Platelet-activating factor (PAF) enhances guinea pig detrusor smooth muscle contractile activities by stimulating voltage-dependent Ca2+ channels and store-operated Ca2+ channels.

We investigated the extracellular Ca2+ influx pathways involved in platelet-activating factor (PAF)-enhanced guinea pig detrusor smooth muscle (DSM) contractile activities. One micromolar PAF-enhanced DSM contractile activities were completely inhibited by extracellular Ca2+ removal and strongly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors. PAF-enhanced DSM contractile activities remaining in the presence of verapamil (10 µM) were not inhibited by LOE-908 (30 µM, an inhibitor of receptor-operated Ca2+ channels (ROCCs)), but were almost completely inhibited by SKF-96365 (30 µM, an inhibitor of store-operated Ca2+ channels (SOCCs) and ROCCs). These results suggest that VDCCs and SOCCs are responsible for PAF-enhanced DSM contractile activities.

Animal toxins: As an alternative therapeutic target following ischemic stroke condition.

Globally, Ischemic stroke (IS) has become the second leading cause of mortality and chronic disability. The process of IS has triggered by the blockages of blood vessels to form clots in the brain which initiates multiple interactions with the key signaling pathways, counting excitotoxicity, acidosis, ionic imbalance, inflammation, oxidative stress, and neuronal dysfunction of cells, and ultimately cells going under apoptosis. Currently, FDA has approved only tissue plasminogen activator therapy, which is effective against IS with few limitations. However, the mechanism of excitotoxicity and acidosis has spurred the investigation of a potential candidate for IS therapy. Acid-sensing ion channels (ASICs) and Voltage-gated Ca2+ channels (VDCCs) get activated and disturb the brain's normal physiology. Animal toxins are novel inhibitors of ASICs and VDCCs channels and have provided neuroprotective insights into the pathophysiology of IS. This review will discuss the potential directions of translational ASICs and VDCCs inhibitors research for clinical therapies.

Molecular Mechanisms in Hippocampus Involved on Object Recognition Memory Consolidation and Reconsolidation.

Acquired information is stabilized into long-term memory through a process known as consolidation. Though, after consolidation, when stored information is retrieved they can be again susceptible, allowing modification, updating and strengthening and to be re-stabilized they need a new process referred to as memory reconsolidation. However, the molecular mechanisms of recognition memory consolidation and reconsolidation are not fully understood. Also, considering that the study of the link between synaptic proteins is key to understanding of memory processes, we investigated, in male Wistar rats, molecular mechanisms in the hippocampus involved on object recognition memory (ORM) consolidation and reconsolidation. We verified that the blockade of AMPA receptors (AMPAr) and L-VDCCs calcium channels impaired ORM consolidation and reconsolidation when administered into CA1 immediately after sample phase or reactivation phase and that these impairments were blocked by the administration of AMPAr agonist and of neurotrophin BDNF. Also, the blockade of CaMKII impaired ORM consolidation when administered 3 h after sample phase but had no effect on ORM reconsolidation and its effect was blocked by the administration of BDNF, but not of AMPAr agonist. So, this study provides new evidence of the molecular mechanisms involved on the consolidation and reconsolidation of ORM, demonstrating that AMPAr and L-VDCCs are necessary for the consolidation and reconsolidation of ORM while CaMKII is necessary only for the consolidation and also that there is a link between BDNF and AMPAr, L-VDCCs and CaMKII as well as a link between AMPAr and L-VDCCs on ORM consolidation and reconsolidation.

Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses.

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1-/-) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 µM). In WT and Panx1-/- mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1-/- mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 µM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1-/- mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1-/- NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 µM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1-/- mice to above the control level.

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals.

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4-aminopyridine (4-AP)-evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca2+ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4-AP-evoked intrasynaptosomal Ca2+ levels, but did not affect the synaptosomal membrane potential. In the presence of N-, P/Q-, and R-type channel blockers, allopregnanolone-mediated inhibition of 4-AP-evoked glutamate release was markedly reduced; however, the intracellular Ca2+ -release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone-mediated inhibition of 4-AP-evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca2+ /calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABAA receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca2+ influx through N-, P/Q-, and R-type Ca2+ channels, through the activation of GABAA receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca2+ -calmodulin/PKA cascade and decreasing 4-AP-evoked glutamate release.

