Hydrogen sulfide-induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice

BACKGROUND AND PURPOSE

Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca_v3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H₂S, we asked whether TRPA1 channels, in addition to Ca_v3.2 channels, contribute to the H₂S-induced mechanical hyperalgesia and allodynia in mice.

EXPERIMENTAL APPROACH

Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca_v3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice.

KEY RESULTS

Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55–0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca_v3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca_v3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons.

CONCLUSIONS AND IMPLICATIONS

Mechanical hyperalgesia and allodynia induced by NaHS/H₂S required activation of both Ca_v3.2 and TRPA1 channels in mice.     

Optimization of ADME Properties for Sulfonamides Leading to the Discovery of a T-Type Calcium Channel Blocker,ABT-639

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Topical application of disodium isostearyl 2-O-L-ascorbyl phosphate, an amphiphilic ascorbic acid derivative, reduces neuropathic hyperalgesia in rats

BACKGROUND AND PURPOSE

Ca_v3.2 T-type calcium channels, targeted by H₂S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca_v3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H₂S donor, and on neuropathic hyperalgesia.

EXPERIMENTAL APPROACH

In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically.

KEY RESULTS

The NaHS-evoked Ca_v3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55–0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP.

CONCLUSIONS AND IMPLICATIONS

Ascorbic acid, known to inhibit Ca_v3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.     

AKAP-dependent sensitization of Cav3.2 channels via the EP4 receptor/cAMP pathway mediates PGE2-induced mechanical hyperalgesia

Background and Purpose

The Ca_v3.2 isoform of T-type Ca²⁺ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE₂-induced hyperalgesia. Here we examined and analysed Ca_v3.2 sensitization via the PGE₂/cAMP pathway in NG108-15 cells that express Ca_v3.2 and produce cAMP in response to PGE₂, and its impact on mechanical nociceptive processing in rats.

Experimental Approach

In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Ca_v3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats.

Key Results

In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE₂ increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE₂ was abolished by RQ-00015986-00, an EP₄ receptor antagonist. AKAP150 was co-immunoprecipitated with Ca_v3.2, regardless of stimulation with db-cAMP, and Ca_v3.2 was phosphorylated by db-cAMP or PGE₂. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE₂ caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl₂, known to inhibit Ca_v3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE₂-induced mechanical hyperalgesia.

Conclusion and Implications

Our findings suggest that PGE₂ causes AKAP-dependent phosphorylation and sensitization of Ca_v3.2 through the EP₄ receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.     

Involvement of NF‐κB in the upregulation of cystathionine‐γ‐lyase,a hydrogen sulfide‐forming enzyme,and bladder pain accompanying cystitis in mice

Hydrogen sulfide (H₂S) is generated from l ‐cysteine by multiple enzymes including cystathionine‐γ‐lyase (CSE), and promotes nociception by targeting multiple molecules such as Ca_v3.2 T‐type Ca²⁺ channels. Bladder pain accompanying cyclophosphamide (CPA)‐induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H₂S/Ca_v3.2 pathway. Therefore, we investigated whether NF‐κB, as an upstream signal of the CSE/H₂S system, contributes to bladder pain in mice with CPA‐induced cystitis. Bladder pain‐like nociceptive behaviour was observed in CPA‐treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β‐Cyano‐l ‐alanine, a reversible selective CSE inhibitor, prevented CPA‐induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF‐κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF‐κB inhibitor. PDTC and curcumin, which inhibits NF‐κB signals, abolished CPA‐induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA‐induced activation of NF‐κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H₂S/Ca_v3.2 signaling. Therefore, NF‐κB‐inhibiting compounds including curcumin may be useful for the treatment of cystitis‐related bladder pain.     

The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels

BACKGROUND AND PURPOSE

Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca_v1.2 channel current (I_Ca1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca_v1.2 channels.

EXPERIMENTAL APPROACH

Twenty-four flavonoids were analysed for their effects on I_Ca1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method.

