Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats / 南方医科大学学报

OBJECTIVE@#To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord.@*METHODS@#The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time.@*RESULTS@#The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 μmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed ( < 0.01), and spontaneous discharge frequency was obviously reduced ( < 0.01, =12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 μmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine ( < 0.01, =7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine ( < 0.01, =6 for each holding potential). During the 6 min of 30.0 μmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures ( < 0.01, =6).@*CONCLUSIONS@#etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.

Suppression of Peripheral Sympathetic Activity Underlies Protease-Activated Receptor 2-Mediated Hypotension

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, omega-conotoxin GVIA (CgTx), a selective N-type Ca2+ channel (I(Ca-N)) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by omega-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of I(Ca-N) which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

Bile Acid Inhibition of N-type Calcium Channel Currents from Sympathetic Ganglion Neurons

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type Ca2+ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type Ca2+ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM Ba2+ as a charge carrier. Cholic acid (CA, 10(-6) M) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type Ca2+ channel, which appeared to be due to an increase in null (no activity) sweeps. For example, the proportion of null sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type Ca2+ channel gating at a concentration as low as 10(-6) M. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type Ca2+ channel function may be beneficial against overexcitation of the synapses.

Huwentoxin-Ⅰ: Antinociceptive Effects and Its Comparison with ω-Conotoxin-MVIIA on Acute Visceral Pain in Rats / 中国生物化学与分子生物学报

The antinociceptive effect of epidural administration of huwentoxin-I was elucidated in a tonic visceral pain rat model produced by acute colon inflammation. The nociceptive behaviors were induced by perendoscopically injecting dilute formalin (50 μl) into the depth of the colonic wall in rats. Both ω-conotoxinMVIIA and morphine hydrochloride were given epidurally as positive control while saline as negative control.Similar to ω-conotoxin-MVIIA and hydrochloride morphine, the epidural administration of HWTX-Ⅰ significantly reduced the nociceptive responses in a dose-dependent manner in tonic visceral pain rat model ( P ＜ 0.05). The suppression effects of both huwentoxin- Ⅰ and ω-conotoxin-MVIIA at 20 μg/kg were kept steady compared with the saline group and reached their maximum effects at the doses of 50 ～ 75 μg/kg within 1 hour when the nociception had been observed. It was also found that at the same doses, huwentoxin- Ⅰ was less effective in antinociception than ω-conotoxin-MVIIA. However, ω-conotoxin-MVIIA, but not huwentoxinⅠ , caused an obvious motor dysfunction at these doses. The action of morphine hydrochloride was initiated faster, but lasted for a shorter time than that of huwentoxin- Ⅰ and ω-conotoxin-MVIIA. Thus, huwentoxinⅠ , a potent blocker of neuronal N-type voltage-sensitive calcium channels, induced a remarkable dosedependent restrain effect similar to ω-conotoxin-MVIIA and morphine on the tonic visceral pain produced by colonic wall injection of formalin in conscious rats.

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.

Effect of Ca2+-channel Blockers on Norepinephrine Release in the Rat Hippocampal Slice and Synaptosome

The aim of this study was to investigate the role of Ca2+-channel blockers in norepinephrine (NE) release from rat hippocampus. Slices and synaptosomes were incubated with [3H]-NE and the releases of the labelled products were evoked by 25 mM KCl stimulation. Nifedipine, diltiazem, nicardipine, flunarizine and pimozide did not affect the evoked and basal release of NE in the slice. But, diltiazem, nicardipine and flunarizine decreased the evoked NE release with a dose-related manner without any change of the basal release from synaptosomes. Also, a large dose of pimozide produced modest decrement of NE release. omega-conotoxin (CTx) GVIA decreased the evoked NE release in a dose-dependent manner without changing the basal release. And omega-CTxMVIIC decreased the evoked NE release in the synaoptosomes without any effect in the slice, but the effect of decrement was far less than that of omega-CTxGVIA. In interaction experiments with omega-CTxGVIA, omega-CTxMVIIC slightly potentiated the effect of omega-CTxGVIA on NE release in the slice and synaptosomal preparations. These results suggest that the NE release in the rat hippocampus is mediated mainly by N-type Ca2+-channels, and that other types such as L-, T- and/or P/Q-type Ca2+-channels could also be participate in this process.

Immunohistochemistry of Voltage-Gated Calcium Channel alpha1B Subunit in Mouse Cerebellum

Secretion of neurotransmitters is initiated by voltagegated calcium influx through presynaptic, voltage- gated N-type calcium channels. However, little is known about their cellular distribution in the mouse cerebellum. In the cerebellum, alpha1B immunoreactivity is found mainly on the cell bodies of all Purkinje cells. In addition, the immunoreactivity was detected on a subset of Purkinje cell dendrites, clustered to form a parasagittal array of bands. In the anterior lobe vermis, immunoreactive Purkinje cell dendrites form narrow stripes separated by broad bands of unstained dendrites. Moving caudally through the vermis, these stripes become thicker as a larger fraction of the Purkinje cell dendrites become immunoreactive. This localization study of the alpha1B pore-forming subunits in mouse cerebellum may guide future investigations of the role of calcium channels in neurological pathways.

