[Show abstract][Hide abstract] ABSTRACT: Lumbar radicular pain is categorized as a type of neuropathic pain, but its pathophysiological mechanisms are not fully understood. The substantia gelatinosa (SG) in the spinal cord dorsal horn receives primary afferent inputs and is considered to be a therapeutic target for treating neuropathic pain. In vivo patch-clamp recording is a useful procedure for analyzing the functional properties of synaptic transmission in SG neurons. Transient receptor potential ankyrin 1 (TRPA1) has been widely identified in the central and peripheral nervous systems, such as in the peripheral nociceptor, dorsal root ganglion, and spinal cord dorsal horn and is involved in synaptic transmission of pain. However, its functional role and mechanism of pain transmission in the spinal cord dorsal horn are not well understood. The purpose of this study was to use in vivo patch-clamp analysis to examine changes in the excitatory synaptic transmission of SG neurons treated with TRPA1 antagonist and to clarify the potential role of TRPA1 in the rat spinal cord dorsal horn.
[Show abstract][Hide abstract] ABSTRACT: Aims: We examined if and how cardiac ion channels are modified by type 2 diabetes mellitus (T2DM). Methods: Subendocardial myocytes (END) and subepicardial myocytes (EPI) were isolated from left ventricles of OLETF, a rat model of T2DM, and LETO, non-diabetic control. END and EPI were used for whole-cell patch-clamp recordings and for protein and mRNA analyses. Results: Action potential durations (APDs) in END and EPI were longer in OLETF than in LETO, and the difference was larger in END. Steady-state transient outward K(+) current (Ito) density was reduced in END, but not in EPI, of OLETF compared with those in LETO, though contribution of the fast component of Ito current recovery from inactivation was smaller in both END and EPI of OLETF than in those of LETO. Kv4.2 protein was reduced only in END in OLETF, though KChIP2 protein levels in both END and EPI were lower in OLETF than in LETO. Corresponding regional differences in mRNA levels of KChIP2 and Kv4.2 were observed between OLETF and LETO. The mRNA level of iroquois homeobox 5 (Irx5) in END was 53% higher in OLETF than in LETO. Densities of inward rectifier K(+) current and L-type Ca(2+) current and mRNA levels of Kv4.3 and Kv1.4 were similar in OLETF and LETO. Conclusion: T2DM induces subendocardium-predominant prolongation of APD via reduction of the fast component of Ito recovery from inactivation and reduced steady-state Ito, in which downregulation of Kv4.2 and KChIP2 may be involved. Increased Irx5 expression may underlie Kv4.2 downregulation in T2DM.
AJP Heart and Circulatory Physiology 01/2014; · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to elucidate the influence of sympathetic nerves on lumbar radiculopathy, we investigated whether sympathectomy attenuated pain behaviour and altered the electrical properties of the dorsal root ganglion (DRG) neurons in a rat model of lumbar root constriction.
Sprague-Dawley rats were divided into three experimental groups. In the root constriction group, the left L5 spinal nerve root was ligated proximal to the DRG as a lumbar radiculopathy model. In the root constriction + sympathectomy group, sympathectomy was performed after the root constriction procedure. In the control group, no procedures were performed. In order to evaluate the pain relief effect of sympathectomy, behavioural analysis using mechanical and thermal stimulation was performed. In order to evaluate the excitability of the DRG neurons, we recorded action potentials of the isolated single DRG neuron by the whole-cell patch-clamp method.
In behavioural analysis, sympathectomy attenuated the mechanical allodynia and thermal hyperalgesia caused by lumbar root constriction. In electrophysiological analysis, single isolated DRG neurons with root constriction exhibited lower threshold current, more depolarised resting membrane potential, prolonged action potential duration, and more depolarisation frequency. These hyperexcitable alterations caused by root constriction were significantly attenuated in rats treated with surgical sympathectomy.
The present results suggest that sympathectomy attenuates lumbar radicular pain resulting from root constriction by altering the electrical property of the DRG neuron itself. Thus, the sympathetic nervous system was closely associated with lumbar radicular pain, and suppressing the activity of the sympathetic nervous system may therefore lead to pain relief.
[Show abstract][Hide abstract] ABSTRACT: Although many researchers have tried to observe the beginning of the heartbeat, no study has shown the beginning of the calcium transient. Here, we evaluate the beginning of the calcium transient in the Wistar rat heart. We first tried to reveal when the heart of the Wistar rat begins to contract because no previous study has evaluated the beginning of the heartbeat in Wistar rats. Observation of embryos transferred to a small incubator mounted on a microscope revealed that the heart primordium, the so-called cardiac crescent, began to contract at embryonic day 9.99-10.13. Observation of embryos loaded with fluo-3 AM revealed that the beginning of the calcium transient precedes the initiation of contraction which precedes the appearance of the linear heart tube. Nifedipine (1 μM), but not ryanodine (1 μM), abolished the calcium transients. These results indicate that calcium transients in the early embryonic period involve exclusively calcium entry through L-type calcium channels in contrast to the situation in mature hearts. This study provides the first demonstration of the relationship between morphological changes in the heart primordium and the beginning of the calcium transient and contraction.