KEY RESULTS

Most of the flavonoids stimulated or inhibited I_Ca1.2 in a concentration- and voltage-dependent manner with EC₅₀ values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC₅₀ values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I_Ca1.2 stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I_Ca1.2 inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2′-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca_v1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I_Ca1.2.

CONCLUSIONS AND IMPLICATIONS

Quercetin-like plant flavonoids were active on vascular Ca_v1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca_v1.2 channels, valuable for the treatment of hypertension and stroke.     

Inhibition of T-type Ca(2+) currents in mouse spermatogenic cells by gossypol, an antifertility compound

Gossypol, a male antifertility compound isolated from cotton, has been proved to inhibit capacitation and the acrosome reaction in human and mammalian sperm. Here, by using whole-cell recording, we observed the effects of gossypol on Ca(2+) and Cl(-) currents in mouse spermatogenic cells obtained by mechanical dissociation. The results showed that gossypol concentration-dependently and irreversibly inhibited T-type Ca(2+) currents in the cells. When the concentration of gossypol was > or =5 microM, the currents were blocked completely. The time to current block was progressively shortened as the gossypol concentration was increased from 5 to 80 microM. Moreover, the drug increased the time constant of inactivation in a concentration-dependent manner, while it did not affect the activation of the current. The inhibitory effect on the T-type Ca(2+) current did not correlate with signaling mediated by G proteins and tyrosine phosphorylation. No obvious effect of gossypol on Cl(-) currents was observed. These data suggest that the gossypol-induced inhibition of T-type Ca(2+) currents could be responsible for the antifertility activity of the compound, indicating a possibility to use gossypol as a local contraceptive drug.     

Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels

There is evidence that nifedipine (Nif) - a dihydropyridine (DHP) Ca(2+)-channel antagonist mostly known for its L-type-specific action--is capable of blocking low voltage-activated (LVA or T-type) Ca(2+) channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 T-channel alpha 1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 expression in Xenopus oocytes or HEK-293 cells (I(alpha 1G), I(alpha 1H) and I(alpha 1I), respectively) and two kinetically distinct, "fast" and "slow", LVA currents in thalamic neurons (I(LVA,f) and I(LVA,s)). At voltages of the maximums of respective currents the drug most potently blocked I(alpha 1H) (IC(50)=5 microM, max block 41%) followed by I(alpha 1G) (IC(50)=109 microM, 23%) and I(alpha 1I) (IC(50)=243 microM, 47%). The mechanism of blockade included interaction with Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 open and inactivated states. Nif blocked thalamic I(LVA,f) and I(LVA,s) with nearly equal potency (IC(50)=22 microM and 28 microM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Ca(v)3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca(2+) channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel alpha 1 subunits mRNA expression patterns in the thalamus point to Ca(v)3.1 and Ca(v)3.3 as the major contributors to thalamic I(LVA,f) and I(LVA,s), respectively.     

Targeting voltage-gated calcium channels: developments in peptide and small-molecule inhibitors for the treatment of neuropathic pain

Chronic pain affects approximately 20% of people worldwide and places a large economic and social burden on society. Despite the availability of a range of analgesics, this condition is inadequately treated, with complete alleviation of symptoms rarely occurring. In the past 30 years, the voltage-gated calcium channels (VGCCs) have been recognized as potential targets for analgesic development. Although the majority of the research has been focused on Ca_v2.2 in particular, other VGCC subtypes such as Ca_v3.2 have recently come to the forefront of analgesic research. Venom peptides from marine cone snails have been proven to be a valuable tool in neuroscience, playing a major role in the identification and characterization of VGCC subtypes and producing the first conotoxin-based drug on the market, the ω-conotoxin, ziconotide. This peptide potently and selectively inhibits Ca_v2.2, resulting in analgesia in chronic pain states. However, this drug is only available via intrathecal administration, and adverse effects and a narrow therapeutic window have limited its use in the clinic. Other Ca_v2.2 inhibitors are currently in development and offer the promise of an improved route of administration and safety profile. This review assesses the potential of targeting VGCCs for analgesic development, with a main focus on conotoxins that block Ca_v2.2 and the developments made to transform them into therapeutics.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

