Propofol facilitates climbing fiber-Purkinje cell synaptic transmission via NMDA receptor in vitro in mice.

Propofol is generally used for the induction and maintenance of anesthesia in clinical procedures via activation of γ -aminobutyric acid A (GABAA) receptors. When administered at the clinical dose, propofol use is associated with movement disorders, including dystonia and ataxia, suggesting that propofol administration impacts the function of cerebellar neuronal circuitry. In this study, we investigated the effect of propofol on climbing fiber (CF)-Purkinje cell (PC) synaptic transmission in mouse cerebellar slices in the absence of GABAergic inhibition using a whole-cell recording technique and pharmacological methods. Our results showed that bath application of propofol enhanced CF-PC synaptic transmission, which was demonstrated by an increased amplitude and area under the curve (AUC) of the excitatory postsynaptic currents (EPSCs) accompanied by a decrease in the paired-pulse ratio (PPR). The propofol-induced increase in the amplitude of P1 was concentration-dependent with a half effective concentration (EC50) of 20.9 µM. The propofol-induced increases in the amplitude and AUC of CF-PC EPSCs were abolished by an N-Methyl-D-aspartate (NMDA) receptor blocker. Furthermore, the application of NMDA enhanced CF-PC EPSCs and overwhelmed the effect of propofol on CF-PC EPSCs. Moreover, intracellular blockade of NMDA receptors attenuated the propofol-induced enhancement of CF-PC synaptic transmission but strengthened the propofol-induced change in the PPR. These results indicate that propofol enhances CF-PC synaptic transmission by activation of NMDA receptors in the mouse cerebellar cortex, suggesting that propofol administration might be involved in propofol-induced dysfunction of the cerebellum via NMDA receptors.

NMDARs contribute to the facial stimuli-evoked mossy fiber-granule cell synaptic transmission in vivo in mice.

N-methyl-D-aspartate receptors (NMDARs) are expressed in granule cell and involve in mossy fiber-granule cell (MF-GC) synaptic transmission in cerebellar cortex. In the absence GABAA receptor activity, we here studied the role of NMDARs during the facial stimulation evoked MF-GC synaptic transmission in urethane-anesthetized mice using electrophysiological recording technique and pharmacological methods. Our results showed that facial stimuli train (20 Hz, 5 pulses) evoked 5 field potential responses (N1-N5) in mouse cerebellar granular layer, which identified MF-GC synaptic transmission. Blocking NMDARs induced significant depression in the amplitude of N2 to N5, accompanied with significant decrease in pulse ratios, area under the curve (AUC) and half-width of N1. A selective GluN2A antagonist, PEAQX (10 µM) also produced significant depression in the amplitude of N2 to N5, and decreases in pulse ratios. However, a selective GluN2B antagonist, TCN-237 (10 µM) did not significantly attenuate the facial stimuli train-induced mossy fiber-granule cell synaptic transmission. Application of NMDA (1 µM) produced increases in the AUC and half-width of Ron, as well the amplitude and AUC of Roff, which was reversed by following application of PEAQX. Our present results indicated that NMDARs, especially GluN2A contribute to the facial stimulation-evoked MF-GC synaptic transmission, suggesting that the NMDARs play an important role during the lateral sensory information synaptic transmission in the cerebellar granular layer in vivo in mice.

Chronic ethanol exposure facilitates facial-evoked MLI-PC synaptic transmission via nitric oxide signaling pathway in vivo in mice.

Ethanol (EtOH) exposure causes alterations of motor coordination, balance, behavior, speech, and certain cognitive functions are considered to be caused partly by impairment of cerebellar circuits function and modulation of synaptic transmission. The cerebellar cortical molecular layer interneuron-Purkinje cell (MLI-PC) synapses are critical for various information integration and transmission, which are sensitive to acute and chronic EtOH exposure. The aim of this study is to investigate the effect of chronic ethanol exposure on the facial stimulation-evoked MLI-PC synaptic transmission in urethane-anesthetized mice, by electrophysiological recording and pharmacological methods. Under current-clamp recording conditions, air-puff stimulation of ipsilateral whisker pad evoked MLI-PC synaptic transmission, which expressed an inhibitory component (P1) followed by a pause of simple spike (SS) firing in cerebellar PCs. Chronic ethanol exposure did not change the latency of the facial stimulation-evoked responses in cerebellar PCs, but induced significant enhancement of the stimulation-evoked MLI-PC synaptic transmission, which expressed increases in amplitude of P1 and pause of SS firing. The amplitude of P1 and pause of SS in ethanol exposure group were significant higher than that in control group. Cerebellar surface application of nitric oxide synthesis (NOS) inhibitor, L-NNA (5 mM) significantly decreased the amplitude of P1 and the pause of SS firing in EtOH exposure group, but did no effect on control group. In contrast, cerebellar surface application of NO donor, SNAP (100 µM) significantly increased the amplitude of P1 and the pause of SS firing in control group, but not in EtOH exposure group. These results indicated that chronic EtOH exposure significantly facilitated the sensory-evoked MLI-PC synaptic transmission via NO signaling pathway in mouse cerebellar cortex.

