Effects of scorpion toxin on excitatory and inhibitory presynaptic terminals

The effects of scorpion toxin (STX) on both spontaneous and evoked glycinergic and glutamatergic postsynaptic currents were studied by using both the mechanically dissociated single SDCN neuron (synaptic bouton preparation) and the ‘focal electrical stimulation technique’. In the experimental condition where Na⁺ channels on postsynaptic soma membrane were blocked by intracellular perfusion of QX-314, STX increased dose-dependently the frequency of spontaneous glycinergic and glutamatergic postsynaptic currents (sIPSC and sEPSC, respectively) without affecting the amplitude, suggesting STX acts on inhibitory and excitatory presynaptic nerve terminal. Such a facilitatory effect of STX on sIPSC was stronger than that on sEPSC. On the other hand, STX significantly enhanced the averaged current amplitude and decreased the failure rate (Rf) of both evoked inhibitory and excitatory postsynaptic currents (eIPSC and eEPSC, respectively), indicating that STX increases not only the release frequency of glycine and glutamate but also the amount of their release from the both presynaptic nerve endings. These effects of STX were completely removed by adding Na⁺ or Ca²⁺ channel blockers, indicating that STX increases Ca²⁺ influx through Ca²⁺ channels triggered by activating voltage-dependent Na⁺ channels on the nerve terminals. In addition, the difference of STX actions on the amplitude of spontaneous and evoked currents was discussed.     

Inhibition of pacemaker currents by nitric oxide via activation of ATP-sensitive K+ channels in cultured interstitial cells of Cajal from the mouse small intestine

We investigated the role of nitric oxide (NO) in pacemaker activity and signal mechanisms in cultured interstitial cells of Cajal (ICC) of the mouse small intestine using whole cell patch-clamp techniques at 30°C. ICC generated pacemaker potential in the current clamp mode and pacemaker currents at a holding potential of –70 mV. (±)-S-nitroso-N-acetylpenicillamine (SNAP; a NO donor) produced membrane hyperpolarization and inhibited the amplitude and frequency of the pacemaker currents, and increased resting currents in the outward direction. These effects were blocked by the use of glibenclamide (an ATP-sensitive K⁺ channel blocker), but not by the use of 5-hydroxydecanoic acid (a mitochondrial ATP-sensitive K⁺ channel blocker). Pretreatment with ODQ (a guanylate cyclase inhibitor) almost blocked the NO-induced effects. The use of cell-permeable 8-bromo-cyclic GMP also mimicked the action of SNAP. However, the use of KT-5823 (a protein kinase G inhibitor) did not block the NO-induced effects. Spontaneous [Ca²⁺]_i oscillations in ICC were inhibited by the treatment of SNAP, as seen in recordings of intracellular Ca²⁺ ([Ca²⁺]_i). These results suggest that NO inhibits pacemaker activity by the activation of ATP-sensitive K⁺ channels via a cyclic GMP dependent mechanism in ICC, and the activation of ATP-sensitive K⁺ channels mediates the inhibition of spontaneous [Ca²⁺]_i oscillations.     

Presynaptic mu-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons

γ-Aminobutyric acid (GABA)-containing interneurons of the ventral tegmental area (VTA) regulate the activity of dopaminergic neurons. These GABAergic interneurons are known to be innervated by synaptic terminals containing enkephalin, an endogenous ligand of μ-opioid receptors. Bath application of μ-opioid receptor agonists inhibits the activity of VTA GABAergic neurons but the mechanism whereby μ-opioid receptors regulate synaptic GABA release from these neurons has not been directly identified. Using cultured VTA neurons we have confirmed that μ-opioid receptor agonists inhibit synaptic GABA release. DAMGO, a selective μ-opioid receptor agonist, had four distinct effects on GABAergic IPSCs: (1) it inhibited the frequency and amplitude of spontaneous IPSCs (sIPSCs), (2) it reduced the amplitude of IPSCs evoked by single action potentials, (3) it inhibited the frequency, but not the amplitude of miniature IPSCs (mIPSCs), and (4) DAMGO inhibited mIPSCs evoked by ionomycin, a Ca²⁺ ionophore. The inhibition of action potential-evoked IPSCs and of spontaneous and ionomycin-evoked mIPSCs by DAMGO was prevented by the K⁺ channel blocker, 4-aminopyridine (4-AP). In conclusion, our work shows that one of the mechanisms through which μ-opioid receptors inhibit GABA release by VTA neurons is through inhibition of the secretory process at the nerve terminal level. In addition, considering that ionomycin stimulates exocytosis through a mechanism that should be insensitive to membrane polarization, our experiments with 4-AP suggest that K⁺ channels are implicated in the inhibition of the efficacy of the secretory process by μ-opioid receptors.     