(+/-)-Naringenin as large conductance Ca(2+)-activated K+ (BKCa) channel opener in vascular smooth muscle cells

BACKGROUND AND PURPOSE. The aim of this study was to investigate, in vascular smooth muscle cells, the mechanical and electrophysiological effects of (+/-)-naringenin. EXPERIMENTAL APPROACH: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. KEY RESULTS: (+/-)-Naringenin induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline (pIC(50) values of 4.74 and 4.68, respectively). Tetraethylammonium, iberiotoxin, 4-aminopyridine and 60 mM KCl antagonised (+/-)-naringenin-induced vasorelaxation, while glibenclamide did not produce any significant antagonism. Naringin [(+/-)-naringenin 7-beta-neohesperidoside] caused a concentration-dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (+/-)-naringenin. In rat tail artery myocytes, (+/-)-naringenin increased large conductance Ca(2+)-activated K(+) (BK(Ca)) currents in a concentration-dependent manner; this stimulation was iberiotoxin-sensitive and fully reversible upon drug wash-out. (+/-)-Naringenin accelerated the activation kinetics of BK(Ca) current, shifted, by 22 mV, the voltage dependence of the activation curve to more negative potentials, and decreased the slope of activation. (+/-)-Naringenin-induced stimulation of BK(Ca) current was insensitive either to changes in the intracellular Ca(2+) concentration or to the presence, in the pipette solution, of the fast Ca(2+) chelator BAPTA. However, such stimulation was diminished when the K(+) gradient across the membrane was reduced. CONCLUSIONS AND IMPLICATIONS: The vasorelaxant effect of the naturally-occurring flavonoid (+/-)-naringenin on endothelium-denuded vessels was due to the activation of BK(Ca) channels in myocytes.     

Analgesic conotoxins: block and G protein-coupled receptor modulation of N-type (Ca(V) 2.2) calcium channels

Conotoxins (conopeptides) are small disulfide bonded peptides from the venom of marine cone snails. These peptides target a wide variety of membrane receptors, ion channels and transporters, and have enormous potential for a range of pharmaceutical applications. Structurally related ω-conotoxins bind directly to and selectively inhibit neuronal (N)-type voltage-gated calcium channels (VGCCs) of nociceptive primary afferent neurones. Among these, ω-conotoxin MVIIA (Prialt) is approved by the Food and Drug Administration (FDA) as an alternative intrathecal analgesic for the management of chronic intractable pain, particularly in patients refractory to opioids. A series of newly discovered ω-conotoxins from Conus catus, including CVID-F, are potent and selective antagonists of N-type VGCCs. In spinal cord slices, these peptides reversibly inhibit excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurones, and in the rat partial sciatic nerve ligation model of neuropathic pain, significantly reduce allodynic behaviour. Another family of conotoxins, the α-conotoxins, are competitive antagonists of mammalian nicotinic acetylcholine receptors (nAChRs). α-Conotoxins Vc1.1 and RgIA possess two disulfide bonds and are currently in development as a treatment for neuropathic pain. It was initially proposed that the primary target of these peptides is the α9α10 neuronal nAChR. Surprisingly, however, α-conotoxins Vc1.1, RgIA and PeIA more potently inhibit N-type VGCC currents via a GABA(B) GPCR mechanism in rat sensory neurones. This inhibition is largely voltage-independent and involves complex intracellular signalling. Understanding the molecular mechanisms of conotoxin action will lead to new ways to regulate VGCC block and modulation in normal and diseased states of the nervous system.     

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.     

