a1-adrenergic receptors accompanied by GATA4 expression are related to proarrhythmic conduction and automaticity in rat interatrial septum

The development of interatrial septum (IAS) is a complicated process, which continues during postnatal life. The hypertrophic signals in developing heart are mediated among others by α-adrenergic pathways. These facts suggest the presence of specific electrophysiological features in developing IAS. This study was aimed to investigate the electrical activity in the tissue preparations of IAS from rat heart in normal conditions and under stimulation of adrenoreceptors. Intracellular recording of electrical activity revealed less negative level of resting membrane potential in IAS if compared to myocardium of left atrium. In normal conditions, non-paced IAS preparations were quiescent, but noradrenaline (10−5 M) and phenylephrine (10−5 M) induced spontaneous action potentials, which could be abolished by α1-blocker prazosin (10−5 M), but not β1-blocker atenolol (10−5 M). Optical mapping showed drastic phenylephrine-induced slowing of conduction in adult rat IAS. The α1-dependent ectopic automaticity of IAS myocardium might be explained by immunohistochemical data indicating the presence of transcription factor GATA4 and abundant α1A-adrenoreceptors in myocytes from adult rat IAS. An elevated sensitivity to adrenergic stimulation due to involvement of α1-adrenergic pathways may underlie increased proarrhythmic potential of adult IAS at least in rats. (AU)

Transdermal iontophoresis delivery system for terazosin hydrochloride: an in vitro and in vivo study.

This study aimed to construct a transdermal iontophoresis delivery system for terazosin hydrochloride (IDDS-TEH), which included a positive and negative electrode hydrogel prescription. Intact guinea pig skin was used as a model for the skin barrier function, and the current intensity, terazosin hydrochloride (TEH) concentration, pH, competitive salt, and transdermal enhancer properties were studied. The blood drug concentration was determined in Sprague-Dawley (SD) rats using HPLC, and the antihypertensive effects of IDDS-TEH were evaluated in spontaneously hypertensive rats (SHRs). The results showed that the steady-state penetration rate of TEH increased (from 80.36 µg·cm-2·h-1 to 304.93 µg·cm-2·h-1), followed by an increase in the current intensity (from 0.10 mA·cm-2 to 0.49 mA·cm-2). The pH values also had a significant influence on percutaneous penetration. The blood concentration of IDDS-TEH was significantly higher (p < .05) than with passive diffusion, which could not be detected. The main pharmacokinetic parameters of the high current group (0.17 mA·cm-2) and the low current group (0.09 mA·cm-2) were AUC0-t: 5873.0 ng·mL-1·h and 2493.7 ng·mL-1·h, respectively. Meanwhile, the pharmacodynamic results showed that IDDS-TEH significantly decreased the blood pressure of SHRs compared with the TEH hydrogel without loading current. Therefore, TEH could be successfully delivered by the transdermal iontophoresis system in vitro and in vivo, and further clinical studies should be explored to develop a therapeutically useful protocol.

Simple and fast LC-MS/MS method for quantification of terazosin in human plasma and application to bioequivalence study.

A simple, fast and sensitive LC-MS/MS method was developed to quantify terazosin in human plasma. The mobile phase consisted of acetonitrile-0.1% (v/v) formic acid (70:30, v/v). Prazosin was used as internal standard (IS). As deproteinization agent, acetonitrile produced a clean sample. A higher response intensity with more symmetrical peak was obtained using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 µm) compared with Kinetex XB-C18 (100 × 2.1 mm, 2.6 µm) column. The response of terazosin and IS were approximately two times in citrate phosphate dextrose (CPD) plasma compared with dipotassium ethylenediaminetetraacetic acid (K2EDTA) plasma. Plasma calibration curve was linear from 1.0 to 100.0 ng/mL, with coefficient of determination r2 ≥ 0.99. The within-run and between-run precision values (CV, %) were <5.2% and <7.8%, while accuracy values were 102.8-112.7% and 103.4-112.2%. The extended run accuracy was 98.6-102.8% and precision (CV, %) 4.3-10.4%. The recovery of analyte was >98% and IS >94%. Terazosin in plasma kept at benchtop was stable for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, for 7 freeze-thaw cycles and in freezer for 140 days. Terazosin and IS stock standard solutions were stable for 140 days at room temperature and in the chiller. The high throughput method was successfully utilized to measure 935 samples in a bioequivalence study of terazosin.

Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Expression and Functional Characterization of Drug Transporters in Brain Microvascular Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

Brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) have been proposed as a new blood-brain barrier model, but their transport function has not been fully clarified. Therefore, in this study, we investigated the gene expression and function of transporters in hiPS-BMECs by means of quantitative reverse transcription-PCR, in vitro transcellular transport studies, and uptake experiments. mRNAs encoding ABC and SLC transporters, such as BCRP, MCT1, CAT1, and GLAST, were highly expressed in hiPS-BMECs. Transcellular transport studies showed that prazosin, [14C]l-lactate, [3H]l-arginine, and [3H]l-glutamate (substrates of BCRP, MCT1, CAT1, and GLAST, respectively) were transported asymmetrically across the hiPS-BMEC monolayer. Substrates of LAT1, OCTN2, CAT1, GLAST, MCT1, and proton-coupled organic cation (H+/OC) antiporter were taken up by hiPS-BMECs in a time-, temperature-, and concentration-dependent manner, and the uptakes were markedly decreased by inhibitors of the corresponding transporter. These results indicate that hiPS-BMECs express multiple nutrient and drug transporters.