Endogenous ET-1 promotes ANP secretion through activation of COX2-L-PGDS-PPARγ signaling in hypoxic beating rat atria.

Endothelin-1 (ET-1) is a potent stimulus for the secretion of atrial natriuretic peptide (ANP) and hypoxia stimulates the release of ET-1, which is involved in the regulation of atrial ANP secretion. However, the precise mechanism of endogenous ET-1 in the regulation of hypoxia-induced ANP secretion is unclear. Therefore, this study aimed to investigate the mechanism of hypoxia-induced endogenous ET-1 regulation of ANP secretion in isolated perfused hypoxic beating rat atria. The results of this study showed that acute hypoxia significantly stimulated ET-1 release and upregulated the expression of its type A as well as type B receptors (ETA and ETB receptors). Endogenous ET-1 induced by hypoxia markedly upregulated the expression of cyclooxygenase 2 (COX2) through activation of its two receptors, leading to an increase in lipocalin-type prostaglandin D synthase (L-PGDS) expression and prostaglandin D2 (PGD2) production. L-PGDS-derived PGD2 activated peroxisome proliferator-activated receptor γ (PPARγ), ultimately promoting hypoxia-induced ANP secretion. Conversely, L-PGDS-derived PGD2 may in turn regulate L-PGDS expression by a nuclear factor erythroid-2-related factor 2 (NRF2)-mediated feedback mechanism. These results indicate that endogenous ET-1 induced by hypoxia promotes hypoxia-induced ANP secretion by activation of COX2-L-PGDS-PPARγ signaling in beating rat atria. In addition, the positive feedback loop between L-PGDS-derived PGD2 and L-PGDS expression induced by hypoxia is part of the mechanism of hypoxia-induced ANP secretion by endogenous ET-1.

Design, synthesis, and negative inotropic evaluation of 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole or piperazine moieties.

In this study, four novel series of 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole or piperazine moieties were designed, synthesized, and evaluated for negative inotropic activity by measuring the left atrium stroke volume in isolated rabbit heart preparations. Almost all of the compounds showed an ability to moderate the cardiac workload by decreasing the heart rate and contractility. Among them, 7h was found to be the most potent with a change in stroke volume of -48.22 ± 0.36% at a concentration of 3 × 10-5  mol/L (metoprolol: -9.74 ± 0.14%). The cytotoxicity of these compounds was evaluated using the human cervical cancer cell line HeLa, the liver cancer cell line Hep3B, and the human normal hepatic cell line LO2. A preliminary study of the mechanism of action for the compound 7h on the regulation of atrial dynamics with ATP-sensitive K+ channel and L-type Ca2+ channel blockers glibenclamide and nifedipine was performed in the isolated perfused beating rabbit atria.

Nicotine excites corticotropin-releasing hormone mRNA-expressing neuron in the hypothalamic paraventricular nucleus in vitro in rats.

Nicotine is known to modulate the activity of the hypothalamic-pituitary-adrenal axis by stimulating corticotropin-releasing hormone (CRH) release from the hypothalamic paraventricular nucleus (PVN). However, the mechanism by which nicotine affects the hypothalamic-pituitary-adrenal axis by modulating PVN CRH neuronal activity is currently unclear. Here, we examined the effects of nicotine on PVN CRH-mRNA-expressing neurons in vitro in rats by whole-cell patch-clamp recordings, biocytin staining, and single-cell reverse transcription-multiplex PCR techniques. Of the 146 PVN putative parvocellular neurons, 17.1% (25/146) coexpressed GAPDH mRNA and CRH mRNA. Under current-clamp recording conditions, application of nicotine (1 µM) induced excitation in 92% (23/25) PVN CRH-mRNA-expressing neurons, which showed a significant increase in the spike firing rate accompanied by a depolarization of the membrane potential. Nicotine induced an increase in the spike firing rate of PVN CRH-mRNA-expressing neurons in a concentration-dependent manner. The half-effective concentration (EC50) of nicotine for increasing the spike firing rate of PVN CRH-mRNA-expressing neurons was 1.6 µM. Extracellular application of ionotropic glutamate receptor antagonist kynurenic acid (1 mM) abolished the nicotine-induced excitation of PVN CRH-mRNA-expressing neurons. Moreover, application of nicotine induced a significant increase in the spontaneous excitatory postsynaptic currents frequency, but without significantly altering the spontaneous excitatory postsynaptic currents amplitude of the CRH-mRNA-expressing neurons. Biocytin staining confirmed that the nicotine-sensitive CRH-mRNA-expressing neurons were located in the PVN parvocellular division. These results indicated that extracellular administration of nicotine indirectly excited PVN CRH-mRNA-expressing neurons, suggesting that nicotine modulated PVN CRH secretion by enhancement of both the presynaptic action potential drive and quantal glutamate release.

cAMP induction by ouabain promotes endothelin-1 secretion via MAPK/ERK signaling in beating rabbit atria.