Differential regulation of Ca(2+)-dependent Cl- currents by FP prostanoid receptor isoforms in Xenopus oocytes

The FP(A) and FP(B) prostanoid receptor isoforms are G-protein-coupled receptors that are activated by prostaglandin F(2alpha) (PGF(2alpha)). Differences in their carboxyl termini prompted us to examine the intracellular calcium (Ca(2+)) signaling of these receptor isoforms using the Xenopus oocyte expression system. Protein expression was determined by immunofluorescence microscopy and whole cell binding with [3H]PGF(2alpha). Positive immunolabeling was observed on the outer membranes of oocytes expressing FLAG-tagged FP receptor isoforms, but not on control (water-injected) oocytes. Intracellular signaling was examined using a two-electrode voltage clamp. Specific whole-cell binding was also detected for both receptor isoforms. Bath application of 10 microM PGF(2alpha) to FP(A)-expressing oocytes produced a chloride (Cl-) current response similar to that of an injection of inositol 1,4,5-trisphosphate (InsP(3)) (5.76+/-0.6 microA, peak current; N=23) that returned to control levels within 25 min. In FP(B)-expressing oocytes the activation of the Cl- current was delayed or completely absent (1.38+/-0.2 microA, peak current; N=18). Control oocytes were not responsive to the application of PGF(2alpha) (0.87+/-0.1 microA, peak current; N=10). Activation of Cl- currents for both FP receptor isoforms was dependent upon intracellular Ca(2+) stores as a 30-min pretreatment with thapsigargin (1 microM; N=5) blocked the PGF(2alpha) induction of the Cl- current. These data indicate that the FP prostanoid receptor isoforms differ in their ability to activate Ca(2+)-dependent Cl- channels when expressed in Xenopus oocytes. The difference appears to be in the ability of the two FP prostanoid receptor isoforms to mobilize intracellular calcium.     

Involvement of soluble guanylate cyclase alpha(1) and alpha(2), and SK(Ca) channels in NANC relaxation of mouse distal colon

In distal colon, both nitric oxide (NO) and ATP are involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission. The role of the soluble guanylate cyclase (sGC) isoforms alpha(1)beta(1) and alpha(2)beta(1), and of the small conductance Ca(2+)-dependent K(+) channels (SK(Ca) channels) in the relaxation of distal colon by exogenous NO and by NANC nerve stimulation was investigated, comparing wild type (WT) and sGCalpha(1) knockout (KO) mice. In WT strips, the relaxation induced by electrical field stimulation (EFS) at 1 Hz but not at 2-8 Hz was significantly reduced by the NO-synthase inhibitor L-NAME or the sGC inhibitor ODQ. In sGCalpha(1) KO strips, the EFS-induced relaxation at 1 Hz was significantly reduced and no longer influenced by L-NAME or ODQ. The SK(Ca) channel blocker apamin alone had no inhibitory effect on EFS-induced relaxation, but combined with ODQ or L-NAME, apamin inhibited the relaxation induced by EFS at 2-8 Hz in WT strips and at 8 Hz in sGCalpha(1) KO strips. Relaxation by exogenous NO was significantly attenuated in sGCalpha(1) KO strips, but could still be reduced further by ODQ. Basal cGMP levels were lower in sGCalpha(1) KO strips but NO still significantly increased cGMP levels versus basal. In conclusion, in the absence of sGCalpha(1)beta(1), exogenous NO is able to partially act through sGCalpha(2)beta(1). NO, acting via sGCalpha(1)beta(1), is the principal neurotransmitter in EFS-evoked responses at 1 Hz. At higher stimulation frequencies, NO, acting at sGCalpha(1)beta(1) and/or sGCalpha(2)beta(1), functions together with another transmitter, probably ATP acting via SK(Ca) channels, with some degree of redundancy.     

A nitric oxide/Ca(2+)/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose:

Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Experimental approach:

In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation.

Key results:

Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca²⁺ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca²⁺]_i.

Conclusions and implications:

These data identified the NO/Ca²⁺/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.     

The ablation of the Ca(v)2.3/E-type voltage-gated Ca2+ channel causes a mild phenotype despite an altered glucose induced glucagon response in isolated islets of Langerhans

Glucagon release upon hypoglycemia is an important homeostatic mechanism utilized by vertebrates to restore blood glucose to normal. Glucagon secretion itself is triggered by Ca2+ influx through voltage-gated ion channels, and the gene inactivation of R-type Ca2+ channels, with Ca(v)2.3 as the ion conducting subunit, has been shown to disturb glucose homeostasis. To understand how glucagon release may be affected in Ca(v)2.3-deficient mice, carbachol, insulin and glucose induced glucagon response was investigated. While the rise of insulin and glucose induced by carbachol is normal, mutant mice show an impaired glucagon-response. Further, the effect of insulin injection on glucagon levels was altered by the loss of the Ca(v)2.3 subunit. Ca(v)2.3-deficient mice are characterized by an impaired glucose suppression of glucagon release. This was most obvious at the level of isolated islets suggesting that Ca(v)2.3 containing R-type voltage-gated Ca2+ channels are involved in the glucose-mediated signalling to glucagon release in mice.     