Voltage-Dependent Calcium Channels, Calcium Binding Proteins, and Their Interaction in the Pathological Process of Epilepsy.

As an important second messenger, the calcium ion (Ca2+) plays a vital role in normal brain function and in the pathophysiological process of different neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and epilepsy. Ca2+ takes part in the regulation of neuronal excitability, and the imbalance of intracellular Ca2+ is a trigger factor for the occurrence of epilepsy. Several anti-epileptic drugs target voltage-dependent calcium channels (VDCCs). Intracellular Ca2+ levels are mainly controlled by VDCCs located in the plasma membrane, the calcium-binding proteins (CBPs) inside the cytoplasm, calcium channels located on the intracellular calcium store (particular the endoplasmic reticulum/sarcoplasmic reticulum), and the Ca2+-pumps located in the plasma membrane and intracellular calcium store. So far, while many studies have established the relationship between calcium control factors and epilepsy, the mechanism of various Ca2+ regulatory factors in epileptogenesis is still unknown. In this paper, we reviewed the function, distribution, and alteration of VDCCs and CBPs in the central nervous system in the pathological process of epilepsy. The interaction of VDCCs with CBPs in the pathological process of epilepsy was also summarized. We hope this review can provide some clues for better understanding the mechanism of epileptogenesis, and for the development of new anti-epileptic drugs targeting on VDCCs and CBPs.

L-type VDCCs participate in behavioral-LTP and memory retention.

Although L-type voltage-dependent calcium channels (VDCCs) have been reported to display different even contrary actions on cognitive functions and long-term potentiation (LTP) formation, there is little information regarding the role of L-type VDCCs in behavioral LTP, a learning-induced LTP model, in the intact brain of freely behaving animals. Here we investigated the effects of verapamil, a non-selective blocker of L-type VDCCs, on behavioral LTP and cognitive functions. Population spikes (PS) were recorded by using electrophysiological methods to examine the role of verapamil in behavioral LTP in the hippocampal dentate gyrus (DG) region. Y-maze assay was used to evaluate the effects of verapamil on learning and memory. Electron microscope was used to observe the changes on synaptic ultrastructural morphology in hippocampal DG area. We found that intrahippocampal verapamil treatments had no significant changes on the PS amplitude during a 90min recordings period. However, intrahippocampal applications of verapamil, including pre- or post-training, reduced behavioral LTP magnitude and memory retention but did not prevent the induction of behavioral LTP and the acquisition of learning. The saline group with behaving trainings showed obvious increases in the number of smile synapses, the length of active zones and the thickness of postsynaptic density as compared to the baseline group, but verapamil with pre-training treatment almost returned these changes to the baseline levels except for the synaptic interface curvature. In conclusion, our results suggest that L-type VDCCs may only contribute to the magnitude of behavioral LTP and the memory maintenance with an activity-independent relationship. L-type VDCCs may be critical to new information long-term storage rather than acquisition in hippocampus.

Relaxing Effect of TSU-68, an Antiangiogenic Agent, on Mouse Airway Smooth Muscle.

BACKGROUND/AIMS: Recently, some small-molecule compounds that were designed for cancer therapy have acquired new roles in the treatment of pulmonary diseases. However, drug screening aimed at abnormal muscle contraction is still limited. TSU-68 is a potent, orally administered, small-molecule agent that can reduce the vascular endothelial growth factor (VEGF)-induced Ca2+ increase in endothelial cells. We questioned whether TSU-68 could also affect calcium influx and relax airway smooth muscle (ASM) cells. The current study aimed to investigate these effects and to explore the underlying mechanisms. METHODS: The effects of TSU-68 on ASM cells were studied in mice using a series of biophysiological techniques, including force measurement and patch-clamp experiments. RESULTS: TSU-68 inhibited high K+ or acetylcholine chloride (ACh)-induced pre-contracted mouse tracheal rings in a concentration-dependent manner. Further research demonstrated that the TSU-68-induced ASM relaxation was mediated by calcium, which was decreased by blocking voltage-dependent Ca2+ channels (VDCCs) and non-selective cation channels (NSCCs). CONCLUSION: Our data indicated that TSU-68 relaxes tense ASM by reducing the intracellular Ca2+ concentration through blocking VDCCs and NSCCs, which suggested that this small molecule might be useful in the treatment of abnormal smooth muscle.

