparable to that on the L-type Ca²⁺ channel. At −100 mV holding potential, the IC₅₀ values for amlodipine block on the L-type and N-type Ca²⁺ channel were 2.4 and 5.8 μM, respectively.
3. The blocking action of amlodipine on the N-type Ca²⁺ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca²⁺ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.
4. The time course of block development by amlodipine was similar for L-type and N-type Ca²⁺ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca²⁺ channel.

     

The antinociceptive effect of combined systemic administration of morphine and the glycine/NMDA receptor antagonist, (+)-HA966 in a rat model of peripheral neuropathy

1. We evaluated the ability of the functional antagonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex, (+)-(1-Hydroxy-3-aminopyrrolodine-2-one) ((+)-HA966), to modulate the antinociceptive action of systemic morphine in a rat model of neuropathic pain produced by chronic constriction injury to the sciatic nerve. Mechanical (vocalization threshold to hindpaw pressure) and thermal (struggle latency to hindpaw immersion into a water bath) stimuli were used.
2. In the mechanical test, morphine (0.05, 0.1 and 0.3 mg kg⁻¹, i.v.) alone produced dose-dependent effects in both neuropathic and uninjured rats. Likewise, morphine (0.1, 0.3 and 1 mg kg⁻¹, i.v.) dose-dependently increased struggle latencies of the nerve-injured hindpaw in the hot noxious (46°C) test but was ineffective in the non-noxious warm (44°C) and cold (10°C) test.
3. Pretreatment with (+)-HA966 (2.5 mg kg⁻¹, s.c.) dose-dependently enhanced the effect of morphine in the mechanical test with the relative potency being nerve-injured hindpaw>contralateral hindpaw>uninjured rat.
4. Likewise, (+)-HA966 dose-dependently enhanced the effect of morphine against a hot (46°C) stimulus and produced, in combination with morphine, a dose-dependent effect against a warm (44°C) stimulus. In the cold (10°C) test, (+)-HA966 reversed the ineffectiveness of the highest dose of morphine.
5. Naloxone blocked the effect of the combination of (+)-HA966 with morphine in all tests. The drug combination produced no motor deficits in animals using the rotarod test.
6. These findings suggest that combined administration of antagonists, acting at the glycine site of the NMDA receptor complex and morphine may be a promising approach in the treatment of neuropathic and acute pain.

     

Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca²⁺, apparently through ryanodine-sensitive Ca²⁺ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca²⁺, by use of Ca²⁺ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells.
2. After phenylephrine-(PE, 10 μM) sensitive Ca²⁺ stores were depleted by maximal PE stimulation in Ca²⁺-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca²⁺ was restored for a 30 min refilling period. At the end of this period, Ca²⁺ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca²⁺ store.
3. In a concentration-dependent manner (30, 100 and 300 μM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca²⁺-free media. This suggests that Ca²⁺ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca²⁺-containing or Ca²⁺-free Krebs solution.
4. Restoring Ca²⁺ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca²⁺. The contraction of tissues pretreated with 300 μM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca²⁺-free and Ca²⁺-containing medium suggesting a rapid reversal of CEP effects.
5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K⁺ in the absence of and after 30 min pre-incubation with 30, 100 and 300 μM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K⁺ (77.6, 41.1 and 10.8% of control).
6. Some arterial rings were pre-incubated with ryanodine (30 μM) for 30 min before the construction of PE concentration-response curves. In Ca²⁺-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 mM K⁺ contraction, n=11) in the presence of external Ca²⁺. EC₅₀ values for PE in ryanodine-treated tissues (1.7±0.25 μM, n=16) were not significantly different from controls (2.5±0.41 μM, n=22). Maximum contractions to PE (118.5±4.4% of 100 mM K⁺ contraction, n=16) were also unaffected by ryanodine when compared to controls (129±4.2%, n=23).
7. When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca²⁺ distribution, it was observed that 100 and 300 μM CEP in Ca²⁺-free medium caused Ca²⁺ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca²⁺ to control values.
8. These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca²⁺ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K⁺. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca²⁺ elevation.

     

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca²⁺ store depletion and Ca²⁺ entry was studied further.
2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca²⁺ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC₄(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody.
3. Depletion of cellular Ca²⁺ pools with inositol 1,4,5-trisphosphate (IP₃), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca²⁺ store depletion, chelating of intra-microsomal Ca²⁺ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca²⁺ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP₃-sensitive stores.
4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca²⁺ entry pathways, membrane hyperpolarization due to K⁺ channel activation or tyrosine kinase activity.
5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca²⁺/Mn²⁺ entry to Ca²⁺ store depletion by 43%, while it did not interfere with intracellular Ca²⁺ release by IP₃ or thapsigargin.
6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization.
7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca²⁺/Mn²⁺ entry (by 105%) and membrane hyperpolarization (by 40%).
8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium.
9. These data demonstrate that endothelial Cyp450 MO is activated by Ca²⁺ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells.
10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca²⁺ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca²⁺ entry activation and increase the driving force for Ca²⁺ entry by membrane hyperpolarization in porcine aortic endothelial cells.

