Atsushi Doi

Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole‐cell patch‐clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current‐clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0–9.5 mV) and suppressed the action potentials. In the voltage‐clamp mode (VH, −70 mV), the application of NA produced an outward current that was blocked by Cs+ and GDP‐β‐S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre‐ and postsynaptic actions of NA. The NA‐induced outward currents showed a clear dose dependency (EC50, 20 μm), and the reversal potential was −88 mV. The outward current was mimicked by an α2‐adrenoceptor agonist, clonidine, and suppressed by an α2‐adrenoceptor antagonist, yohimbine, but not by α1‐ and β‐antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli‐induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G‐protein‐mediated activation of K+ channels through α2‐adrenoceptors, thereby producing an antinociceptive effect.

Slow oscillation of membrane currents mediated by glutamatergic inputs of rat somatosensory cortical neurons: In vivo patch-clamp analysis

Using in vivo patch‐clamp technique, the slow oscillation of membrane currents was characterized by its synaptic nature, correlation with electroencephalogram (EEG) and responses to different anesthetic agents, in primary somatosensory cortex (SI) neurons in urethane‐anesthetized rats. In more than 90% of the SI neurons, the slow oscillation of the inward currents (0.1–2.5 Hz) with the duration of several hundreds of a millisecond was observed at the holding membrane potential of −70 mV. The reversal potential of the inward currents was approximately 0 mV and was suppressed by application of an α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) receptor antagonist. In most cases (> 90%) the inward current was synchronized with positive wave of the surface EEG recorded from ipsilateral and even contralateral cortical regions. The frequency as well as duration of the slow oscillation decreased by a volatile anesthetic agent, isoflurane (1.5–5.0%), and excitatory postsynaptic currents (EPSCs) were almost abolished at the highest concentration. Intraperitoneal injection of pentobarbital (25 mg/kg) also decreased the frequency of the slow oscillation without affecting short EPSCs. When γ‐aminobutyric acid A (GABAA) receptors were activated by local microinjection of muscimol (3 × 10−3 m, 1–10 µL) into the thalamus, the frequency of the slow oscillation markedly decreased, but was not abolished completely. These findings suggest that the slow oscillation of the inward currents is generated by the summation of glutamatergic EPSCs, and affected by isoflurane and pentobarbital differently. In addition, GABAergic system in the thalamus can affect the frequency, but is not essentially implicated in the genesis of the slow oscillation.

An electrophysiological study of muscarinic and nicotinic receptors of rat paratracheal ganglion neurons and their inhibition by Z‐338

To study the mechanisms involved in the action of Z‐338, a newly synthesized gastroprokinetic agent, experiments were performed with the paratracheal ganglion cells acutely dissociated from 2‐week‐old Wistar rats. The effects of Z‐338 on both nicotinic and muscarinic responses of the ganglion cells were studied by nystatin perforated patch recording configuration under the current‐ and voltage‐clamp conditions. Acetylcholine (ACh) or nicotine, and muscarine or oxotremorine‐M (OX‐M) induced membrane depolarization with rapid and slow time courses respectively, followed by repetitive generation of action potentials in the ganglion cell. Corresponding to the membrane depolarization induced by cholinergic agents, ACh induced biphasic inward currents with rapid and slow time courses under the voltage‐clamp condition. Nicotine and muscarine or OX‐M evoked inward currents with rapid and slow time courses, respectively. The rapid and slow inward currents were accompanied by increase and decrease in the membrane conductance, respectively. In addition, OX‐M dose‐dependently suppressed the M‐type K+ current evoked in response to hyperpolarizing voltage‐steps from VH of −25 mV to −50 mV, indicating that the activation of muscarinic acetylcholine receptors inhibits M‐type K+ current, thus inducing inward current in the ganglion cell. Z‐338 competitively suppressed the inward currents induced by OX‐M through M1 ACh receptor, and uncompetitively suppressed the currents induced by nicotine. The inhibitory actions of Z‐338 on the membrane depolarization and corresponding inward currents mediated by M1‐muscarinic and neuronal nicotinic ACh receptors in the isolated ganglion cells were discussed in relation to the inhibitory actions on autoreceptors in the parasympathetic nerve terminals, which would explain the gastroprokinetic actions of Z‐338.