Role of histamine H3 receptor in glucagon-secreting αTC1.6 cells.

Pancreatic α-cells secrete glucagon to maintain energy homeostasis. Although histamine has an important role in energy homeostasis, the expression and function of histamine receptors in pancreatic α-cells remains unknown. We found that the histamine H3 receptor (H3R) was expressed in mouse pancreatic α-cells and αTC1.6 cells, a mouse pancreatic α-cell line. H3R inhibited glucagon secretion from αTC1.6 cells by inhibiting an increase in intracellular Ca(2+) concentration. We also found that immepip, a selective H3R agonist, decreased serum glucagon concentration in rats. These results suggest that H3R modulates glucagon secretion from pancreatic α-cells.

Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice.

Nobiletin, a polymethoxylated flavonoid found in citrus fruit peel, reportedly improves memory impairment in rodent models. Here we report its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor and cognitive deficits. Nobiletin administration (50mg/kg i.p.) for 2 consecutive weeks improved motor deficits seen in MPTP-induced Parkinson model mice by 2weeks, an effect that continued until 2weeks after drug withdrawal. Drug treatment promoted similar rescue of MPTP-induced cognitive impairment at equivalent time points. Nonetheless, nobiletin treatment did not block loss of dopaminergic neurons seen in the MPTP-treated mouse midbrain, nor did it rescue decreased tyrosine hydroxylase (TH) protein levels seen in the striatum or hippocampal CA1 region of these mice. Interestingly, nobiletin administration (50mg/kg i.p.) rescued reduced levels of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation at Thr-34 of dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32) in striatum and hippocampal CA1 to levels seen in sham-operated mice. Likewise, CaMKII- and cAMP kinase-dependent TH phosphorylation was significantly restored by nobiletin treatment. MPTP-induced reduction of dopamine contents in the striatum and hippocampal CA1 region was improved by nobiletin administration (50mg/kg i.p.). Acute intraperitoneal administration of nobiletin also enhanced dopamine release in striatum and hippocampal CA1, an effect partially inhibited by treatment with nifedipine (a L-type Ca(2+) channel inhibitor) or NNC 55-0396 (a T-type Ca(2+) channel inhibitor) and completely abolished by combined treatment with both. Overall, our study describes a novel nobiletin activity in brain and suggests that nobiletin rescues motor and cognitive dysfunction in MPTP-induced Parkinson model mice, in part by enhancing dopamine release.

Cholinergic modulation of primary afferent glutamatergic transmission in rat medullary dorsal horn neurons.

Although muscarinic acetylcholine (mACh) receptors are expressed in trigeminal ganglia, it is still unknown whether mACh receptors modulate glutamatergic transmission from primary afferents onto medullary dorsal horn neurons. In this study, we have addressed the cholinergic modulation of primary afferent glutamatergic transmission using a conventional whole cell patch clamp technique. Glutamatergic excitatory postsynaptic currents (EPSCs) were evoked from primary afferents by electrical stimulation of trigeminal tract and monosynaptic EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices. Muscarine and ACh reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, muscarine reduced the frequency of miniature EPSCs without affecting the current amplitude, suggesting that muscarine acts presynaptically to decrease the probability of glutamate release onto medullary dorsal horn neurons. The muscarine-induced decrease of glutamatergic EPSCs was significantly occluded by methoctramine or AF-DX116, M2 receptor antagonists, but not pirenzepine, J104129 and MT-3, selective M1, M3 and M4 receptor antagonists. The muscarine-induced decrease of glutamatergic EPSCs was highly dependent on the extracellular Ca2+ concentration. Physostigmine and clinically available acetylcholinesterase inhibitors, such as rivastigmine and donepezil, significantly shifted the concentration-inhibition relationship of ACh for glutamatergic EPSCs. These results suggest that muscarine acts on presynaptic M2 receptors to inhibit glutamatergic transmission by reducing the Ca2+ influx into primary afferent terminals, and that M2 receptor agonists and acetylcholinesterase inhibitors could be, at least, potential targets to reduce nociceptive transmission from orofacial tissues.