     

Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca²⁺-permeable AMPA receptors during the incubation of cocaine craving

Cue-induced cocaine seeking intensifies or incubates after withdrawal from extended access cocaine self-administration, a phenomenon termed incubation of cocaine craving. The expression of incubated craving is mediated by Ca²⁺-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Thus, CP-AMPARs are a potential target for therapeutic intervention, making it important to understand mechanisms that govern their accumulation. Here we used subcellular fractionation and biotinylation of NAc tissue to examine the abundance and distribution of AMPAR subunits, and GluA1 phosphorylation, in the incubation model. We also studied two transmembrane AMPA receptor regulatory proteins (TARPs), γ-2 and γ-4. Our results, together with earlier findings, suggest that some of the new CP-AMPARs are synaptic. These are probably associated with γ-2, but they are loosely tethered to the PSD. Levels of GluA1 phosphorylated at serine 845 (pS845 GluA1) were significantly increased in biotinylated tissue and in an extrasynaptic membrane-enriched fraction. These results suggest that increased synaptic levels of CP-AMPARs may result in part from an increase in pS845 GluA1 in extrasynaptic membranes, given that S845 phosphorylation primes GluA1-containing AMPARs for synaptic insertion and extrasynaptic AMPARs supply the synapse. Some of the new extrasynaptic CP-AMPARs are likely associated with γ-4, rather than γ-2. The maintenance of CP-AMPARs in NAc synapses during withdrawal is accompanied by activation of CaMKII and ERK2 but not CaMKI. Overall, AMPAR plasticity in the incubation model shares some features with better described forms of synaptic plasticity, although the timing of the phenomenon and the persistence of related neuroadaptations are significantly different.This article is part of a Special Issue entitled ‘Synaptic Plasticity and Addiction’.     

Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurones

1. The effect of adenosine 5′-triphosphate (ATP) on glutamatergic synaptic transmission in hippocampus was examined by an indicator of intracellular Ca²⁺ oscillations. These oscillations were postsynaptic responses by glutamate released from presynaptic sites. ATP completely inhibited the oscillations in a concentration-dependent manner.
2. The ATP-induced inhibition was mediated via P2-purinoceptors since ATP exhibited the inhibitory action even in the presence of P1-purinoceptor antagonists. Also non-hydrolysable ATP analogues and uridine 5′-triphosphate (UTP) inhibited the oscillation.
3. The rank order of agonist potency of ATP analogues for inhibition of the Ca²⁺ oscillation was as follows: 2-methyl-thio-adenosine 5′-triphosphate⩾ATP>adenosine 5′-O-(3-thiotriphosphate)>UTP>α,β-methylene-adenosine 5′-triphosphate. These inhibitory effects were insensitive to suramin. Judging from this rank order of potency, the inhibitory P2-purinoceptor could be assigned to a subclass of GTP-binding protein coupled-type receptors.
4. The site of action of ATP was thought to be presynaptic since ATP did not affect the postsynaptic Ca²⁺ responses by glutamate. These results suggest the existence of a presynaptic inhibitory P2-receptor that inhibits glutamate release in the hippocampus.

     

Effect of rolipram and dibutyryl cyclic AMP on resequestration of cytosolic calcium in FMLP-activated human neutrophils

1. We have investigated the effects of the selective phosphodiesterase (PDE) type 4 inhibitor, rolipram (0.01–1 μM) on cytosolic Ca²⁺ fluxes in FMLP-activated human neutrophils, as well as on superoxide production by, and release of elastase from, these cells.
2. Cytosolic Ca²⁺ fluxes were measured by use of fura-2 spectrofluorimetry in combination with a radiometric procedure that enables distinction between net efflux and influx of the cation. Superoxide production and elastase release were measured by lucigenin-enhanced chemiluminescence and a colorimetric procedure, respectively.
3. Pretreatment of neutrophils with rolipram did not affect the FMLP-activated release of Ca²⁺ from intracellular stores, but was associated with dose-related acceleration of the rate of decline in fura-2 fluorescence and with decreased efflux, as well as store-operated influx of ⁴⁵Ca²⁺, indicative of enhancement of resequestration of the cation by the endo-membrane Ca²⁺-ATPase.
4. Inhibition of superoxide production and elastase release was observed at concentrations of rolipram which accelerated the clearance of Ca²⁺ from the cytosol of FMLP-activated neutrophils.
5. These effects of rolipram on FMLP-activated Ca²⁺ fluxes, superoxide generation and elastase release were mimicked by pretreatment of neutrophils with dibutyryl cyclic AMP (0.5–4 mM), while theophylline (10–150 μM), a non-specific PDE inhibitor, as well as the β₂-agonist, salbutamol, were less effective.
6. We conclude that rolipram deactivates FMLP-stimulated human neutrophils by enhancement of cyclic AMP-dependent resequestration of cytosolic Ca²⁺.