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3′,5′-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca²⁺ imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N^G-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K⁺ channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca²⁺ ([Ca²⁺]_i) oscillations revealed that ODQ and L-NAME increased [Ca²⁺]_i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca²⁺]_i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca²⁺ release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.     

Li+ enhances GABAergic inputs to granule cells in the rat hippocampal dentate gyrus

Defects in GABAergic interneurons are thought to be involved in the pathophysiology of bipolar disorder, and Li+ has been used as a primary therapeutic agent in the treatment. We used the patch clamp technique to investigate whether Li+ affects on spontaneous GABAergic synaptic inputs to granule cells (GCs) in hippocampal dentate gyrus. Extracellularly applied Li+ (25 mM) markedly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs), an effect completely blocked by picrotoxin or bicuculline. Li+ increased sIPSCs frequency in the presence of tetrodotoxin (TTX), but to a lesser extent than its absence. Li+ caused no change in the cumulative amplitude distribution of miniature IPSCs, indicating that a presynaptic mechanism is involved. When TTX was added in the presence of Li+, large-amplitude sIPSCs (>30 pA) were abolished specifically with no effect on small-amplitude sIPSCs (<20 pA). Intracellular Li+ (6 mM) applied via the patch pipette depolarized the resting membrane potential in fast-spiking interneurons, resulting in an increase in spontaneous action potential (AP) firing. This change, however, was not observed in GCs. These results suggest that Li(+)-induced spontaneous AP firing in GABAergic interneurons contributes to the increase in GABAergic synaptic inputs to GCs.     

Effects of nitric oxide on large‐conductance Ca2+‐activated K+ currents in human cardiac fibroblasts through PKA and PKG‐related pathways

The human cardiac fibroblast (HCF) is the most abundant cell type in the myocardium, and HCFs play critical roles in maintaining normal cardiac function. However, unlike cardiomyocytes, the electrophysiology of HCFs is not well established. In the cardiovascular system, Ca²⁺‐activated K⁺ (KCa) channels have distinct physiological and pathological functions, and nitric oxide (NO) plays a key role. In this study, we investigated the potential effects of NO on KCa channels in HCFs. We recorded strong oscillating, well‐maintained outward K⁺ currents without marked inactivation throughout the test pulse period and detected outward rectification in the I‐V curve; these are all characteristics that are typical of KCa currents. These currents were blocked with iberiotoxin (IBTX, a BKCa blocker) but not with TRAM‐34 (an IKCa blocker). The amplitudes of the currents were increased with SNAP (an NO donor), and these increases were inhibited with IBTX. The SNAP‐stimulating effect on the BKCa currents was blocked by pretreatment with KT5823 (a protein kinase G [PKG] inhibitor) or 1 H‐[1,‐2, ‐4] oxadiazolo‐[4,‐3‐a] quinoxalin‐1‐one (ODQ; a soluble guanylate cyclase inhibitor). Additionally, 8‐bromo‐cyclic guanosine 3’,5’‐monophosphate (8‐Br‐cGMP) stimulated the BKCa currents, and pretreatment with KT5720 (a protein kinase A [PKA] inhibitor) and SQ22536 (an adenylyl cyclase inhibitor) blocked the NO‐stimulating effect on the BKCa currents. Furthermore, 8‐bromo‐cyclic adenosine 3’,5’‐monophosphate (8‐Br‐cAMP) activated the BKCa currents. These data suggest that BKCa current is the main subtype of the KCa current in HCFs and that NO enhances these currents through the PKG and PKA pathways.     