Intracisternal Administration of Voltage Dependent Calcium Channel Blockers Attenuates Orofacial Inflammatory Nociceptive Behavior in Rats

Voltage dependent calcium channel (VDCC), one of the most important regulator of Ca2+ concentration in neuron, play an essential role in the central processing of nociceptive information. The present study investigated the antinociceptive effects of L, T or N type VDCC blockers on the formalin-induced orofacial inflammatory pain. Experiments were carried out on adult male Sprague-Dawley rats weighing 220-280 g. Anesthetized rats were individually fixed on a stereotaxic frame and a polyethylene (PE) tube was implanted for intracisternal injection. After 72 hours, 5% formalin (50 microL) was applied subcutaneously to the vibrissa pad and nociceptive scratching behavior was recorded for nine successive 5 min intervals. VDCC blockers were administered intracisternally 20 minutes prior to subcutaneous injection of formalin into the orofacial area. The intracisternal administration of 350 or 700 microg of verapamil, a blocker of L type VDCC, significantly decreased the number of scratches and duration in the behavioral responses produced by formalin injection. Intracisternal administration of 75 or 150 microg of mibefradil, a T type VDCC blocker, or 11 or 22 microg of cilnidipine, a N type VDCC blocker, also produced significant suppression of the number of scratches and duration of scratching in the first and second phase. Neither intracisternal administration of all VDCC blockers nor vehicle did not affect in motor dysfunction. The present results suggest that central VDCCs play an important role in orofacial nociceptive transmission and a targeted inhibition of the VDCCs is a potentially important treatment approach for inflammatory pain originating in the orofacial area.

Influence of Cilnidipine on Catecholamine Release in the Perfused Rat Adrenal Medulla

The present study was attempted to investigate the effect of cilnidipine (FRC-8635), which is a newly synthesized novel dihydropyridine (DHP) type of organic Ca2 channel blockers, on secretion of catecholamines (CA) evoked by acetylcholine (ACh), high K, DMPP and McN-A-343 from the isolated perfused rat adrenal gland. Cilnidipine (1~10microM) perfused into an adrenal vein for 60 min produced relatively dose- and time-dependent inhibition in CA secretory responses evoked by ACh (5.32 10 3 M), DMPP (10 4 M for 2 min) and McN-A-343 (10 4 M for 2 min). However, lower dose of cilnidipine did not affect CA secretion by high K (5.6 10 2 M), higher dose of it reduced greatly CA secretion of high K. Cilnidipine itself did fail to affect basal catecholamine output. In the presence of cilnidipine (10microM), the CA secretory responses evoked by Bay-K-8644 (10microM), an activator of L-type Ca2 channels and cyclopiazonic acid (10microM), an inhibitor of cytoplasmic Ca2 -ATPase were also inhibited. Moreover, omega-conotoxin GVIA (1microM), a selective blocker of the N-type Ca2 channels, given into the adrenal gland for 60 min, also inhibited time-dependently CA secretory responses evoked by Ach, high K, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid. Taken together, these results demostrate that cilnidipine inhibits CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors from the isolated perfused rat adrenal gland without affecting the basal release. However, at lower dose, cilnidipine did not affect CA release by membrane depolarization while at larger dose inhibited that. It seems likely that this inhibitory effect of cilnidipine is exerted by blocking both L- and N-type voltage-dependent Ca2 channels (VDCCs) on the rat adrenomedullary chromaffin cells, which is relevant to inhibition of both the Ca2 influx into the adrenal chromaffin cells and intracellular Ca2 release from the cytoplasmic store. It is thought that N-type VDCCs may play an important role in regulation of CA release from the rat adrenal medulla.