Increasing evidence suggests that the complex interactions among multiple cell types including neuronal, glial, and vascular cells, are critical for maintaining adequate cerebral blood flow that is necessary for normal brain function and survival. The disturbance of these interactions contributes to the pathogenesis of central nervous system disorders such as stroke and Alzheimer’s disease. The retina is part of the central nervous system, and the properties of vasculature in the retina are similar to those in the brain. The interactions among multiple cell types in the retina also play an important role in the maintenance of tissue homeostasis, and the impairment of interactions can contribute to the onset and/or progression of retinal diseases. In this review, we describe the neurovascular interactions in the retina and alternations of interactions in pathological conditions such as diabetic retinopathy and glaucoma.
The endothelin (ET) system consists of two G protein coupled–receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca2+ concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca2+ entry and receptor-operated Ca2+ entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca2+ sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1–induced Ca2+ mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca2+ signaling and to GIPs in the signal transduction, modification, and degradation of ETRs.
Curcumin is a major yellow pigment and active component of turmeric widely used as dietary spice and herbal medicine. This compound has been reported to be a promising antitumor agent, although the underlying molecular mechanisms are not fully understood yet. In this study, we reported that curcumin inhibited growth of lung adenocarcinoma cells, but had no cytotoxic activity to IMR-90 normal lung fibroblast cells. Curcumin induced autophagy in the A549 human lung adenocarcinoma cell line, evidenced by LC3 immunofluorescence analysis and immunoblotting assays on LC3 and SQSTM1. Moreover, the autophagy inhibitor 3-MA partly blocked the inhibitory effect of curcumin on the growth of A549 cells. Curcumin markedly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetylCoA carboxylase in A549 cells. At last, pharmacological blockade of the AMPK signaling pathway by compound C and genetic disruption of the AMPK signaling pathway with siRNA-mediated AMPKα1 knockdown impaired the autophagy-inducing effect of curcumin. Collectively, our data suggests that curcumin induces autophagy via activating the AMPK signaling pathway and the autophagy is important for the inhibiting effect of curcumin in lung adenocarcinoma cells.
Hyperlipidemia is referred to as hypercholesterolemia, hypertriglyceridemia, or both in combined hyperlipidemia. Here, a novel mouse model of combined hyperlipidemia is described. Mice were orally given a single dose of a modeling agent (MA) made of a mixture of schisandrin B/cholesterol/bile salts (1/2/0.5 g/kg) suspended in olive oil. MA treatment increased serum triglycerides (TG) and total cholesterol (TC) (up to 422% and 100% at 12 – 96 h post-treatment, respectively) and hepatic TG and TC (up to 220% and 26%, respectively) in a time- and dose-dependent manner, associated with elevation of high-density lipoprotein and low-density lipoprotein levels. Serum alanine/aspartate aminotransferase activities, indicators of liver cell damage, were also elevated (up to 198%) at 48 and 72 h post-MA treatment. Fenofibrate blocks MA-induced hyperlipidemia, lipid accumulation in the liver, as well as liver injury. Oral administration of a mixture of schisandrin B, cholesterol, and bile salt could generate an interesting mouse model of combined hyperlipidemia associated with hepatic steatosis and steatohepatitis.
The present study aims to determine the estrogenicity of Millettia macrophylla, a Cameroonian medicinal plant, in ovariectomized rats and to investigate the underlying mechanisms, in order to justify scientifically its traditional use. To accomplish this objective, we used dichloromethane (DCM) and methanol (MeOH) extracts of the stem bark of M. macrophylla. In the cell culture based assay, the MeOH extract significantly transactivated estrogen receptor α (ERα) and estrogen receptor β (ERβ); in addition, the estrogen-like effects of both, DCM and MeOH extracts, could be inhibited in vitro by the pure ER antagonist ICI 182,780, indicating that these effects were primarily mediated through ERs. In animal experiments, both DCM and MeOH extracts significantly increased the uterine and vaginal epithelial heights in the 3-day treatment assay, while only the MeOH extract exhibited such effects in the sub-chronic treatment regimen. Furthermore, the MeOH extract significantly decreased fasting serum triglycerides, total cholesterol levels and artherogenic risk in the sub-chronic treatment. These results indicate that M. macrophylla extracts have estrogen-like effects supporting their traditional use in Cameroon to alleviate some menopausal problems (See graphical abstract in Supplementary Fig. 1, available in the online version only).
Bisphenol A (BPA) is an estrogenic endocrine disruptor used for producing polycarbonate plastics and epoxy resins. This study investigated the effects of perinatal BPA exposure on learning and memory, general activity, and emotionality in male Sprague Dawley rats using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) used to assess spatial learning and memory. Mother rats were orally administered BPA (50 or 500 μg∙kg−1/day) or vehicle (1 ml∙kg−1/day) from gestational day 10 to postnatal day 14. In the MAZE test, compared to the offspring of vehicle-treated rat mothers, male offspring of mothers exposed to 50 μg∙kg−1/day of BPA, but not those of mothers exposed to 500 μg∙kg−1/day of BPA, needed significantly more time to reach the reward. Although male offspring of mothers exposed to 50 μg∙kg−1/day of BPA showed an increase in a behavioral measure of wariness after repeated testing in the open-field test, no significant effects were observed in locomotor activities. No significant differences were observed in any other behavioral test including the elevated plus-maze test. The present study suggests that perinatal exposure to low-dose BPA specifically and non-monotonically impairs spatial learning and memory in male offspring rats.
