Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

Synthesis of siRNAs incorporated with cationic peptides R8G7 and R8A7 and the effect of the modifications on siRNA properties.

Small interfering RNA (siRNA) can be used as an innovative next-generation drug. However, there are several challenges in the therapeutic application of siRNAs, including their low cell membrane permeability. In this study, we designed and synthesized siRNAs, incorporating the cationic peptides R8G7 and R8A7 to improve cell membrane permeability of siRNAs. Thermal denaturation studies revealed that R8G7 and R8A7 modifications increased the thermal stability of the siRNA duplexes. Incorporating these peptides at the 3'-ends of the siRNA passenger strands increased the stability of the siRNAs in a buffer containing bovine serum. Further, we found that the peptide-siRNA conjugates did not show sufficient RNA interference (RNAi) activity in the absence of the transfection reagent; however, when the transfection reagent was used, the peptide-siRNA conjugates preserved their RNAi activity.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats.

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Involvement of GABAB receptor in the antihypersensitive effect in anterior cingulate cortex of partial sciatic nerve ligation model.

The role of the GABAB receptor in the anterior cingulate cortex (ACC) of neuropathic pain is unclear. Injection of a GABAB receptor antagonist CGP35348 into the ACC induced mechanical hypersensitivity in normal rats. Activation of the GABAB receptor injected by a GABAB receptor agonist baclofen into the ACC attenuated mechanical hypersensitivity in partial sciatic nerve ligation (PSNL) rats. Co-microinjection of CGP35348 with a muscarinic M1 receptor agonist McN-A-343 into the ACC significantly inhibited McN-A-343-induced antihypersensitivity in PSNL rats. These results suggest that the GABAB receptor in the ACC contributes to mechanical hypersensitivity and is involved in muscarinic M1 receptor-mediated antihypersensitivity.

Complex formation between the vasopressin 1b receptor, ß-arrestin-2, and the µ-opioid receptor underlies morphine tolerance.

Chronic morphine exposure upregulates adenylate cyclase signaling and reduces analgesic efficacy, a condition known as opioid tolerance. Nonopioid neurotransmitters can enhance morphine tolerance, but the mechanism for this is poorly understood. We show that morphine tolerance was delayed in mice lacking vasopressin 1b receptors (V1bRs) or after administration of V1bR antagonist into the rostral ventromedial medulla, where transcripts for V1bRs and µ-opioid receptors are co-localized. Vasopressin increased morphine-binding affinity in cells expressing both V1bR and µ-opioid receptors. Complex formation among V1bR, ß-arrestin-2, and µ-opioid receptor resulted in vasopressin-mediated upregulation of ERK phosphorylation and adenylate cyclase sensitization. A leucine-rich segment in the V1bR C-terminus was necessary for the association with ß-arrestin-2. Deletion of this leucine-rich segment increased morphine analgesia and reduced vasopressin-mediated adenylate cyclase sensitization. These findings indicate that inhibition of µ-opioid-receptor-associated V1bR provides an approach for enhancing morphine analgesia without increasing analgesic tolerance.

Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission.

Background Cholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods. Results We found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex. Conclusions These results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior cingulate cortex.

Glutamate functions in stomatal closure in Arabidopsis and fava bean.

Guard cells are indispensable for higher plants because they control gas exchange and water balance to maintain photosynthetic activity. The signaling processes that govern their movement are controlled by several factors, such as abscisic acid (ABA), blue light, pathogen-associated molecular patterns (PAMPs), and carbon dioxide. Herein, we demonstrated that the amino acid glutamate (Glu), a well-known mammalian neurotransmitter, functions as a novel signaling molecule in stomatal closure in both Arabidopsis and fava bean (Vicia faba L.). Pharmacological and electrophysiological analyses provided important clues for the participation of Glu-receptors, Ca(2+), and protein phosphorylation during the signaling process. Genetic analyses using Arabidopsis ABA-deficient (aba2-1) and ABA-insensitive (abi1-1 and abi2-1) mutants showed that ABA is not required for Glu signaling. However, loss-of-function of the Arabidopsis gene encoding Slow Anion Channel-Associated 1 (SLAC1) and Calcium-Dependent Protein Kinase 6 (CPK6) impaired the Glu response. Moreover, T-DNA knockout mutations of the Arabidopsis Glu receptor-like gene (GLR), GLR3.5, lost their sensitivity to Glu-dependent stomatal closure. Our results strongly support functional Glu-signaling in stomatal closure and the crucial roles of GLRs in this signaling process.

8-Mercapto-Cyclic GMP Mediates Hydrogen Sulfide-Induced Stomatal Closure in Arabidopsis.

Plants are exposed to hydrogen sulfide (H2S) both exogenously, as it exists as a pollutant gas in the environment, and endogenously, as it is synthesized in cells. H2S has recently been found to function as a gaseous signaling molecule, but its signaling cascade remains unknown. Here, we examined H2S-mediated guard cell signaling in Arabidopsis. The H2S donor GYY4137 (morpholin-4-ium-4-methoxyphenyl [morpholino] phosphinodithioate) induced stomatal closure, which peaked after 150 min at 1 µM or after 90 min at 10 and 100 µM. After reaching maximal closure, stomatal apertures gradually increased in size in response to further exposure to GYY4137. GYY4137 induced nitric oxide (NO) generation in guard cells, and GYY4137-induced stomatal closure was reduced by an NO scavenger and inhibitors of NO-producing enzymes. Mass spectrometry analyses showed that GYY4137 induces the synthesis of 8-nitro-cGMP and 8-mercapto-cGMP and that this synthesis is mediated by NO. In addition, 8-mercapto-cGMP triggered stomatal closure. Moreover, inhibitor and genetic studies showed that calcium, cADP ribose and slow anion channel 1 act downstream of 8-mercapto-cGMP. This study therefore demonstrates that 8-mercapto-cGMP mediates the H2S signaling cascade in guard cells.