Human adenosine A(3) receptor leads to intracellular Ca(2+) mobilization but is insufficient to activate the signaling pathway via phosphoinositide 3-kinase gamma in mice

Selective antagonists for the adenosine A(3) receptor (A3AR), a member of the G protein-coupled receptors, have been indicated as potential drugs for anti-asthma or anti-inflammation. However, potent antagonists for the rodent A3AR have not been identified. To evaluate the pharmacological effects of human A3AR antagonists in mice, we here generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. The expression levels of human A3AR in the A3AR-humanized mice were equivalent to those of mouse A3AR in wild-type mice. Elevation of the intracellular Ca(2+) concentration induced by an A3AR agonist was observed in bone marrow-derived mast cells from the A3AR-humanized mice and this Ca(2+) mobilization was completely antagonized by a human A3AR antagonist. However, antigen-dependent degranulation was not potentiated by the A3AR agonist in the mast cells from A3AR-humanized mice. The agonist-stimulated human A3AR did not lead to the phosphorylation of either extracellular signal-regulated kinase 1/2 or protein kinase B in A3AR-humanized mice. The rate of human A3AR internalization in the mast cells was also markedly decreased compared with that of mouse A3AR in the mast cells. These results demonstrate that the human A3AR is insufficient to activate phosphoinositide 3-kinase gamma-dependent signaling pathways in mice, probably due to the uncoupling of member(s) of the G proteins, which are capable of activating phosphoinositide 3-kinase gamma, to the human A3AR, despite the mouse G protein(s) responsible for the Ca(2+) elevation are coupled with the human A3AR.     

Dopamine D(2) receptor-induced COX-2-mediated production of prostaglandin E(2) in D(2)-transfected Chinese hamster ovary cells without simultaneous administration of a Ca(2+)-mobilizing agent

We have earlier demonstrated that dopamine stimulates the liberation of the prostaglandin E(2) (PGE(2)) precursor, arachidonic acid, in Chinese hamster ovary cells transfected with the rat dopamine D(2) receptor (long isoform), also without concomitant administration of a Ca(2+)-releasing agent [Nilsson et al., Br J Pharmacol 1998;124:1651-8]. In the present report, we show that dopamine, under the same conditions, also induces a concentration-dependent increase in the production of PGE(2), with a maximal effect of 235% at approximately 100 microM, and with an EC(50) of 794 nM. The effect was counteracted by the D(2) antagonist eticlopride, pertussis toxin, the inhibitor of intracellular Ca(2+) release TMB-8, incubation in Ca(2+)-free experimental medium, and PKC desensitization obtained by chronic pretreatment with the phorbol ester TPA. It was also antagonized by the non-specific cyclooxygenase (COX) inhibitor, indomethacin, and by the selective COX-2 inhibitor, NS-398, but not by the specific COX-1 inhibitor, valeryl salicylate. Both the non-specific phospholipase A(2) inhibitor, quinacrine, and an inhibitor of cPLA(2) and iPLA(2), AACOF3, counteracted the effect; in contrast, a selective iPLA(2) inhibitor, BEL, and a selective sPLA(2) inhibitor, TAPC, were ineffective. No effects of dopamine were obtained in control cells mock-transfected with the p3C vector only. The results reinforce previous assumptions that dopamine may interact with eicosanoid metabolism by means of D(2) receptor activation, and implicate an involvement of cPLA(2) and COX-2 in this effect. It is suggested that measurement of dopamine-induced PGE(2) production may serve as a convenient way to study D(2) receptor function in vitro.