The Journal of Physiological Sciences 03/2011; 61(2):141-9. · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Postganglionic neurons in the sympathetic nervous system reportedly are involved in lumbar radicular pain and release norepinephrine (NE), a neurotransmitter. Increased numbers of sympathetic nerve fibers have been found in dorsal root ganglion (DRG) neurons in a root constriction model. Whether this is a reasonable model for pain, however, is unclear
We asked whether: (1) painful behaviors occurred in the root constriction model; (2) NE enhanced the excitability of DRG neurons in the root constriction model; and (3) which adrenoceptors were related to the mediation of the NE effects.
The L5 root was sutured proximal to the DRG as the root constriction model. Behavioral tests were performed until 28 days after surgery. At 10 to 14 days after the root constriction, DRG neurons were quickly excised and digested with collagenase for electrophysiologic studies. Action potentials were recorded from single DRG neurons using a whole-cell patch clamp technique. NE (10 μmol/L) was directly applied to the DRG neurons. The adrenergic sensitivity was examined in combination with antagonists.
The rats with root constriction exhibited painful behavior. NE increased the excitability of DRG neurons in the root constriction model. The effects of NE were inhibited by pretreatment with an α-antagonist and α(2)-antagonist but not an α(1)-antagonist.
Our observations suggest NE plays an important role in generating lumbar radicular pain mainly via α(2)-adrenoceptors.
An α(2)-antagonist may be an appropriate agent for trials to treat lumbar radicular pain.
Clinical Orthopaedics and Related Research 02/2011; 469(9):2568-76. · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently reported that the heart-beat in rat embryo initiated between E9.99 and E10.13 using CCD camera system, and also L-type Ca2+ channel (Cav1.2, Cavβ2), Na+/Ca2+ exchanger (NCX1), and voltage-gated K+ channels (ERG1, MiRP1) contribute to action potential (AP) configuration in the period of heart-beat initiation in the early rat embryo. Furthermore, we found that the Ca2+ transient firstly began a part of cardiac crescent, then the contraction starts about several minutes late (Kobayashi, et al. J Physiol Sci 2011). In the present study, we studied mRNA expression of L-type Ca2+ channel Cav1.3 that is important for the generation of spontaneous AP in SA node cells by participating to diastolic depolarization. To investigate delay of contraction after initiation of excitation, we also examined mRNA expression of sarcomeric-related proteins; cardiac α-actin (Actc1) and cardiac troponin C (TnnC1). Whole mount in situ hybridization and RT-PCR showed that Actc1 and TnnC1 mRNA expressed at day E9.8. Interestingly, expression of Cav1.3 mRNA was not observed at day E9.8, and was up-regulated at day E10.0. These results suggest that mRNA expression of Cav1.3 contribute to the initiation of excitation, and also mRNA expression of Actc1 and TnnC1 are necessary to the initiation of contraction in the early rat embryo.
Journal of Arrhythmia 01/2011; 27(Supplement):PJ2_044.
[Show abstract][Hide abstract] ABSTRACT: Although there is evidence that the volatile anaesthetic desflurane directly relaxes preconstricted airway smooth muscle in vitro, the anaesthetic increases the lung resistance in vivo. The constrictive mechanisms of desflurane are, however, still unknown. This study was conducted to clarify the increasing mechanisms of desflurane on lung resistance by examining the vagal nerve reflexes in guinea pigs.
The effects of desflurane and sevoflurane on total lung resistance (R(L)) and dynamic lung compliance (C(Dyn)) were investigated in animals that were either untreated, pretreated with atropine or vagotomy, pretreated with the tachykinin receptor antagonists sendide or MEN-10376, or given chronic pretreatment with capsaicin.
Desflurane biphasically and dose-dependently increased R(L) (by 180% and 230% at the first and second peaks, respectively, at 2 minimum alveolar concentration) concomitant with a decrease in C(Dyn). However, sevoflurane had little effect on either R(L) or C(Dyn). Although vagotomy partially inhibited the first peak of R(L) by 30%, neither atropine nor vagotomy had any effect on the other respiratory responses to desflurane. Antagonization of tachykinin receptors of airway smooth muscles completely diminished the increase in R(L) induced by desflurane. Desflurane also had little effect on respiratory parameters after the capsaicin pretreatment, in which tachykinin containing afferent C-fibres was desensitized.