Aldosterone-sensitive nucleus tractus solitarius neurons regulate sensitivity of the baroreceptor reflex in high sodium-loaded rats.

We examined the role of aldosterone-sensitive neurons in the nucleus tractus solitarius (NTS) in the arterial baroreceptor reflex (baroreflex) function. Baroreflex sensitivity was induced by phenylephrine in high sodium-loaded rats and was significantly reduced. This baroreflex sensitivity was reversed by microinjection of the mineralocorticoid receptor (MR) antagonist eplerenone into the NTS. 11beta-Hydroxysteroid dehydrogenase type 2 neurons and MR were also identified in the NTS. These data suggest that the aldosterone-sensitive neurons in the NTS may have an important role in baroreflex function.

Involvement of Na+/Ca2+ exchanger in pentylenetetrazol-induced convulsion by use of Na+/Ca2+ exchanger knockout mice.

Involvement of Na+/Ca2+ exchanger (NCX) in pentylenetetrazol (PTZ)-induced convulsion by use of NCX knockout mice and the selective ligand SEA0400 to NCX was examined. In the SEA0400-administered group, the latency to clonic convulsion was extended into 210 s, although the latency to clonic convulsion was observed until 100 s in control group. SEA0400 had little effect on bicuculline-induced clonic seizure nicotine-induced wild running and 4-aminopyridine-induced tonic flexion, respectively. Tonic flexion convulsion was occurred three fifth in the wild type mice group by administration of PTZ, but tonic flexion was not observed in NCX1 knockout mice groups. These results suggest that NCX is involved in inhibitory action in PTZ-induced convulsion.

Modulation of P2X receptors in dorsal root ganglion neurons of streptozotocin-induced diabetic neuropathy.

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X(2) and P2X(3) receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X(2), P2X(3) and/or P2X(2/3) receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.

New topics in vasopressin receptors and approach to novel drugs: involvement of vasopressin V1a and V1b receptors in nociceptive responses and morphine-induced effects.

Arginine vasopressin (AVP) receptors have been classified into V1a, V1b, and V2 subtypes. Recent studies have demonstrated the involvement of AVP in anti-nociception and in morphine-induced anti-nociception. However, the roles of individual AVP-receptor subtypes have not been fully elucidated. Here, we have summarized the role of V1-receptor subtypes in behavioral responses to noxious stimuli and to morphine. In this review, we focus on studies using mice lacking the V1a receptor (V1a(-/-) mice) and the V1b receptor (V1b(-/-) mice).

The spinal muscarinic M(1) receptors and GABA(A) receptors contribute to the McN-A-343-induced antinociceptive effects during thermal stimulation of mice.

The present study was undertaken to clarify how spinal muscarinic receptors are involved in the antinociceptive effects in thermal stimulation. Intrathecal (i.t.) injection of the muscarinic agonist McN-A-343 inhibited the tail-flick response to noxious thermal stimulation in a dose-dependent manner (31.5 - 63.0 nmol). This McN-A-343-induced antinociceptive effect was dose-dependently inhibited by intrathecal (i.t.) injection of a nonselective muscarinic receptor antagonist atropine, the selective muscarinic M(1) antagonist pirenzepine, or the M(4) antagonist himbacine. The inhibition of pirenzepine was greater than that of himbacine. In contrast, the selective muscarinic M(2) antagonist methoctramine did not inhibit the antinociceptive effects of McN-A-343. In addition, the McN-A-343-induced antinociceptive effect was attenuated by i.t. injection of the GABA(A) antagonist bicuculline, but not by injection of the GABA(B) antagonist CGP35348. These results suggest that McN-A-343 produces its antinociceptive effect on the response to thermal stimulation via spinal muscarinic M(1) receptors and, at least in part, through neuronal pathways involving spinal GABA(A) receptors in mice.

A high-sodium diet in streptozotocin-induced diabetic rats impairs endothelium-derived hyperpolarizing factor-mediated vasodilation.

We examined endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation of mesenteric arteries in high-sodium loaded streptozotocin (STZ)-induced diabetic rats. The study shows that acetylcholine (ACh)-induced, EDHF-mediated relaxation is relatively maintained in STZ-induced diabetic rats, but after a high-sodium diet was given, the function was significantly impaired in STZ-induced diabetic rats.

Contribution of Ca(2+) -dependent protein kinase C in the spinal cord to the development of mechanical allodynia in diabetic mice.

In this paper, we directly demonstrate, for the first time, the activation of Ca(2+)-dependent protein kinase C (PKC) in the spinal cord of diabetic mice. In streptozotocin (STZ)-treated (200 mg/kg, i.v.) diabetic mice, hypersensitivity (allodynia) to mechanical stimulation appeared 7 d after STZ injection. This mechanical allodynia was inhibited by intrathecal injection of the PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, but not the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). The activity of membrane-associated Ca(2+)-dependent PKC in the spinal cords of STZ-induced diabetic mice was significantly higher than that observed in non-diabetic mice. These results suggest that activation of Ca(2+)-dependent PKC in the spinal cord, contributes to the mechanical allodynia in the pain associated with diabetic neuropathy.

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells.

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.