Electrophysiological studies were performed to determine whether serotonergic modulation in the nucleus accumbens (NAcc) was affected after repeated methamphetamine (MAP) administration. NAcc slices (400 μm) from Wistar rats administered MAP (5 mg/kg) or saline once daily for 5 days were prepared 1, 5, or 10 days after the final injection. Population spikes (PS) induced by local stimulation of NAcc were recorded. PS inhibition by serotonin was significantly attenuated in the MAP group at 5 days but did not differ at 1 or 10 days. We next analyzed the effects of serotonin receptor subtype (5-HT1A,2,3,4,6,7)-selective agonists of PS. Differences between saline and MAP groups in 5-HT1A,2,3,4,6 receptor agonist–induced changes of PS were small or not significant. Interestingly, 5-HT7 receptor agonists significantly enhanced PS in the MAP group. Changes in the secondary messenger system related to 5-HT7 receptors were also investigated. Adenylate cyclase activator–induced PS enhancements were significantly larger in the MAP group. However, dibutyryl-cAMP–induced PS enhancement was not significantly different. In conclusion, 5-HT–induced inhibition of PS in NAcc was attenuated 5 days after repeated MAP treatment: the change in the effect of 5-HT was probably due to enhancement of the excitatory modulation via the 5-HT7 receptor with adenylate cyclase signal transduction systems.
To develop a simple screening system for blockers of voltage-gated Kv1.3 and Kv1.5 channels, new cell lines co-expressing mutated Nav1.5 (IFM/Q3), Kir2.1 (Kir), and Kv1.3 or Kv1.5 were introduced as IFM/Q3+Kir+Kv1.3 and IFM/Q3+Kir+Kv1.5, respectively. Electrical stimulation (ES) of a cell line, IFM/Q3+Kir, induced prolonged action potentials due to the slow inactivation of IFM/Q3 and subsequent cell death. Additional co-expression of Kv1.3 or Kv1.5 to IFM/Q3+Kir shortened the evoked action potentials and prevented cell death. In the presence of margatoxin, a selective Kv1.3-blocker, ES induced cell death in IFM/Q3+Kir+Kv1.3, but not in IFM/Q3+Kir+Kv1.5. In the presence of 4-aminopyridine, a non-selective Kv-channel blocker, ES application elicited cell death in both cell lines. The IC50s of acacetin, a Kv1.5-blocker, was 10.2 μM in IFM/Q3+Kir+Kv1.3 and almost identical to that in IFM/Q3+Kir+Kv1.5 (7.6 μM). The IC50s of citalopram, a 5-HT uptake-inhibitor, were 1.8 μM in IFM/Q3+Kir+Kv1.3 and 1.5 μM in IFM/Q3+Kir+Kv1.5, respectively. These IC50s were comparable to those determined electrophysiologically. In conclusion, acacetin and citalopram block both Kv1.3 and Kv1.5 without selectivity. The Kv1.3 or Kv1.5 channel inhibition assay using these new cell lines may be applicable to high–throughput screening because of its simplicity, accuracy, and high cost-performance.
In the present study, we explored the effects of demethylation in a cigarette smoke extract (CSE)-induced mouse emphysema model. Animals were randomly assigned to the control group, CSE group, 5-aza-2′-deoxycytidine (AZA) group, and CSE+AZA group (n = 10 per group). The mitochondrial transcription factor A (mtTFA) promoter methylation increased over 4-fold in the CSE group compared with the control group, which was reversed by AZA. The mtTFA and the cytochrome c oxidase subunit II (COX II) mRNA and protein levels were decreased approximately 3-fold in the CSE group compared with the control group, which was largely restored by AZA. Histological analysis showed that the CSE group exhibited emphysema compared with the control, which was alleviated by AZA. In addition, CSE significantly induced lung cell apoptosis and decreased lung function and lung mitochondrial COX activity, which was mostly restored by AZA. In conclusion, we for the first time provide evidence that demethylation therapy with AZA can effectively improve emphysema, lung function, lung cell apoptosis, and lung mitochondrial COX activity in a CSE-induced mouse emphysema model, which adds fresh insight into the therapeutic potential of demethylating agents in the prevention and treatment of cigarette smoke-induced emphysema.
Developmental changes in excitation–contraction mechanisms were examined in the ventricular myocardium from fetal, neonatal, and 1-, 2-, and 4-week-old mice. In isolated tissue, the negative inotropic effect of nifedipine decreased, while that of ryanodine increased with age. Action potential duration decreased with age, especially during the late fetal period. In ventricular cardiomyocytes, fluorescence imaging revealed that the sarcoplasmic reticulum increases progressively during pre- and postnatal development. t-Tubules were absent in the fetus and neonate, were observed only in the subsarcolemmal region at 1 week after birth, and were present throughout the cytoplasm at 2 and 4 weeks after birth. The amplitude of Ca2+ transients, as well as its ryanodine-sensitive component, increased with age. In the neonate and 1-week-old mice, Ca2+ at the cell center showed slower rise than the subsarcolemmal region, but in 2- and 4-week-old mice, Ca2+ increased simultaneously across the entire width of the cell. These results suggest that in the mouse ventricular myocardium, the shortening of the action potential during the late fetal period and the development of t-tubule–sarcoplasmic reticulum coupling during the second postnatal week largely contribute to the developmental increase in the dependence of contraction on sarcoplasmic reticulum function.
The conventional method for the real-time assessment of murine colitis requires a large number of animals. The 13C-butyrate breath test could be useful for evaluating disease activity and the amelioration of human ulcerative colitis non-invasively. The purpose of this study was to investigate whether this test can be used to assess the phase of inflammation in murine colitis. We investigated the excretion of 13CO2 measured by the 13C-butyrate breath test after rectal instillation of butyrate in the DSS colitis model. The colon length, MPO activity, and histological damage were analyzed as parameters. The efficacy of salicylazosulfa-pyridine (SASP) on 13CO2 excretion was also studied. The 13CO2 excretion curves in the 0.5% DSS– and 0.75% DSS–treated groups were significantly lower than those in the normal group (P < 0.01, P < 0.01). Good correlation between the results of the breath test and the inflammation parameters was observed. The 13CO2 excretion curve in DSS murine colitis after the administration of SASP was significantly higher than in the normal group (P < 0.01). The 13C-butyrate breath test can be used to evaluate the inflammatory phase of DSS murine colitis, and it may be a new non-invasive method for assessing murine colitis.
To clarify the matrix metalloproteinase (MMP)-9 inhibitory effect of an angiotensin-converting enzyme (ACE) inhibitor in vivo, we evaluated the effect of an ACE inhibitor against elastase-induced abdominal aortic aneurysm (AAA) progression in mice. Molecular models showed that imidapril bound directly to the mouse MMP-9 active center. An active form of imidapril, imidaprilat, dose-dependently inhibited MMP-9 activity in the extract from elastase-induced AAA in wild-type mice. Imidapril (10 mg/kg per day) was administered to wild-type or angiotensin II type 1 (AT1) receptor knockout mice. Blood pressure was significantly lower in AT1 receptor–knockout mice than in wild-type mice, but imidapril did not affect blood pressure in AT1 receptor–knockout mice. The aortic diameter was significantly expanded after elastase application, but the expansion was significantly lower in AT1 receptor–knockout mice than in wild-type mice. In AT1 receptor–knockout mice, the aortic expansion was further attenuated by imidapril. MMP-9 activity in aorta was significantly augmented after elastase application. The MMP-9 activity was significantly lower in AT1 receptor–knockout mice than in wild-type mice, and it was further attenuated by imidapril. In conclusion, MMP-9 inhibition by imidapril might contribute to the attenuation of AAA progression in AT1 receptor–knockout mice.
We investigated the anticonvulsant effect of acute Fuzi total alkaloid (FTA) in seizure induced by the GABAA-receptor antagonist pentylenetetrazole (PTZ). FTA significantly increased the seizure latency and decreased the mortality in PTZ-treated mice. Administration of PTZ increased c-Fos expression in the hippocampus, medial prefrontal cortex, and piriform cortex; and this PTZ-induced effect was inhibited by FTA in a dose-dependent manner. Furthermore, the effects of FTA on PTZ-induced seizure and c-Fos expression were reversed by the GABAA/benzodiazepine receptor–selective antagonist flumazenil. These findings suggest that the anticonvulsant effects of FTA may be related to modulation of GABAA–benzodiazepine receptor complex.
We investigated whether milnacipran, a serotonin–noradrenaline reuptake inhibitor, exhibits an antipruritic effect through the spinal action in mice. Intrathecal injections of milnacipran (0.1 – 10 μg/site) significantly suppressed serotonin-induced biting, which is an itch-related response. However, such an effect was not observed with fluvoxamine (10 μg/site), which is a selective serotonin reuptake inhibitor. Furthermore, an intraperitoneal injection of milnacipran (10 mg/kg) inhibited serotonin-induced biting. When phentolamine (1.0 μg/site), a non-selective α-adrenoceptor antagonist, was intrathecally injected, it inhibited the above response of milnacipran. These results suggest that milnacipran suppresses itching through the inhibition of noradrenaline reuptake in the spinal cord.
Lamotrigine (LTG) is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder and it has been known that LTG targets voltage-dependent sodium channels (VGSCs). In this study, we investigated the effect of LTG on the Nav1.4 Na+ current using HEK293 cells expressing mouse Nav1.4 VGSCs. By the treatment of LTG, Nav1.4 Na+ current was inhibited in a dose-dependent manner. Moreover, 100 μM LTG decreased Nav1.4 Na+ current around 40% and shifted the V1/2 of the inactivation curve to the hyperpolarization side by 20.96 mV. These findings suggest that LTG inhibits Nav1.4 Na+ current and modifies the kinetics of the inactivated state.