Distinct effects of CGRP on typical and atypical smooth muscle cells involved in generating spontaneous contractions in the mouse renal pelvis

Background and purpose:

We investigated the cellular mechanisms underlying spontaneous contractions in the mouse renal pelvis, regulated by calcitonin gene-related peptide (CGRP).

Experimental approach:

Spontaneous contractions, action potentials and Ca²⁺ transients in typical and atypical smooth muscle cells (TSMCs and ATSMCs) within the renal pelvis wall were recorded separately using tension and intracellular microelectrode recording techniques and Fluo-4 Ca²⁺ imaging. Immunohistochemical and electron microscopic studies were also carried out.

Key results:

Bundles of CGRP containing transient receptor potential cation channel, subfamily V, member 1-positive sensory nerves were situated near both TSMCs and ATSMCs. Nerve stimulation reduced the frequency but augmented the amplitude and duration of spontaneous phasic contractions, action potentials and Ca²⁺ transients in TSMCs. CGRP and agents increasing internal cyclic adenosine monophosphate (cAMP) mimicked the nerve-mediated modulation of TSMC activity and suppressed ATSMCs Ca²⁺ transients. Membrane hyperpolarization induced by CGRP or cAMP stimulators was blocked by glibenclamide, while their negative chronotropic effects were less affected. Glibenclamide enhanced TSMC Ca²⁺ transients but inhibited ATSMC Ca²⁺ transients, while both 5-hydroxydecanoate and diazoxide, a blocker and opener of mitochondrial ATP-sensitive K⁺ channels, respectively, reduced the Ca²⁺ transient frequency in both TSMCs and ATSMCs. Inhibition of mitochondrial function blocked ATSMCs Ca²⁺ transients and inhibited spontaneous excitation of TSMCs.

Conclusions and implications:

The negative chronotropic effects of CGRP result primarily from suppression of ATSMC Ca²⁺ transients rather than opening of plasmalemmal ATP-sensitive K⁺ channels in TSMCs. The positive inotropic effects of CGRP may derive from activation of TSMC L-type Ca²⁺ channels. Mitochondrial Ca²⁺ handling in ATSMCs also plays a critical role in generating Ca²⁺ transients.     

Nicotinic receptor activation on primary sensory afferents modulates autorhythmicity in the mouse renal pelvis

BACKGROUND AND PURPOSE

The modulation of the spontaneous electrical and Ca²⁺ signals underlying pyeloureteric peristalsis upon nicotinic receptor activation located on primary sensory afferents (PSAs) was investigated in the mouse renal pelvis.

EXPERIMENTAL APPROACH

Contractile activity was followed using video microscopy, electrical and Ca²⁺ signals in typical and atypical smooth muscle cells (TSMCs and ASMCs) within the renal pelvis were recorded separately using intracellular microelectrodes and Fluo-4 Ca²⁺ imaging.

KEY RESULTS

Nicotine and carbachol (CCh; 1–100 μM) transiently reduced the frequency and increased the amplitude of spontaneous phasic contractions in a manner unaffected by muscarininc antagonists, 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) and pirenzipine (10 nM) or L-NAME (L-Nω-nitroarginine methyl ester; 200 μM), inhibitor of NO synthesis, but blocked by the nicotinic antagonist, hexamethonium or capsaicin, depletor of PSA neuropeptides. These negative chronotropic and delayed positive inotropic effects of CCh on TSMC contractions, action potentials and Ca²⁺ transients were inhibited by glibenclamide (Glib; 1 μM), blocker of ATP-dependent K (KATP) channels. Nicotinic receptor-evoked inhibition of the spontaneous Ca²⁺ transients in ASMCs was prevented by capsaicin but not Glib. In contrast, the negative inotropic and chronotropic effects of the non-selective COX inhibitor indomethacin were not prevented by Glib.

CONCLUSIONS AND IMPLICATIONS

The negative chronotropic effect of nicotinic receptor activation results from the release of calcitonin gene-related peptide (CGRP) from PSAs, which suppresses Ca²⁺ signalling in ASMCs. PSA-released CGRP also evokes a transient hyperpolarization in TSMCs upon the opening of KATP channels, which reduces contraction propagation but promotes the recruitment of TSMC Ca²⁺ channels that underlie the delayed positive inotropic effects of CCh.     