Desflurane but not sevoflurane increased R(L) concomitant with a decrease in C(Dyn) in guinea pigs. The increase in lung resistance by desflurane might be due to antidromic tachykinin release from afferent C-fibres but not acetylcholine release from parasympathetic efferent nerves.
BJA British Journal of Anaesthesia 04/2009; 102(5):704-13. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation. On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 microm to 10 microm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia. The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly. We consider that the sympathetic nervous system plays an important role in the generation of radicular pain.
The Bone & Joint Journal 01/2008; 89(12):1666-72. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The beta subunits of voltage-dependent calcium channels bind the pore-forming alpha(1) subunit and play an important role in the regulation of calcium channel function. Recently, we have identified a new splice variant of the beta(4) subunit, which we have termed the beta(4d) subunit. The beta(4d) subunit is a truncated splice variant of the beta(4b) subunit and lacks parts of the guanylate kinase (GK) domain and the C-terminus. The calcium current in BHK cells expressing alpha(1C) and alpha(2)delta with the beta(4d) subunit was as small as that without the beta(4d) subunit. Western blot analysis revealed that beta(4d) protein was expressed to a lesser extent that the beta(4b) protein. In addition, a GST pull down assay showed that the beta(4d) subunit could not interact with the alpha(1) subunit of the calcium channel. Collectively, our results suggest that the GK domain of the beta subunit is essential for the expression of the functional calcium channel.
Biochemical and Biophysical Research Communications 09/2007; 360(3):679-83. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ca(V)1.2 (alpha(1c)) is a pore-forming subunit of the voltage-dependent L-type calcium channel and is expressed in many tissues. The beta and alpha(2)/delta subunits are auxiliary subunits that affect the kinetics and the expression of Ca(V)1.2. In addition to the beta and alpha(2)/delta subunits, several molecules have been reported to be involved in the regulation of Ca(V)1.2 current. Calmodulin, CaBP1 (calcium-binding protein-1), CaMKII (calcium/calmodulin-dependent protein kinase II), AKAPs (A-kinase anchoring proteins), phosphatases, Caveolin-3, beta(2)-adrenergic receptor, PDZ domain proteins, sorcin, SNARE proteins, synaptotagmin, CSN5, RGK family, and AHNAK1 have all been reported to interact with Ca(V)1.2 and the beta subunit. This review focuses on the effect of these molecules on Ca(V)1.2 current.
Journal of Pharmacological Sciences 05/2007; 103(4):347-53. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DRG neuron was electrophysiologically investigated using a rat model with constriction of the proximal site of the DRG.
To investigate the pathomechanisms of lumbar radiculopathy, we established a rat model with constriction of the proximal site of the DRG. And to characterize the DRG neurons in the rat model of lumbar radiculopathy, the physiologic properties regarding action potential, Na, and K current of the DRG neurons were analyzed through the use of patch clamp recordings.
In lumbar root constriction models, properties of secondary afferent neurons in the dorsal horn have been investigated. However, the electrical properties of DRG neuron have not been well investigated.
To compare the excitability of DRG neurons between root constriction models and sham, we examined the threshold current, action potential (AP) threshold, resting membrane potential (RMP), afterhyperpolarization (AHP), action potential duration 50 (APD50), action potential amplitude, maximum rise time of AP, and pattern of discharges evoked by depolarizing current. We also examined the peak Na current and steady-state Na and K currents with the voltage clamp technique.
The rats in the root constriction group demonstrated mechanical allodynia and thermal hyperalgesia. In measurement of the action potential, lower threshold current, more depolarized RMP, larger AHP, and prolonged APD50 were measured in the root constriction neurons compared with the sham group. The incidence of sustained burst was significantly higher in root constriction neurons. The Na current in root constriction neurons was markedly larger. There were no significant differences in K current density and voltage dependency.
The constriction of lumbar root increased excitability and Na current amplitude of DRG neurons. These findings indicate that lumbar radicular pain may be associated with increased excitability of involved DRG neurons.
[Show abstract][Hide abstract] ABSTRACT: Using isolated perfused lungs of normal or monocrotaline (MCT: 50 mg/kg)-induced pulmonary hypertensive rats, we tested the hypothesis that the pulmonary vascular effects of propofol depend on activation of the alpha-adrenoreceptor.
Changes in pulmonary perfusion pressure induced by propofol (10(-5) to 10(-4) M) were measured with or without phenylephrine (10(-6) M) pretreatment. Before phenylephrine administration, we assessed the effects of inhibitors of nitric oxide synthase (N(omega)-nitro-l-arginine methylester: 10(-4) M), cyclooxygenase (indomethacin: 10(-5) M), and protein kinase C inhibitor, bisindolylmaleimide I (10(-6) M) or calphostin C (10(-6) M).