Presynaptic inhibition of GABAergic miniature currents by metabotropic glutamate receptor in the rat CNS

The modulation of spontaneous miniature GABAergic inhibitory postsynaptic currents (mIPSC) by the metabotropic glutamate receptors was investigated in the mechanically dissociated rat nucleus basalis of Meynert neurons using the conventional whole-cell patch recording configuration. An application of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD) reversibly reduced the frequency of mIPSC without affecting the current amplitude distribution. The application of K+ channel blockers such as 4-aminopyridine, Cs+, Ba2+ or tetraethylammonium increased the mIPSC frequency, but failed to inhibit the tACPD action on mIPSC. Although the removal of Ca2+ from the extracellular solution reduced the mIPSC frequency, the inhibitory effect of tACPD on mIPSC was unaltered. These results suggested that neither voltage-dependent K+ or Ca2+ channels are involved in the inhibitory effect of tACPD on mIPSC frequency. Forskolin, an activator of adenylate cyclase, facilitated the mIPSC frequency in a concentration-dependent manner and inhibited the tACPD-induced suppression of mIPSC frequency. 8-Br-cAMP, a membrane permeable analog of cAMP, also prevented the inhibitory action of tACPD. However, Sp-cAMP, an activator of protein kinase A, could not prevent the inhibitory action of tACPD. L-CCG-I and (2R,4R)-APDC, group II mGluR agonists, mimicked the tACPD action on mIPSC frequency, but L-AP4, a group III mGluR agonist, had no such effect. MCCG, a group II mGluR antagonist, fully blocked the tACPD action. It was concluded that the activation of group II mGluR on the GABAergic presynaptic nerve terminals projecting to the rat nucleus basalis of Meynert neurons therefore inhibits the GABA release by reducing the activity of the cAMP-dependent pathway.

Revival of effective and safe high-dose mizoribine for the kidney transplantation*

Abstract:  We investigated whether high‐dose Mizoribine (MIZ: a water‐soluble anti‐metabolite), 4–6 mg/kg/d was as effective and safe as mycophenolate mofetil (MMF) for patients after kidney transplantation. Between January 2001 and December 2005, 36 recipients at a stable phase more than one month passed after transplantation underwent conversion from MMF to MIZ, two from Azathioprine to MIZ, and two cases on MIZ from the beginning. There were 24‐male and 16‐female patients whose average age was 43.3 yr old and average weight was 54.0 kg. The types of transplantations were living donor renal transplantation 25, cadaveric renal transplantation 11, and simultaneous pancreas–kidney transplantation four examples. Of these, 33 patients were on Tacrolimus‐based triple regimen and seven patients on Cyclosporine A base. The drugs used together with MIZ were basically the same as those before conversion. The reasons for conversion to MIZ were infection in 18 cases (45.0%), bone marrow suppression in nine cases (22.5%) and diarrhea in eight cases (20.0%), and post‐transplant lymphoproliferative disorder in one case (2.5%). We initiated 4–6 mg/kg/d of MIZ divided twice a day depending on the serum creatinine (sCr) value of each patient.

Modulation of glycine-induced currents by zinc and other metal cations in neurons acutely dissociated from the dorsal motor nucleus of the vagus of the rat

Effects of Zn2+ and other polyvalent cations on glycine-induced currents in the freshly dissociated rat dorsal motor nucleus of the vagus neurons were investigated under voltage-clamp conditions by the use of the nystatin-perforated patch recording configuration. Glycine evoked a Cl- current in a concentration-dependent manner with a half-maximum effective concentration (EC50) of 3.3x10-5 M. Strychnine inhibited the 3x10-5 M glycine-induced current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 6.8x10-7 M. At low concentrations (3x10-8 M-3x10-6 M), Zn2+ potentiated the current elicited by 3x10-5 M glycine. On the other hand, at concentrations higher than 10-5 M, Zn2+ inhibited the glycine response. The biphasic action of Zn2+ was mimicked by Ni2+, but La3+ and Co2+ had only potentiating effect. Zn2+ shifted the concentration-response curve for the glycine-induced current without changing the maximum response, and the EC50 values for the glycine response in the absence and presence of 10-6 M and 10-4 M Zn2+ were 3.3x10-5 M, 1.3x10-5 M and 1.3x10-4 M, respectively. These results suggest that the biphasic modulation of glycine response by Zn2+ results from changes in apparent glycine affinity.

