N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Calcium channels involved in neurotransmitter release at adult,neonatal and P/Q-type deficient neuromuscular junctions (Review)

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (Ica) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca(2+) inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca(2+)-inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.     

Calcium channels involved in neurotransmitter release at adult,neonatal and P/Q-type deficient neuromuscular junctions (Review)

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (I Ca ) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca 2+ inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca 2+ -inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.     

A novel series of pyrazolylpiperidine N-type calcium channel blockers

Selective blockers of the N-type calcium channel have proven to be effective in animal models of chronic pain. However, even though intrathecally delivered synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been approved for the treatment of chronic pain in humans, its mode of delivery and narrow therapeutic window have limited its usefulness. Therefore, the identification of orally active, small-molecule N-type calcium channel blockers would represent a significant advancement in the treatment of chronic pain. A novel series of pyrazole-based N-type calcium channel blockers was identified by structural modification of a high-throughput screening hit and further optimized to improve potency and metabolic stability. In vivo efficacy in rat models of inflammatory and neuropathic pain was demonstrated by a representative compound from this series.     

Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.     

beta-Adrenergic receptor activation induces internalization of cardiac Cav1.2 channel complexes through a beta-arrestin 1-mediated pathway

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of beta-adrenergic receptors (beta-ARs), which leads to an increase in calcium current (I(Ca-L)) density through cardiac Ca(v)1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I(Ca-L) density and kinetics in heart failure often occur in the absence of changes in Ca(v)1 channel expression, arguing for the importance of post-translational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained beta-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and beta-arrestin 1 (beta-Arr1) into clathrin-coated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G(i/o) mediates this response. A peptide that selectively disrupts the interaction between Ca(V)1.2 and beta-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G(i) of the beta-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure.     

Molecular and immunohistochemical studies in expression of voltage-dependent Ca2+ channels in dorsal root ganglia from streptozotocin-induced diabetic mice

We have recently demonstrated that intrathecal injection of a selective P/Q-type blocker of the voltage-dependent Ca(2+) channels (VDCCs) significantly inhibited the mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice, its antinociceptive effect being greater than in controls. In this study, to further clarify the underlying mechanism of the STZ-induced hyperalgesia, we investigated the expression level of the VDCC alpha1A and alpha1B subunits in the dorsal root ganglia (DRGs) and the dorsal spinal cord under this hyperalgesia. Real-time PCR analysis showed mRNA expression of alpha1A (P/Q-type), but not alpha1B (N-type), was significantly increased in the DRGs from the STZ-induced diabetic mice. On the other hand, gene expression of both alpha1 subunits was not changed in the dorsal part of the spinal cord. In diabetic DRG neurons, the number of large nerve cells was significantly reduced, whereas small neurons were significantly increased. Immunohistochemical study demonstrated the alpha1A-positive neurons, but not alpha1B-positive neurons, increased significantly greater in diabetic DRGs than in control in all cell size. These results indicate that an alteration in expression of P/Q-type VDCCs, especially in the small and medium-diameter primary afferent fibers, in pain pathways ascending input to the spinal cord may be involved in hypersensitivity in STZ-induced diabetes.     

Voltage-dependent calcium channels in the corpora allata of the adult male loreyi leafworm, Mythimna loreyi