Association of urinary transforming growth factor- ?_1, laminin and type Ⅳ collagen with diabetic nephropathy in type 2 diabetic patients / 中华内分泌代谢杂志

Urinary excretions of transforming grow th factor-? 1 (TGF-? 1), laminin (LN) and type Ⅳ collagen were determined i n 182 type 2 diabetic patients. Urinary excretions of TGF-? 1, LN and type Ⅳ collagen were increased in type 2 diabetic patients, and these findings were fa irly well correlated with severity of diabetic nephropathy (DN). Urinary TGF-? 1 seems to be the early index of DN, urinary LN and type Ⅳ collagen appear to be the indices of DN severity.

N-type calcium channels

The early studies of cardiac and smooth muscle cells provided evidence for two different calcium channels, the L-type (also called high-voltage activated (HVA)) and the T-type (low-voltage activated (LVA)). These calcium channels provided calcium for muscle contractions and pace-making activities. As might be expected, the number of different calcium channels increased when researchers studied neurons and the identification of the neuronal calcium channel has proven to be much more difficult than with the muscle calcium channels. There are two reasons for this difficulty; (1) a larger number of different calcium channels in neurons and (2) many of the different calcium channels have similar kinetic properties. This review uses the N-type calcium channel to illustrate the difficulties in identifying and characterizing calcium channels in neurons. It shows that the discovery of toxins that can specifically block single calcium channel types has made it possible to easily and rapidly discern the physiological roles of the different calcium channels in the neuron. Without these toxins it is unlikely that progress would have been as rapid.

Chromosomal Mapping and Brain Distribution of alpha1 Subunit of N-type Voltage Dependent Calcium Channel / 대한해부학회지

Voltage dependent calcium channels mediate wide variety of physiological functions including neurotransmitter release, neurite outgrowth, and gene expression in neurons. omega-Conotoxin-sensitive N-type calcium channels are exclusively expressed in nervous system and involved in the control of neurotransmitter release from neurons. In this experiment, I have investigated human chromosomal location and rat neuronal distribution of N-type voltage dependent calcium channel alpha1, subunit [alpha1B]. I have localized human alpha1B subunit gene to the long arm of chromosome 9[9q34] by fluorescent in situ hybridization. The distribution of rat alphaB1 subunit mRNA has been examined in the rat brain by in situ hybridization histochemistry and high level of alpha1B subunit mRNA has been observed in olfactory bulb, anterior olfactory nucleus, cerebral cortex, piriform cortex, hippocampus, dentate gyrus, parabrachial nucleus, and cerebellum and low level of expression was also found in other areas of rat brain.

Analgesic effect of intrathecal SO3 on rats with chronic constriction injury and its regulation of intracellular calcium ion concentration of spinal dorsal root ganglion neurons / 中国药理学通报

Aim To evaluate the effect of intrathecal administration of ?-conopeptide SO3,an neotype N-type calcium channel blocker,on pain related behaviors and changes in intracellular calcium ion concentration of the DRG neurons in a rat model of chronic constriction injury(CCI).Methods 40 male SD rats weighing 230~270 g were randomly divided into five groups of 8 animals each.Rats in the N group served as unoperated controls;in group C the rats were placed four loose ligatures around the right sciatic nerve;in group CN 20?l normal Saline was administered 14 days after operation;in group CS1 SO3 600 ng was administered the same time as group CN;in group CS7 SO3(30 ng?h~(-1)) was chronically administered for 7 days from the 7th day post-operation.Thermal hyperalgesia and mechanical allodynia were tested by measuring paw withdrawal latency(PWL) in response to thermal stimulation and by von-Frey filaments.The animals were decapitated on time.The DRGs(L_4-L_6) were homogenized into single cells and intracellular calcium ion concentration([Ca~(2+)]_i) was measured by flow cytometry.Results The CCI procedure leads to the development of spontaneous pain,heat hyperalgesia and tactile allodynia in the hind foot innervated by the injured nerve.Intrathecal of SO3 produced significant antinociceptive effects on both thermal hyperalgesia and mechanical allodynia.In CCI rats,significant increases were observed in [Ca~(2+)]_i on the contralateral side and ipsilateral side of DRGs on day 14 after operation.I.t NS did not show any difference in [Ca~(2+)]_i to those in group C.The treatment with i.t SO3 600 ng suppressed the increases in [Ca~(2+)]_i of bilateral side of DRGs.Chronically administration of SO3 suppressed the increases in [Ca~(2+)]_i of bilateral side of DRGs further.Conclusion Selective N-type VSCC blockers are potent and efficacious antinociceptive agents when they are administered by the spinal route,and they are effective in a rat model of neuropathic pain.The antinociceptive effect provided by intrathecal SO3 is associated with N-type calcium channel,which suppresses the increases in [Ca~(2+)]_i in the DRGs.