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na(+)-K(+)-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 µmol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 µmol/L), an inhibitor for reverse mode of Na(+)-Ca(2+) exchangers (NCX), but did not by L-type Ca(2+) channel blocker nifedipine (1.0 µmol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 µmol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 µmol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 µmol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 µmol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

Effects of ethanol on sensory stimulus-evoked responses in the cerebellar molecular layer in vivo in mice.

Overdose intake of ethanol can impair cerebellar cortical neurons to integrate and transfer external information, resulting in a dysfunction of cerebellar motor regulation or cerebellar ataxia. However, the mechanisms underlying ethanol-impaired transfer of sensory information from cerebellar cortical molecular layer neurons remain unclear. In the present study, we investigated the effects of ethanol on sensory stimulation-evoked responses in the cerebellar molecular layer of urethane-anesthetized mice, by electrophysiological and pharmacological methods. Our results demonstrated that air-puff stimulation (30 ms, 50-60 psi) of the ipsilateral whisker-pad evoked field potential responses in the molecular layer of the cerebellar cortex folium Crus II, which expressed a negative component (N1) followed by a gamma-aminobutyric acid receptor A (GABAA)-mediated positive component (P1). Cerebellar surface perfusion of ethanol between 2 and 5mM did not change the latency of the evoked responses and the amplitude of N1, but enhanced the amplitude and the area under the curve of P1. Interestingly, high concentrations (>20mM) of ethanol induced a significantly decrease in the amplitude and area under the curve of P1. Furthermore, high concentration ethanol (300 mM) significantly decreased the rise in tau and tau decay value of P1, whereas low concentration ethanol (2-5mM) significantly increased these values of P1. Inhibition of GABAA receptor activity reversed P1 and also abolished the effects of ethanol on sensory stimulation-evoked responses. These results indicated that ethanol induced a bidirectional effect on the sensory stimulation-evoked GABAergic responses in the cerebellar cortical molecular layer, suggesting that acute alcohol intake impacted the sensory information processing of cerebellar cortex.

Synthesis and positive inotropic evaluation of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted piperazine moieties.

Four series of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted piperazine moieties were synthesized and evaluated for their positive inotropic activity by measuring the left atrium stroke volume in isolated rabbit-heart preparations. Several compounds were developed and showed favorable activities compared to the standard drug milrinone, with (4-([1,2,4]triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)(p-tolyl)methanone (5g) being identified as the most potent with an increased stroke volume of 19.15±0.22% (milrinone: 2.46±0.07%) at a concentration of 3×10(-5) M. A preliminary study of mechanism of action revealed that 5g displayed its positive inotropic effect may be related to the PDE-cAMP-PKA signaling pathway. Compounds exhibiting inotropic effects were also evaluated in terms of the chronotropic effects.

MAPK and PI3K pathways regulate hypoxia-induced atrial natriuretic peptide secretion by controlling HIF-1 alpha expression in beating rabbit atria.

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are pivotal and intensively studied signaling pathways in hypoxic conditions. However, the roles of MAPK and PI3K in the regulation of hypoxia-induced atrial natriuretic peptide (ANP) secretion are not well understood. The purpose of the present study was to investigate the mechanism by which the MAPK/ERK (extracellular signal-regulated kinase) and PI3K signaling pathways regulate the acute hypoxia-induced ANP secretion in isolated beating rabbit atria. An acute hypoxic perfused beating rabbit atrial model was used. The ANP levels in the atrial perfusates were measured by radioimmunoassay, and the hypoxia-inducible factor-1α (HIF-1α) mRNA and protein levels in the atrial tissue were determined by RT-PCR and Western blot. Acute hypoxia significantly increased ANP secretion and HIF-1α mRNA and protein levels. Hypoxia-induced ANP secretion was markedly attenuated by the HIF-1α inhibitors, rotenone (0.5µmol/L) and CAY10585 (10µmol/L), concomitantly with downregulation of the hypoxia-induced HIF-1α mRNA and protein levels. PD098059 (30µmol/L) and LY294002 (30µmol/L), inhibitors of MAPK and PI3K, markedly abolished the hypoxia-induced ANP secretion and atrial HIF-1α mRNA and protein levels. The hypoxia-suppressed atrial dynamics were significantly attenuated by PD098059 and LY294002. Acute hypoxia in isolated perfused beating rabbit atria, markedly increased ANP secretion through HIF-1α upregulation, which was regulated by the MAPK/ERK and PI3K pathways. ANP appears to be part of the protective program regulated by HIF-1α in the response to acute hypoxic conditions.