In the corpora allata (CA) of the adult male loreyi leafworm, Mythimna loreyi, juvenile hormone acid (JHA) biosynthesis and release show a dose dependence on extracellular Ca(2+) concentration. Maxima are obtained with Ca(2+) concentrations of 2-10 mM, and synthesis and release are significantly inhibited under a Ca(2+)-free condition. The Ca(2+)-free inhibition of JHA release can be reversed by returning the glands to medium at 5 mM Ca(2+). The cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which was measured with fura-2, in individual CA cells also shows a dose dependence on extracellular Ca(2+) concentration, with significant [Ca(2+)](i) depression being observed in the absence of extracellular Ca(2+).High K(+) significantly increases the JHA release and causes a transient [Ca(2+)](i) increase within seconds in CA cells. High-K(+)-stimulated JHA release is partially inhibited by the benzothiazepine (BTZ)-, dihydropyridine (DHP)- and phenylalkylamine (PAA)-sensitive L-type voltage-dependent calcium channel (VDCC) antagonists diltiazem, nifedipine and verapamil, respectively; by the N- and P/Q-type VDCC antagonist omega-conotoxin (omega-CgTx) MVIIC; and by the T-type VDCC antagonist amiloride. The N-type antagonist omega-CgTx GVIA is the most potent in inhibiting the high-K(+)-stimulated JHA release. No inhibitory effect is shown by the P-type antagonist omega-agatoxin TK (omega-Aga TK). The high-K(+)-induced transient [Ca(2+)](i) increase is largely inhibited by the L-type antagonists (diltiazem, nifedipine, verapamil), by the N- and P/Q-type antagonist omega-CgTx MVIIC and by the T-type antagonist amiloride, and is totally inhibited by the N-type antagonist omega-CgTx GVIA. No inhibitory effect is shown by the P-type antagonist omega-Aga TK.We hypothesize that L-type, N-type and T-type VDCCs may be involved to different degrees in the high-K(+)-stimulated JHA release and transient [Ca(2+)](i) increase in the individual CA cells of the adult male M. loreyi, and that the N-type VDCCs may play important roles in these cellular events.     

Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels

N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain.     

In Vivo Analysis of Voltage-Dependent Calcium Channels

The molecular cloning of calcium channel subunits has identified an unexpectedly large number of genes and splicing variants, many of whichhave complex expression patterns: a central problem of calcium channel biology is to understand the functional significance of this genetic complexity. The genetic analysis of voltage-dependent calcium channels (VDCCs) provides an approach to defining channel function that is complimentary to pharmacological, electrophysiological, and other molecular methods. By discovering or creating alleles of VDCC genes, one can gain an understanding of the VDCC function at the whole animal level. Of particular interest are mutations in the alpha1 genes that encode the pore forming subunits, as they define the specific channel subtypes. In fact, a variety of calcium channelopathies and targeted mutations have been described for these genes in the last 6 years. The mutant alleles described below illustrate how phenotype analysis of these alleles has uncovered very specific functional roles that can be localized to specific synapses or cells.     

Pain perception in mice lacking the beta3 subunit of voltage-activated calcium channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.     

Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.     

Structure-activity relationships of trimethoxybenzyl piperazine N-type calcium channel inhibitors

We previously reported the small organic N-type calcium channel blocker NP078585 that while efficacious in animal models for pain, exhibited modest L-type calcium channel selectivity and substantial off-target inhibition against the hERG potassium channel. Structure-activity studies to optimize NP078585 preclinical properties resulted in compound 16, which maintained high potency for N-type calcium channel blockade, and possessed excellent selectivity over the hERG (~120-fold) and L-type (~3600-fold) channels. Compound 16 shows significant anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain and is also efficacious in the rat formalin model of inflammatory pain.     

The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1)

Mutations in VMD2, encoding bestrophin (best-1), cause Best vitelliform macular dystrophy (BMD), adult-onset vitelliform macular dystrophy (AVMD), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). BMD is distinguished from AVMD by a diminished electrooculogram light peak (LP) in the absence of changes in the flash electroretinogram. Although the LP is thought to be generated by best-1, we find enhanced LP luminance responsiveness with normal amplitude in Vmd2-/- mice and no differences in cellular Cl- currents in comparison to Vmd2+/+ littermates. The putative Ca2+ sensitivity of best-1, and our recent observation that best-1 alters the kinetics of voltage-dependent Ca2+ channels (VDCC), led us to examine the role of VDCCs in the LP. Nimodipine diminished the LP, leading us to survey VDCC beta-subunit mutant mice. Lethargic mice, which harbor a loss of function mutation in the beta4 subunit of VDCCs, exhibited a significant shift in LP luminance response, establishing a role for Ca2+ in LP generation. When stimulated with ATP, which increases [Ca++]I, retinal pigment epithelial cells derived from Vmd2-/- mice exhibited a fivefold greater response than Vmd2+/+ littermates, indicating that best-1 can suppress the rise in [Ca2+]I associated with the LP. We conclude that VDCCs regulated by a beta4 subunit are required to generate the LP and that best-1 antagonizes the LP luminance response potentially via its ability to modulate VDCC function. Furthermore, we suggest that the loss of vision associated with BMD is not caused by the same pathologic process as the diminished LP, but rather is caused by as yet unidentified effects of best-1 on other cellular processes.     

Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.     

Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation

Voltage-gated Ca2+ channels (VDCCs) are heteromultimeric proteins that mediate Ca2+ influx into cells upon membrane depolarization. These channels are involved in various cellular events, including gene expression, regulation of hormone secretion and synaptic transmission. Kir/Gem, Rad, Rem, and Rem2 belong to the RGK family of Ras-related small G proteins. RGK proteins interact with the beta-subunits and downregulate VDCC activity. Kir/Gem was proposed to prevent surface expression of functional Ca2+ channels, while for Rem2 the mechanism remains controversial. Here, we have analyzed the mechanism by which Rad and Rem regulate VDCC activity. We show that, similar to Kir/Gem and Rem2, 14-3-3 and CaM binding regulate the subcellular distribution of Rad and Rem, which both inhibit Ca2+ channel activity by preventing its expression on the cell surface. This function is regulated by calmodulin and 14-3-3 binding only for Rad and not for Rem. Interestingly, nuclear targeting of Rad and Rem can relocalize and sequester the beta-subunit to the nucleus, thus providing a novel mechanism for Ca2+ channel downregulation.     

钠离子通道NaV1.7与神经病理性疼痛

钠通道NaV1.7是电压门控性钠通道的亚型之一。大多数钠离子通道NaV1.7表达在背根神经节(DRG)小C纤维的伤害性感受器上,具有缓慢开放和缓慢关闭失活的特点。它能够产生大量的斜坡电流,降低感觉神经元中动作电位产生的阈值,放大外来小的缓慢的去极化斜坡电流,从而增加神经元兴奋性,对疼痛的产生、传递、调节具有关键性作用。随着遗传学研究的不断深入,钠离子通道NaV1.7的功能获得性突变和功能缺失性突变,使其成为了新型镇痛疗法中一个的特别有吸引力的药物靶点,受到人们的广泛关注。而研究发现,NaV1.7通道在不同因素引起的神经病理性疼痛中通过不同途径提高神经元兴奋性,参与神经病理性疼痛,给NaV1.7选择性抑制剂研发带来了巨大阻碍。目前,虽然已有的NaV1.7选择性抑制剂具备有效镇痛作用,且无明显副作用或成瘾问题,但寻找NaV1.7选择性配体极其困难。此外,现有的NaV1.7选择性抑制剂也因神经病理性疼痛类型的不同在抑制效力、靶向性、安全性以及可行性等方面存在差异。提示寻找NaV1.7通道作用于不同神经病理性疼痛的普遍机制或NaV1.7通道特有的受体结合位点,可能是未来NaV1.7选择性抑制剂研发的主要方向。本文就NaV1.7通道在不同因素引起的神经病理性疼痛中的主要作用进行简要综述。     

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety,and oral analgesic efficacy in rat pain models

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.     

Cyclic AMP-dependent modulation of cardiac Ca channels expressed in Xenopus laevis oocytes.

Cyclic AMP-dependent modulation of cardiac L-type voltage-dependent Ca channel (VDCC) has been probed in Xenopus laevis oocytes injected with poly(A+) RNA from rat heart. A 2 to 3 fold increase of the Ba current amplitude was routinely obtained upon microinjection of cAMP (50-500 microM). Inhibition of protein kinase A (PKA) dramatically reduced the Ba current amplitude, indicating that cAMP-dependent modulation plays an important role in maintaining the basal activity of expressed Ca channels. Moreover, the effects of the DHP agonist Bay K 8644 on kinetic properties of expressed Ba current (IBa,C) were dependent on PKA activation. The results suggest that most expressed cardiac L-type VDCCs are phosphorylated and demonstrate that reconstitution in Xenopus oocytes is a suitable approach to address how phosphorylation regulates VDCC activity.