Idebenone inhibition of glutamate release from rat cerebral cortex nerve endings by suppression of voltage-dependent calcium influx and protein kinase A

The present study was aimed at investigating the effect and the possible mechanism of idebenone on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Idebenone inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration dependent. Inhibition of glutamate release by idebenone was prevented by chelating extracellular Ca²⁺, or by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to DL-threo-beta-benzyl-oxyaspartate, a glutamate transporter inhibitor. Idebenone decreased the depolarization-induced increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_C),whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The inhibitory effect of idebenone on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. Furthermore, the idebenone effect on 4-AP-evoked Ca²⁺ influx and glutamate release was completely abolished by the protein kinase A (PKA) inhibitors, H89 and KT5720. On the basis of these results, it was concluded that idebenone inhibits glutamate release from rat cortical synaptosomes and this effect is linked to a decrease in [Ca²⁺]_C contributed by Ca²⁺ entry through presynaptic voltage-dependent Ca²⁺ channels and to the suppression of PKA signaling cascade.     

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca²⁺ entry through the voltage-dependent Ca²⁺ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca²⁺ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca²⁺ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca²⁺]_i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca²⁺ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 µM) of nifedipine, a L-type Ca²⁺ channel blocker, produced a decrease in the amplitude of the spontaneous Ca²⁺ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (α1G, α1H) of the T-type Ca²⁺ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca²⁺ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca²⁺ transients consistent with the reduction of the frequency. It is concluded that T-type Ca²⁺ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.     

Effects of A2 type botulinum toxin on spontaneous miniature and evoked transmitter release from the rat spinal excitatory and inhibitory synapses

We observed effects of newly developed A2 type botulinum toxin (A2NTX) on spontaneous miniature and evoked transmitter release from inhibitory (glycinergic or GABAergic), or excitatory (glutamatergic) nerve terminals in rat spinal cord, by use of ‘synaptic bouton’ preparations, under voltage-clamp condition. A2NTX (0.1–1 pM) initially augmented and then decreased amplitude and frequency of spontaneous miniature release of glycine or GABA (mIPSCs) concentration-dependently. At an increased concentration (1–10 pM), A2NTX suppressed the amplitude of glutamatergic mEPSCs. The rank order of the inhibitory effects was glycinergic > GABAergic >> glutamatergic synapses. Focal electrical stimulation of ‘synaptic boutons’ elicited eIPSC or eEPSC with larger amplitude and low failure rate (Rf). A2NTX (0.01–1 pM) initially enhanced the amplitude or decreased the failure rate of eIPSC or eEPSC, and then almost completely abolished the generation of eIPSC or eEPSC. The action of A2NTX on the evoked transmitter release was partially reversible. The rank order of the inhibitory effects on the amplitude or Rf were glycinergic eIPSC ≥ GABAergic eIPSC > glutamatergic eEPSCs. Excess extracellular K⁺ or Ca²⁺ (excess [K⁺]_o or [Ca²⁺]_o), and 4-AP restored spontaneous miniature glycinergic, GABAergic or glutamatergic postsynaptic currents suppressed by A2NTX. We conclude that A2NTX inhibits spontaneous miniature release at 0.1–10 pM and evoked release at 0.01–1 pM in rat spinal cord, and the inhibition was much efficient in the evoked rather than the spontaneous miniature release. Excess [K⁺]_o, 4-AP and excess [Ca²⁺]_o, which can raise the intracellular Ca²⁺ concentration via the activation of voltage-dependent Ca²⁺ channels, rescue the transmission suppressed by A2NTX poisoning, suggesting the transmitter release machinery became less sensitive to intracellular Ca²⁺ in A2NTX poisoned ‘synaptic boutons’.     

Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.     

Inhibitory effect of glutamate release from rat cerebrocortical nerve terminals by α2 adrenoceptor agonist dexmedetomidine

The present study examined the effect of dexmedetomidine, an α₂ adrenoceptor agonist, on endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes). We also explored the possible mechanism that triggers dexmedetomidine to act. Dexmedetomidine dose-dependently inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine. Presynaptic α_2A adrenoceptors were involved in this release inhibition, with the α_2A antagonist (but not by the α_2B/C antagonist) blocking the dexmedetomidine-mediated inhibition. The effect of dexmedetomidine on the evoked glutamate release was prevented by the chelating extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on the action of dexmedetomidine. Dexmedetomidine decreased the degree of depolarization-induced increase in the intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. The inhibitory effect of dexmedetomidine on evoked glutamate release was abolished by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but was insensitive to the endoplasmic reticulum ryanodine receptors or mitochondrial Na⁺/Ca²⁺ exchange. In addition, the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors prevented dexmedetomidine from inhibiting glutamate release. Further, western blotting showed that dexmedetomidine decreased the 4-aminopyridine-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 and synapsin I, the main presynaptic target of mitogen-activated protein kinase. Thus, we concluded that dexmedetomidine acts at α_2A adrenoceptors present on cerebrocortical nerve terminals inhibit the release of glutamate. We further concluded that this effect is linked to the suppression of voltage-dependent Ca²⁺ channels and mitogen-activated protein kinase activity.     

Dantrolene and neutromuscular junction: Evidence for intracellular calcium stores

Dantrolene sodium (DaNa) markedly depresses the frequency of spontaneous miniature potentials at the frog neuromuscular junction. Its action is still observed at low [Ca²⁺]_o and is little affected by temperature; its effect is not readily reversible. Theophylline antagonises the action of DaNa. DaNa prevents the stimulatory effect of the ionophore A23187 at 25°C, but is ineffective in antagonising the action of X537A. DaNa and theophylline do not affect the functioning and integrity of mitochondria in vitro and it is suggested that DaNa acts at intracellular Ca²⁺ stores (other than the mitochondria) in the presynaptic terminals.     

一氧化氮诱导心肌细胞预适应早期保护作用的信号转导途径

目的：探讨一氧化氮(NO)诱导心肌细胞预适应早期保护作用及可能的信号转导途径。方法：体外培养新生大鼠心肌细胞，分别以NO合成前体L-精氨酸(L-Arg)和NO供体SNAP处理细胞，观察心肌细胞在随后6h的缺氧损伤程度，以明确NO是否诱导心肌细胞预适应早期保护作用；分别以cGMP阻断剂亚甲基蓝、蛋白激酶C(PKC)抑制剂D-鞘氨醇、钙拮抗剂拉西地平和ATP敏感的钾通道[K(ATP)通道]阻断剂格列苯脲作用心肌细胞30min后加入SNAP作用60min，观察心肌细胞缺氧损伤程度，检测指标为心肌细胞存活率及乳酸脱氢酶(LDH)活性。结果：SNAP和L-Arg均可诱导心肌细胞预适应早期保护作用，一氧化氮合酶抑制剂L-NAME可阻断L-Arg的保护作用，亚甲基蓝可完全取消SNAP对缺氧心肌细胞的保护作用，D-鞘氨醇、拉西地平和格列苯脲均可减弱SNAP的作用。结论：NO可能通过cGMP依赖途径诱导心肌细胞预适应早期保护作用，而PKC的活化和钙通道、K(ATP)通道的开放是其下游重要的环节。     

Exogenous and Endogenous Cannabinoids Suppress Inhibitory Neurotransmission in the Human Neocortex

Activation of CB₁ receptors on axon terminals by exogenous cannabinoids (eg, Δ⁹-tetrahydrocannabinol) and by endogenous cannabinoids (endocannabinoids) released by postsynaptic neurons leads to presynaptic inhibition of neurotransmission. The aim of this study was to characterize the effect of cannabinoids on GABAergic synaptic transmission in the human neocortex. Brain slices were prepared from neocortical tissues surgically removed to eliminate epileptogenic foci. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in putative pyramidal neurons using patch-clamp techniques. To enhance the activity of cannabinoid-sensitive presynaptic axons, muscarinic receptors were continuously stimulated by carbachol. The synthetic cannabinoid receptor agonist WIN55212-2 decreased the cumulative amplitude of sIPSCs. The CB₁ antagonist rimonabant prevented this effect, verifying the involvement of CB₁ receptors. WIN55212-2 decreased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, but did not change their amplitude, indicating that the neurotransmission was inhibited presynaptically. Depolarization of postsynaptic pyramidal neurons induced a suppression of sIPSCs. As rimonabant prevented this suppression, it is very likely that it was due to endocannabinods acting on CB₁ receptors. This is the first demonstration that an exogenous cannabinoid inhibits synaptic transmission in the human neocortex and that endocannabinoids released by postsynaptic neurons suppress synaptic transmission in the human brain. Interferences of cannabinoid agonists and antagonists with synaptic transmission in the cortex may explain the cognitive and memory deficits elicited by these drugs.     

Dual effect of 2,4-dinitrophenol on the spontaneous release of transmitter at the frog neuromuscular junction.

The uncoupling agent 2,4-Dinitrophenol (DNP) has a dual effect on the spontaneous release of transmitter at the frog neuromuscular junction, causing an initial fall in miniature endplate potential (MEPP) frequency followed by a dramatic rise. The latter effect is probably associated with the release of Ca²⁺ from the mitochondria. The initial fall in MEPP rate is independent of [Ca²⁺]₀ but is largely suppressed by pretreatment with theophylline; it is suggested that DNP also combines with another intracellular Ca²⁺-store, so reducing Ca²⁺ leakage and causing a fall in the steady-state level of [Ca²⁺]_i. The results confirm that MEPP frequency is largely determined by [Ca²⁺]_i at the presynaptic terminals.     

Cellular and Molecular Mechanisms of Nitric Oxide–Induced Heart Muscle Relaxation

• 1.The nitric oxide (NO) donor S-nitro-N-acetyl-penicillamine (SNAP) inhibits Helix aspersa heart activity and relaxes muscles.
• 2.K-free saline and ouabain both depress SNAP-induced relaxation in most experiments, but in a few preparations they either had no effect or potentiated SNAP-induced relaxation.
• 3.Na-K pump reactivation following preincubation in K-free saline leads to the pronounced transient relaxation of heart muscle, the magnitude of which depends on the duration of preincubation.
• 4.0.1 mM SNAP inhibited the ouabain sensitive part of ⁸⁶Rb uptake, which reflects Na-K pump activity. This inhibition is potentiated by phospholipase C.
• 5.SNAP increased cGMP levels in the heart.
• 6.These results indicate that SNAP-induced relaxation depends on Na and Ca gradients across the membrane, which suggests that Na:Ca exchange is involved in the mechanisms of SNAP-induced relaxation. It is postulated that SNAP elicits its inhibitory effect on the heart through a cGMP-dependent Na:Ca exchange.

     

Stress Impairs GABAergic Network Function in the Hippocampus by Activating Nongenomic Glucocorticoid Receptors and Affecting the Integrity of the Parvalbumin-Expressing Neuronal Network

Stress facilitates the development of psychiatric disorders in vulnerable individuals. It affects physiological functions of hippocampal excitatory neurons, but little is known about the impact of stress on the GABAergic network. Here, we studied the effects of stress and a synthetic glucocorticoid on hippocampal GABAergic neurotransmission and network function focusing on two perisomatic interneurons, the parvalbumin (PV)- and the cholecystokinin (CCK)-positive neurons. In acute hippocampal slices of rat, application of the potent glucocorticoid receptor (GR) agonist dexamethasone (DEX) caused a rapid increase in spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons. This effect was mediated by a nongenomic GR that evoked nitric oxide (NO) release from pyramidal neurons. Retrograde NO signaling caused the augmentation of GABA release from the interneurons and increased CCK release, which in turn further enhanced the activity of the PV-positive cells. Interestingly, chronic restraint stress also resulted in increased sIPSCs in CA1 pyramidal neurons that were Ca²⁺-dependent and an additional DEX application elicited no further effect. Concomitantly, chronic stress reduced the number of PV-immunoreactive cells and impaired rhythmic sIPSCs originating from the PV-positive neurons. In contrast, the CCK-positive neurons remained unaffected. We therefore propose that, in addition to the immediate effect, the sustained activation of nongenomic GRs during chronic stress injures the PV neuron network and results in an imbalance in perisomatic inhibition mediated by the PV and CCK interneurons. This stress-induced dysfunctional inhibitory network may in turn impair rhythmic oscillations and thus lead to cognitive deficits that are common in stress-related psychiatric disorders.