Changes in pulmonary perfusion pressure by phenylephrine after pretreatment of nitric oxide synthase inhibitor and indomethacin in normal rats were significant (5 +/- 3 and 7 +/- 2 mm Hg), whereas that after pretreatment of bisindolylmaleimide I were small in MCT-rats (2 +/- 1 mm Hg). Propofol caused pulmonary vasoconstriction after phenylephrine pretreatment both in normal and MCT-treated rats. In normal rats, the propofol-induced increase in pulmonary perfusion pressure after indomethacin pretreatment was slightly smaller than that in the non-pretreated lungs (P < 0.05). In MCT-treated rats, the propofol-induced increases in pulmonary perfusion pressure after both protein kinase C inhibitors were smaller than that in the non-pretreated lungs (P < 0.05).
Propofol may increase pulmonary vascular resistance during alpha-adrenoreceptor activation.
Anesthesia and analgesia 01/2007; 104(1):112-8. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.
[Show abstract][Hide abstract] ABSTRACT: L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.
Journal of Molecular and Cellular Cardiology 05/2006; 40(4):562-9. · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gating properties of Na(+) channels are the critical determinants for the state-dependent block by class I antiarrhythmic drugs; however, recent site-directed mutagenesis studies have shown that the Na(+) channel selectivity filter region controls drug access to and dissociation from the binding site. To validate these observations, we have exploited a naturally occurring cardiac Na(+) channel mutation, S1710L, located next to the putative selectivity filter residue of domain 4, and evaluated the pharmacological properties to mexiletine using whole-cell, patch-clamp recordings. Consistent with the large negative shift of steady-state inactivation and the enhanced slow inactivation, the S1710L channel showed greater mexiletine tonic block than wild-type (WT) channel. In contradiction, S1710L showed attenuated use-dependent block by mexiletine and accelerated recovery from block, suggesting that the drug escape though the external access path is facilitated. Extracellularly applied QX-314, a membrane-impermeant derivative of lidocaine, elicited significantly enhanced tonic block in S1710L similar to mexiletine. However, recovery from internally applied QX-314 was accelerated by 4.4-fold in S1710L compared with WT. These results suggest that the drug access to and dissociation from the binding site through the hydrophilic path are substantially altered. Moreover, K(+) permeability was 1.9-fold increased in S1710L, verifying that the mutated residue is located in the ion-conducting pore. We propose that the Na(+) channel selectivity filter region is a structural determinant for the antiarrhythmic drug sensitivity in addition to gating properties of the indigenous Na(+) channels that govern the state-dependent drug block.
[Show abstract][Hide abstract] ABSTRACT: We recently cloned the beta(2c) subunit of the L-type Ca(2+) channel as a functional type of beta subunit from the rat heart. In order to clarify the contribution of the beta(2c) subunit to native Ca(2+) channel function, we investigated the single-channel properties of Ca(2+) channels reconstituted with beta(2a) or beta(2c) subunits and compared them with the properties of native channels. In contrast to the Ca(2+) channel with beta(2a) subunit, long-lasting closings were dominant in the Ca(2+) channel with beta(2c) subunit and the native channel. The ensemble-averaged current of the cells with beta(2c) subunits was comparable to that of the native cardiomyocytes. Many high P(o) sweeps (mode 2) were observed in the cells with beta(2a) subunits, while only a few high P(o) sweeps were observed in the cells with beta(2c) subunits and the native cells. These findings suggest that the beta(2c) subunit is one of the functional beta subunits in the rat heart.
European Journal of Pharmacology 04/2004; 487(1-3):37-45. · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the effects of transforming growth factor (TGF)-betas on morphological and receptor phenotypes, as well as proliferation of four currently established human prostatic myofibroblast cell lines and one commercially available prostatic stromal cell line.
The effects of TGF-betas on morphological changes and proliferation of the cells were studied by immunohistochemistry and bromodeoxyuridine assay, respectively. The expression of alpha 1-receptor subtypes was measured by real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and the radioligand binding assay for the receptors was also performed.
TGF-betas 1, 2, and 3 induced expression of desmin and myosin of cells of the established cell lines, and significantly inhibited their growth. The alpha 1a-receptor was expressed only in the commercially available cell line and alpha 1b and 1d, in all cell lines. TGF-beta 1 suppressed the expression of all three subtypes of the alpha 1-receptor. The binding sites of cells of all the cell lines were reduced by treatment with this growth factor.
TGF-betas may induce human prostatic stromal cells to express the smooth muscle phenotype and inhibited their growth. However, the growth factor reduced the binding sites of the receptor and suppressed mRNA expression of its subtypes, suggesting that morphological and receptor phenotypes may be regulated via more than one pathway by TGF-beta(s).
The Prostate 03/2004; 58(2):174-82. · 3.57 Impact Factor