Aconitine facilitates spontaneous transmitter release at rat ventromedial hypothalamic neurons

The effects of aconitine, an Aconitum alkaloid, on spontaneous inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs respectively) were investigated in the mechanically dissociated rat ventromedial hypothalamic (VMH) neurons in which native presynaptic nerve terminals remained intact. Under current‐clamp conditions, aconitine (3×10−6 M) depolarized the neuron with generating the action potentials. The aconitine‐induced depolarization was markedly suppressed in the presence of CNQX but it was facilitated in the presence of bicuculline, suggesting that release of excitatory and inhibitory neurotransmitters may be involved in the aconitine action in addition to its direct action on postsynaptic membrane. Under the voltage‐clamp conditions, aconitine reversibly increased the frequency of spontaneous IPSC and EPSC frequency, but it did not alter their amplitude distribution. Tetrodotoxin (TTX, 3×10−7 M) completely abolished the aconitine action on spontaneous IPSC frequency. Likewise removal of extracellular Na+ completely suppressed the aconitine action. Both Ca2+‐free external solution or addition of 10−4 M Cd2+ to normal solutions eliminated the facilitatory effect of aconitine on the IPSC frequency. Overall these results suggest that aconitine depolarizes the presynaptic membrane by activating voltage‐dependent Na+ channels. Increase of intraterminal Ca2+ concentration via an activation of voltage‐dependent Ca2+ channels in turn enhances the spontaneous transmitter release from presynaptic nerve terminals. The presynaptic action of aconitine may play a crucial role for membrane excitability of rat VMH neurons.

Availability of Pancreatic Allograft Biopsies Via a Laparotomy

OBJECTIVE The aim of this study was to evaluate the availability of a pancreatic allograft biopsy via a laparotpmy. PATIENTS AND METHODS From September 2004 to November 2007, 17 pancreas transplantations were performed: 15 simultaneous pancreas and kidney transplantations (SPK), 1 pancreas transplant alone (PTA), and one pancreas after kidney transplantation (PAK). Thirteen pancreatic allograft biopsies were obtained via an open laparotomy. This study evaluated the complications associated with this procedure, the rate of obtaining an adequate sample, and the relationship between biopsy-proven rejections and laboratory markers. In SPK cases we evaluated the synchronization between pancreas and kidney rejection. The pancreatic samples were diagnosed according to the Drachenberg classification. RESULTS No complications resulted from the procedure. The rate of obtaining adequate samples was 84.6%. Pancreas rejection correlated with elevation of the laboratory markers in 71.4%. Simultaneous pancreas and kidney rejection occurred in 62.5%, only kidney in 25%, and only pancreas in 12.5%. CONCLUSION A pancreas graft biopsy was absolutely imperative to improve the outcome in PTA, and even in SPK cases. A pancreatic allograft biopsy via a laparotomy was a safe, necessary and easy procedure to obtain an accurate diagnosis of rejection among pancreas transplantation patients.

Alterations of hepatic metabolism in chronic kidney disease via D-box binding protein aggravate the renal dysfunction.

Chronic kidney disease (CKD) is associated with an increase in serum retinol; however, the underlying mechanisms of this disorder are poorly characterized. Here, we found that the alteration of hepatic metabolism induced the accumulation of serum retinol in 5/6 nephrectomy (5/6Nx) mice. The liver is the major organ responsible for retinol metabolism; accordingly, microarray analysis revealed that the hepatic expression of most CYP genes was changed in 5/6Nx mice. In addition, D-box-binding protein (DBP), which controls the expression of several CYP genes, was significantly decreased in these mice. Cyp3a11 and Cyp26a1, encoding key proteins in retinol metabolism, showed the greatest decrease in expression in 5/6Nx mice, a process mediated by the decreased expression of DBP. Furthermore, an increase of plasma transforming growth factor-β1 (TGF-β1) in 5/6Nx mice led to the decreased expression of the Dbp gene. Consistent with these findings, the alterations of retinol metabolism and renal dysfunction in 5/6Nx mice were ameliorated by administration of an anti-TGF-β1 antibody. We also show that the accumulation of serum retinol induced renal apoptosis in 5/6Nx mice fed a normal diet, whereas renal dysfunction was reduced in mice fed a retinol-free diet. These findings indicate that constitutive Dbp expression plays an important role in mediating hepatic dysfunction under CKD. Thus, the aggravation of renal dysfunction in patients with CKD might be prevented by a recovery of hepatic function, potentially through therapies targeting DBP and retinol.

Inhibition of G0/G1 Switch 2 Ameliorates Renal Inflammation in Chronic Kidney Disease

Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we show that inhibition of G0/G1 switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD.