3,3'-O-dimethylquercetin: A bi-functional vasodilator isolated from green propolis of the Caatinga Mimosa tenuiflora.

In the search for novel, bi-functional compounds acting as CaV1.2 channel blockers and K+ channel stimulators, which represent an effective therapy for hypertension, 3,3'-O-dimethylquercetin was isolated for the first time from Brazilian Caatinga green propolis. Its effects were investigated through electrophysiological, functional, and computational approaches. In rat tail artery myocytes, 3,3'-O-dimethylquercetin blocked Ba2+ currents through CaV1.2 channels (IBa1.2) in a concentration-dependent manner, with the inhibition being reversed upon washout. The compound also shifted the voltage dependence of the steady-state inactivation curve to more negative potentials without affecting the slope of the inactivation and activation curves. Furthermore, the flavonoid stimulated KCa1.1 channel currents (IKCa1.1). In silico simulations provided additional evidence for the binding of 3,3'-O-dimethylquercetin to KCa1.1 and CaV1.2 channels and elucidated its mechanism of action. In depolarized rat tail artery rings, the flavonoid induced a concentration-dependent relaxation. Moreover, in rat aorta rings its antispasmodic effect was inversely related to the transmembrane K+ gradient. In conclusion, 3,3'-O-dimethylquercetin demonstrates effective in vitro vasodilatory properties, encouraging the exploration of its scaffold to develop novel derivatives for potential use in the treatment of hypertension.

Neurochemical phenotypes of huntingtin-associated protein 1 in reference to secretomotor and vasodilator neurons in the submucosal plexuses of rodent small intestine.

Huntingtin-associated protein 1(HAP1) is an immunohistochemical marker of the stigmoid body (STB). Brain and spinal cord regions with lack of STB/HAP1 immunoreactivity are always neurodegenerative targets, whereas STB/HAP1 abundant regions are usually spared from neurodegeneration. In addition to the brain and spinal cord, HAP1 is abundantly expressed in the excitatory and inhibitory motor neurons in myenteric plexuses of the enteric nervous system (ENS). However, the detailed expression of HAP1 and its neurochemical characterization in submucosal plexuses of ENS are still unknown. In this study, we aimed to clarify the expression and neurochemical characterization of HAP1 in the submucosal plexuses of the small intestine in adult mice and rats. HAP1 was highly expressed in the submucosal plexuses of both rodents. The percentage of HAP1-immunoreactive submucosal neurons was not significantly varied between the intestinal segments of these rodents. Double immunofluorescence results revealed that almost all the cholinergic secretomotor neurons containing ChAT/ CGRP/ somatostatin/ calretinin, non-cholinergic secretomotor neurons containing VIP/NOS/TH/calretinin, and vasodilator neurons containing VIP/calretinin expressed HAP1. Our current study is the first to clarify that STB/HAP1 is expressed in secretomotor and vasodilator neurons of submucosal plexuses, suggesting that STB/HAP1 might modulate or protect the secretomotor and vasodilator functions of submucosal neurons in ENS.

Effects of bisphenol A on human umbilical arteries.

Bisphenol A (BPA) is an endocrine-disrupting chemical widely used in the plastics industry, including food container, toys, and medical equipment. We analyzed the effect of BPA in human umbilical artery contractility and expression of some proteins modulating this function, such as ionic channels and proteins involved in the cGMP pathway. Using standard organ bath technique, rings of human umbilical arteries without endothelium were contracted by 5-HT (1 µM) and histamine (10 µM) and the effect of different concentrations of BPA (1 nM-100 µM) was analyzed. The results showed that BPA is a vasodilator of these arteries in a concentration-dependent way. Besides, qPCR studies on human umbilical smooth muscle cells (HUSMC) allowed to analyze the effects of BPA on gene expression. Thus, 12-h exposition to BPA induced reduction of expression of L-type calcium channels (LTCC), alpha subunit of BKCa channels, and Kvß1 and Kvß3 from Kv channels. BPA also decreased the expression of soluble guanylate cyclase (sGC) and natriuretic peptide receptor type A (NPRA), meanwhile increasing that of PKG, proteins involved in vasodilation of human umbilical arteries (HUA) by cGMP. Further studies will be necessary to increase knowledge about the implications of these changes induced by BPA exposure.

Structural identification of vasodilator binding sites on the SUR2 subunit.

ATP-sensitive potassium channels (KATP), composed of Kir6 and SUR subunits, convert the metabolic status of the cell into electrical signals. Pharmacological activation of SUR2- containing KATP channels by class of small molecule drugs known as KATP openers leads to hyperpolarization of excitable cells and to vasodilation. Thus, KATP openers could be used to treat cardiovascular diseases. However, where these vasodilators bind to KATP and how they activate the channel remains elusive. Here, we present cryo-EM structures of SUR2A and SUR2B subunits in complex with Mg-nucleotides and P1075 or levcromakalim, two chemically distinct KATP openers that are specific to SUR2. Both P1075 and levcromakalim bind to a common site in the transmembrane domain (TMD) of the SUR2 subunit, which is between TMD1 and TMD2 and is embraced by TM10, TM11, TM12, TM14, and TM17. These KATP openers synergize with Mg-nucleotides to stabilize SUR2 in the NBD-dimerized occluded state to activate the channel.

Endothelium-dependent vasorelaxant effects of praeruptorin a in isolated rat thoracic aorta.

Praeruptorin A (PA) is a natural coumarin compound from the roots of Radix Peucedani and is commonly used in the treatment of certain respiratory diseases and hypertension. Although previous studies identified relaxant effects of PA on tracheal and arterial preparations, little is known about its vasodilative effects and underlying mechanisms. Here, an organ bath system and tension recording methods were used to prepare and analyze isolated rat thoracic aorta artery rings. Aorta artery rings were pre-contracted with phenylephrine and then incubated with PA, and the possible mechanism of relaxation was investigated by adding inhibitors of nitric oxide synthase (NG-nitro-L-arginine methyl ester, L-NAME), endothelial nitric oxide synthase (L-NG-nitroarginine, L-NNA), cyclooxygenase (indomethacin), guanylyl cyclase (1 H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one, ODQ), and KCa channels (tetraethylammonium, TEA). Our study showed that PA-induced vasodilation was blocked by L-NAME, L-NNA, and ODQ, while CaCl2-induced vasoconstriction was countered by PA. Thus, PA may exert a vasodilatory effect by influencing the amounts of endothelium-derived relaxing factors through endothelial-dependent NO-cGMP and prostacyclin pathways (such as NO and prostacyclin 2). In the rat thoracic aorta, PA reduces vasoconstriction by inhibiting Ca2+ inflow.

Cardiovascular characterization of the novel organic mononitrate NDIBP in rats.

Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 µM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 µM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle.

EETs/sEHi alleviates nociception by blocking the crosslink between endoplasmic reticulum stress and neuroinflammation in a central poststroke pain model.

BACKGROUND: Central post-stroke pain (CPSP) is a chronic and intolerable neuropathic pain syndrome following a cerebral vascular insult, which negatively impacts the quality of life of stroke survivors but currently lacks efficacious treatments. Though its underlying mechanism remains unclear, clinical features of hyperalgesia and allodynia indicate central sensitization due to excessive neuroinflammation. Recently, the crosslink between neuroinflammation and endoplasmic reticulum (ER) stress has been identified in diverse types of diseases. Nevertheless, whether this interaction contributes to pain development remains unanswered. Epoxyeicosatrienoic acids (EETs)/soluble epoxy hydrolase inhibitors (sEHi) are emerging targets that play a significant role in pain and neuroinflammatory regulation. Moreover, recent studies have revealed that EETs are effective in attenuating ER stress. In this study, we hypothesized that ER stress around the stroke site may activate glial cells and lead to further inflammatory cascades, which constitute a positive feedback loop resulting in central sensitization and CPSP. Additionally, we tested whether EETs/sEHi could attenuate CPSP by suppressing ER stress and neuroinflammation, as well as their vicious cycle, in a rat model of CPSP. METHODS: Young male SD rats were used to induce CPSP using a model of thalamic hemorrhage and were then treated with TPPU (sEHi) alone or in combination with 14,15-EET or 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, the EET antagonist), tunicamycin (Tm, ER stress inducer), or 4-PBA (ER stress inhibitor). Nociceptive behaviors, ER stress markers, JNK and p38 (two well-recognized inflammatory kinases of mitogen-activated protein kinase (MAPK) signaling) expression, and glial cell activation were assessed. In addition, some healthy rats were intrathalamically microinjected with Tm or lipopolysaccharide (LPS) to test the interaction between ER stress and neuroinflammation in central pain. RESULTS: Analysis of the perithalamic lesion tissue from the brain of CPSP rats demonstrated decreased soluble epoxy hydrolase (sEH) expression, which was accompanied by increased expression of ER stress markers, including BIP, p-IRE, p-PERK, and ATF6. In addition, inflammatory kinases (p-p38 and p-JNK) were upregulated and glial cells were activated. Intrathalamic injection of sEHi (TPPU) increased the paw withdrawal mechanical threshold (PWMT), reduced hallmarks of ER stress and MAPK signaling, and restrained the activation of microglia and astrocytes around the lesion site. However, the analgesic effect of TPPU was completely abolished by 14,15-EEZE. Moreover, microinjection of Tm into the thalamic ventral posterior lateral (VPL) nucleus of healthy rats induced mechanical allodynia and activated MAPK-mediated neuroinflammatory signaling; lipopolysaccharide (LPS) administration led to activation of ER stress along the injected site in healthy rats. CONCLUSIONS: The present study provides evidence that the interaction between ER stress and neuroinflammation is involved in the mechanism of CPSP. Combined with the previously reported EET/sEHi effects on antinociception and neuroprotection, therapy with agents that target EET signaling may serve as a multi-functional approach in central neuropathic pain by attenuating ER stress, excessive neuroinflammation, and subsequent central sensitization. The use of these agents within a proper time window could not only curtail further nerve injury but also produce an analgesic effect.

Ferulic Acid Derivatives and Avenanthramides Modulate Endothelial Function through Maintenance of Nitric Oxide Balance in HUVEC Cells.

Wholegrain oats contain a variety of phenolic compounds thought to help maintain healthy vascular function, through the maintenance of local levels of the vasodilator nitric oxide (NO). Thus, the full molecular mechanisms involved are not yet clear. With this work we aim to understand the possible cellular mechanisms by which avenanthramides and ferulic acid derivatives, present in oats, may help maintain a healthy vascular function through the modulation of the NO pathway. Primary Human Umbilical Vein Endothelial Cells (HUVEC) were exposed to ferulic acid, isoferulic acid, hydroferulic acid, ferulic acid 4-O-glucuronide, isoferulic acid 3-O-sulfate, dihydroferulic acid 4-O-glucuronide, avenanthramide A, avenanthramide B and avenanthramide C (1 µM) or vehicle (methanol) for 24 h. Apocynin and Nω-Nitro-L-arginine (L-NNA) were additionally included as controls. NO and cyclic GMP (cGMP) levels, superoxide production and the activation of the Akt1/eNOS pathway were assessed. The statistical analysis was performed using one-way ANOVA followed by a Tukey post-hoc t-test. Apocynin and all phenolic compounds increased NO levels in HUVEC cells (increased DAF2-DA fluorescence and cGMP), and significantly reduced superoxide levels. Protein expression results highlighted an increase in the Akt1 activation state, and increased eNOS expression. Overall, our results indicated that the glucuronide metabolites do not enhance NO production through the Akt1/eNOS pathway, thus all compounds tested are able to reduce NO degradation through reduced superoxide formation.

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling.

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.

Pharmacological characterization of MT-1207, a novel multitarget antihypertensive agent.

Hypertension is a serious public health problem worldwide. MT-1207, chemically named 3-(4-(4-(1H-benzotriazole-1-yl)butyl)piperazine-1-yl) benzisothiazole hydrochloride, is a new chemical entity that has entered into clinical trial as antihypertensive agent in China. In this paper we report the pharmacological profile of MT-1207 regarding its acute, subacute, and long-term effects on hypertensive animal models, and its actions on isolated organs in vitro as well as its molecular targets. Blood pressure (BP) was measured in conscious animals; amlodipine was taken as a positive control drug. We showed that both single dose of MT-1207 (1.25-20 mg/kg, ig) in spontaneously hypertensive rats (SHR) and MT-1207 (0.25-6 mg/kg, ig) in two-kidney one-clip (2K1C) dogs dose-dependently decreased BP. MT-1207 quickly decreased BP within 5 min after administration; the hypotensive effect lasted for 8 and 12 h, respectively, in SHR and 2K1C dogs without reflex increase in heart rate. Multiple doses of MT-1207 (5 mg · kg-1 · d-1 in SHR; 2 mg · kg-1 · d-1 in 2K1C dogs, for 7 days) significantly decreased BP, slightly reduced heart rate, and both of them recovered after withdrawal. Long-term administration of MT-1207 (10 mg · kg-1 · d-1 for 4 months or more time) produced a stable BP reduction, improved baroreflex sensitivity, reduced renal and cardiovascular damage in SHR, and delayed stroke occurrence and death in stroke-prone SHR. In isolated rat aortic rings precontracted by adrenaline, KCl, noradrenaline or 5-hydroxytryptamine (5-HT), MT-1207 (10-9-10-4 M) caused concentration-dependent relaxation. In a panel of enzyme activity or radioligand binding assays of 87 molecular targets, MT-1207 potently inhibited adrenergic α1A, α1B, α1D, and 5-HT2A receptors with Ki < 1 nM. The antagonism of MT-1207 against these receptors was confirmed in isolated rabbit arteries. We conclude that MT-1207 is a novel and promising single-molecule multitarget agent for hypertension treatment to reduce hypertensive organ damage and stroke mortality.

Vasodilatory effects of a variety of positive allosteric modulators of GABAA receptors on rat thoracic aorta.

Different subtypes of GABAA (gamma-aminobutyric acid A) receptors, through their specific regional and cellular localization, are involved in the manifestation of various functions, both at the central and peripheral levels. We hypothesized that various non-neuronal GABAA receptors are expressed on blood vessels, through which positive allosteric modulators of GABAA receptors exhibit vasodilatory effects. This study involved two parts: one to determine the presence of α1-6 subunit GABAA receptor mRNAs in the rat thoracic aorta, and the other to determine the vasoactivity of the various selective and non-selective positive GABAA receptor modulators: zolpidem (α1-selective), XHe-III-074 (α4-selective), MP-III-022 (α5-selective), DK-I-56-1 (α6-selective), SH-I-048A and diazepam (non-selective). Reverse transcription-polymerase chain reaction (RT-PCR) analysis data demonstrated for the first time the expression of α1, α2, α3, α4 and α5 subunits in the rat thoracic aorta tissue. Tissue bath assays on isolated rat aortic rings revealed significant vasodilatory effects of diazepam, SH-I-048A, XHe-III-074, MP-III-022 and DK-I-56-1, all in terms of achieved relaxations (over 50% of relative tension decrease), as well as in terms of preventive effects on phenylephrine (PE) contraction. Diazepam was the most efficient ligand in the present study, while zolpidem showed the weakest vascular effects. In addition, diazepam-induced relaxations in the presence of antagonists PK11195 or bicuculline were significantly reduced (P < 0.001 and P < 0.05, respectively) at lower concentrations of diazepam (10-7 M and 3 × 10-7 M). The present work suggests that the observed vasoactivity is due to modulation of "vascular" GABAA receptors, which after further detailed research may provide a therapeutic target.

Metabolic Profiling of Vasorelaxant Extract from Malvaviscus arboreus by LC/QTOF-MS.

We aimed to develop a standardized methodology to determine the metabolic profile of organic extracts from Malvaviscus arboreus Cav. (Malvaceae), a Mexican plant used in traditional medicine for the treatment of hypertension and other illnesses. Also, we determined the vasorelaxant activity of these extracts by ex vivo rat thoracic aorta assay. Organic extracts of stems and leaves were prepared by a comprehensive maceration process. The vasorelaxant activity was determined by measuring the relaxant capability of the extract to decrease a contraction induced by noradrenaline (0.1 µM). The hexane extract induced a significant vasorelaxant effect in a concentration- and endothelium-dependent manner. Secondary metabolites, such as polyunsaturated fatty acids, terpenes and one flavonoid, were annotated by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in positive ion mode. This exploratory study allowed us to identify bioactive secondary metabolites from Malvaviscus arboreus, as well as identify potentially-new vasorelaxant molecules and scaffolds for drug discovery.

A multitarget semi-synthetic derivative of the flavonoid morin with improved in vitro vasorelaxant activity: Role of CaV1.2 and KCa1.1 channels.

CaV1.2 channels play a fundamental role in the regulation of vascular smooth muscle tone. The aim of the present study was to synthesize morin derivatives bearing the nitrophenyl moiety of dihydropyridine Ca2+ antagonists to increase the flavonoid vasorelaxant activity. The effects of morin and its derivatives were assessed on CaV1.2 and KCa1.1 channels, both in vitro and in silico, as well as on the contractile responses of rat aorta rings. All compounds were effective CaV1.2 channel blockers, positioning in the α1C subunit region where standard blockers bind. Among the four newly synthesized morin derivatives, the penta-acetylated morin-1 was the most efficacious Ca2+ antagonist, presenting a vasorelaxant profile superior to that of the parent compound and, contrary to morin, antagonized also the release of Ca2+ from the sarcoplasmic reticulum; surprisingly, it also stimulated KCa1.1 channel current. Computational analysis demonstrated that morin-1 bound close to the KCa1.1 channel S6 segment. In conclusion, these findings open a new avenue for the synthesis of valuable multi-functional, vasorelaxant morin derivatives capable to target several pathways underpinning the pathogenesis of hypertension.

Chuanxiongdiolides R4 and R5, phthalide dimers with a complex polycyclic skeleton from the aerial parts of Ligusticum chuanxiong and their vasodilator activity.

Chuanxiongdiolides R4-R6 (1-3), three novel phthalide dimers featuring two classes of unreported monomeric units (ligustilide/senkyunolide A and ligustilide/neocnidilide) with an unprecedented linkage style (3a,7'/7a,7'a), were isolated from the aerial parts of Ligusticum chuanxiong, together with three pairs of enantiomeric phthalide dimers [(-)/(+)-4a/4b, 5a/5b, and 6a/6b]. The bioassays revealed that compounds 1, 3, 4, 5, and 6 showed significant vasodilation effects, and the mechanism may be attributed to Cav1.2 activation blockade. Based on the established compounds library, the structure activity relationship of the phthalides was proposed. Our findings afford possible leads for developing new vasodilator against cardiovascular and cerebrovascular diseases such as hypertension and ischemic stroke.

Cerebral Vasodilator Property of Poly(ADP-Ribose) Polymerase Inhibitor (PJ34) in the Neonatal and Adult Mouse Is Mediated by the Nitric Oxide Pathway.

The poly(ADP-ribose) polymerase (PARP) inhibitor PJ34 has been reported to improve endothelial dysfunction in the peripheral system. We addressed the role of PJ34 on the vascular tone and vasoreactivity during development in the mouse brain. Blood flows were measured in the basilar trunk using ultrasonography. Cerebral vasoreactivity or vasodilation reserve was estimated as a percentage increase in mean blood flow velocities (mBFV) recorded under normoxia-hypercapnia in control and after PJ34 administration. Non-selective and selective eNOS and nNOS inhibitors were used to evaluate the role of NO-pathway into the hemodynamic effects of PJ34. PJ34 increased mBFVs from 15.8 ± 1.6 to 19.1 ± 1.9 cm/s (p = 0.0043) in neonatal, from 14.6 ± 1.4 to 16.1 ± 0.9 cm/s (p = 0.0049) in adult, and from 15.7 ± 1.7 to 17.5 ± 2.0 cm/s (p = 0.0024) in aged mice 48 h after administration. These PJ34 values were similar to those measured in age-matched control mice under normoxia-hypercapnia. This recruitment was mediated through the activation of constitutive NO synthases in both the neonatal (38.2 ± 6.7 nmol/min/mg protein) and adult (31.5 ± 4.4 nmol/min/mg protein) brain, as compared to age-matched control brain (6.9 ± 0.4 and 6.3 ± 0.7 nmol/min/mg protein), respectively. In addition, quite selective eNOS inhibitor was able to inhibit the recruitment. PJ34 by itself is able to increase cerebral blood flow through the NO-pathway activation at least over 48 h after a single administration.

The Role of Losartan as a Potential Neuroregenerative Pharmacological Agent after Aneurysmal Subarachnoid Haemorrhage.

BACKGROUND: Cerebral vasospasm (CVS) remains a major cause of delayed cerebral ischaemia following aneurysmal subarachnoid haemorrhage (SAH), making it a life-threatening type of stroke with high morbidity and mortality. Endothelin-1 is known as key player mediating a strong vasocontractile effect. Interestingly, losartan restores the impaired vasorelaxative ET(B1) receptor function in a non-competitive direct fashion. With this study, we aimed to investigate a potential losartan-dependent vasodilatory effect vice versa by inhibiting NO release through L-NAME, thus pushing forward concepts to alleviate vasospasm and possibly prevent ischaemia and neurodegeneration. METHODS: Cerebral vasospasm was induced by the use of an established double-injection rat model. Sprague-Dawley rats were culled on Day 3 after the ictus, and the vasospastic basilar artery was harvested for isometric investigations of the vessel tone. Ring segments were preincubated with and without L-NAME and/or losartan. RESULTS: Preincubation with L-NAME induced dose-dependent vasoconstriction via endothelin-1 in the non-SAH cohort, which was dose-dependently reduced by losartan. After SAH and dose-dependent endothelin-1 administration, maximal contraction was achieved in the control group without losartan. Furthermore, this maximal contraction was significantly decreased in the losartan group and was reversed by L-NAME. CONCLUSIONS: After SAH, losartan was shown to positively influence the ET(B1) receptor pathway in a non-competitive direct agonistic and indirect fashion. Losartan alleviated the maximum contraction triggered by endothelin-1. This effect was resolved due to NO inhibition by L-NAME. Considering this spasmolytic effect of losartan besides its already well-known effects (attenuating cerebral inflammation, restoring cerebral autoregulation and reducing epileptogenic activity) and alleviating early brain injury, losartan seems to have potential as a promising pharmacological agent after SAH.

P-glycoprotein overactivity in epileptogenic developmental lesions measured in vivo using (R)-[11 C]verapamil PET.

OBJECTIVE: Overexpression of the drug transporter P-glycoprotein (P-gp) is thought to be involved in drug-resistance in epilepsy by extrusion of antiepileptic drugs (AEDs). We used positron emission tomography (PET) and the P-gp substrate radiotracer (R)-[11 C]verapamil (VPM) together with the third-generation P-gp inhibitor tariquidar (TQD) to evaluate P-gp function in individuals with drug-resistant epileptogenic developmental lesions. METHODS: Twelve healthy controls (7 male, median age 45, range 35-55 years), and two patients with epileptogenic developmental lesions (2 male, aged 24 and 62 years) underwent VPM-PET scans before and 60 minutes after a 30-minute infusion of 2 and 3 mg/kg TQD. The influx rate constant, VPM-K1 , was estimated from the first 10 minutes of dynamic data using a single-tissue compartment model with a VPM plasma input function. Statistical parametric mapping (SPM) analysis was used to compare individual patients with the healthy controls. RESULTS: At baseline, SPM voxel-based analysis revealed significantly lower uptake of VPM corresponding to the area of the epileptogenic developmental lesion compared to 12 healthy controls (P < .048). This was accentuated following P-gp inhibition with TQD. After TQD, the uptake of VPM was significantly lower in the area of the epileptogenic developmental lesion compared to controls (P < .002). SIGNIFICANCE: This study provides further evidence of P-gp overactivity in patients with drug-resistant epilepsy, irrespective of the type of lesion. Identifying P-gp overactivity as an underlying contributor to drug-resistance in individual patients will enable novel treatment strategies aimed at overcoming or reversing P-gp overactivity.

Suitability and functional characterization of two Calu-3 cell models for prediction of drug permeability across the airway epithelial barrier.

The Calu-3 cell line has been largely investigated as a physiological and pharmacological model of the airway epithelial barrier. Its suitability for prediction of drug permeability across the airway epithelia, however, has not been yet evaluated by using large enough set of model drugs. We evaluated two Calu-3 cell models (air-liquid and liquid-liquid) for drug permeability prediction based on the recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. Bidirectional permeability assays using 22 model drugs and several zero permeability markers, as well as using ABC transporter substrates were conducted. Functional activity of P-gp, but not of BCRP was revealed. The potential of the Calu-3 cells to be used as a model of the nasal epithelial barrier, despite their different anatomical origin, has been demonstrated by the obtained excellent correlation with the fully differentiated 3D human nasal epithelial model (MucilAir™) for 11 model drugs, as well as by the good correlation obtained with the human nasal epithelial cell line RPMI 2650. In addition, the permeability values determined in the two Calu-3 models correlated well with the intestinal permeability model Caco-2.

Oleuropein promotes hippocampal LTP via intracellular calcium mobilization and Ca2+-permeable AMPA receptor surface recruitment.

Oleuropein (OLE), a major phenolic compound in olive oil, has been demonstrated to possess several pharmacological properties, including neuroprotection. However, the cognitive effects of OLE and its action mechanism have remained unclear. Here, we examined the effect of OLE on long-term potentiation (LTP) using field excitatory postsynaptic potential recorded in the CA1 region of both wild-type and 5XFAD mouse hippocampal slice preparations. In initial experiments with wild-type mice, 100 µM/1 h of OLE produced significant enhancements in the LTPs of Schaffer collateral synapses in the CA1 regions of treated mice, as compared to the vehicle-treated controls. As assessed by surface biotinylation and Western blot analysis, OLE caused a significant increase in protein kinase A (PKA)-mediated phosphorylation and the surface expression of GluA1 containing calcium permeable- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) in the hippocampus. Furthermore, we found that OLE enhanced LTP induction, while GluA1 phosphorylation occurred in an N-methyl-d-aspartate receptors (NMDARs)-independent manner. The OLE-induced CP-AMPAR trafficking resulted from elevated intracellular Ca2+ levels via regulation of phospholipase C (PLC). Consistently, we also found involvement of NMDAR-independent LTP and GluA1 phosphorylation in 5XFAD transgenic mice hippocampal slices treated with OLE. Together, our findings indicate that OLE may regulate beneficial effects on memory through the facilitation of CP-AMPAR trafficking and synaptic transmission.

Target-responsive vasoactive probes for ultrasensitive molecular imaging.

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.