Palladium nanoparticles in electrochemical sensing of trace terazosin in human serum and pharmaceutical preparations.

In this approach, palladium nanoparticle film was simply fabricated on the surface of carbon paste electrode by electrochemical deposition method. The film was characterized using scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The prepared electrode exhibited an excellent electrocatalytic activity toward detection of trace amounts of terazosin, which is an antihypertensive drug. Under the optimum experimental conditions, a linear range of 1.0×10-8-1.0×10-3molL-1 with a detection limit of 1.9×10-9molL-1 was obtained for determination of terazosin using differential pulse voltammetry as a sensitive method. The efficiency of palladium nanoparticle film on the surface of carbon paste electrode successfully proved for determination of terazosin in pharmaceutical sample and human serum sample with promising recovery results. The effect of some foreign species has been studied.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

AIM: Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. METHODS: Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). RESULTS: BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 µmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. CONCLUSION: UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Drug effects on the CVS in conscious rats: separating cardiac output into heart rate and stroke volume using PKPD modelling.

BACKGROUND AND PURPOSE: Previously, a systems pharmacology model was developed characterizing drug effects on the interrelationship between mean arterial pressure (MAP), cardiac output (CO) and total peripheral resistance (TPR). The present investigation aims to (i) extend the previously developed model by parsing CO into heart rate (HR) and stroke volume (SV) and (ii) evaluate if the mechanism of action (MoA) of new compounds can be elucidated using only HR and MAP measurements. EXPERIMENTAL APPROACH: Cardiovascular effects of eight drugs with diverse MoAs (amiloride, amlodipine, atropine, enalapril, fasudil, hydrochlorothiazide, prazosin and propranolol) were characterized in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats following single administrations of a range of doses. Rats were instrumented with ascending aortic flow probes and aortic catheters/radiotransmitters for continuous recording of MAP, HR and CO throughout the experiments. Data were analysed in conjunction with independent information on the time course of the drug concentration following a mechanism-based pharmacokinetic-pharmacodynamic modelling approach. KEY RESULTS: The extended model, which quantified changes in TPR, HR and SV with negative feedback through MAP, adequately described the cardiovascular effects of the drugs while accounting for circadian variations and handling effects. CONCLUSIONS AND IMPLICATIONS: A systems pharmacology model characterizing the interrelationship between MAP, CO, HR, SV and TPR was obtained in hypertensive and normotensive rats. This extended model can quantify dynamic changes in the CVS and elucidate the MoA for novel compounds, with one site of action, using only HR and MAP measurements. Whether the model can be applied for compounds with a more complex MoA remains to be established.

Preparation and transdermal diffusion evaluation of the prazosin hydrochloride-loaded electrospun poly(vinyl alcohol) fiber mats.

This study reports on the use of electrospun polyvinyl alcohol (PVA) nanofiber mats loaded with prazosin hydrochloride (PRH) as a transdermal drug delivery system, investigating the morphology of electrospun PVA nanofibers, the in vitro release characteristics of the drug from the as-spun fibers, and the influence of permeation enhancer (water-resoluble azone, WSA) on transdermal diffusion of PRH through a rat skin. The same was also conducted on the PRH -loaded as-cast PVA films for comparison. Results indicated that the morphology of PRH-loaded PVA fibers observed by scanning electron microscopy (SEM) relied on the electrospinning processing parameters, and the addition of WSA had obvious effects on the diameter and morphology of electrospun PVA fibers. The PRH-loaded electrospun PVA fiber mats exhibited much higher accumulated release dose and release rate of PRH than as-cast PVA films. And WAS can improve the release amount and rate of PRH from drug-loaded samples. The content of PRH in receiver was more than that in the stratum corneum and in the dermis. It was concluded that the PRH-loaded electropun PVA fiber mats as a transdermal patches can be a promising candidate for the conventional preparation.

Concordance between criteria for covariate model building.

When performing a population pharmacokinetic modelling analysis covariates are often added to the model. Such additions are often justified by improved goodness of fit and/or decreased in unexplained (random) parameter variability. Increased goodness of fit is most commonly measured by the decrease in the objective function value. Parameter variability can be defined as the sum of unexplained (random) and explained (predictable) variability. Increase in magnitude of explained parameter variability could be another possible criterion for judging improvement in the model. The agreement between these three criteria in diagnosing covariate-parameter relationships of different strengths and nature using stochastic simulations and estimations as well as assessing covariate-parameter relationships in four previously published real data examples were explored. Total estimated parameter variability was found to vary with the number of covariates introduced on the parameter. In the simulated examples and two real examples, the parameter variability increased with increasing number of included covariates. For the other real examples parameter variability decreased or did not change systematically with the addition of covariates. The three criteria were highly correlated, with the decrease in unexplained variability being more closely associated with changes in objective function values than increases in explained parameter variability were. The often used assumption that inclusion of covariates in models only shifts unexplained parameter variability to explained parameter variability appears not to be true, which may have implications for modelling decisions.

Brief maternal separation affects brain α1-adrenoceptors and apoptotic signaling in adult mice.

Exposure to adversity during early life is a risk factor for the development of different mood and psychiatric disorders, including depressive-like behaviors. Here, neonatal mice were temporarily but repeatedly (day 1 to day 13) separated from mothers and placed in a testing environment containing a layer of odorless clean bedding (CB). We assessed in adult animals the impact of this early experience on binding sites and mRNA expression of α1-adrenergic receptor subtypes, heat shock proteins (HSPs) and proapoptotic and antiapoptotic members of the Bcl-2 family proteins in different brain regions involved in processing of olfactory information and rewarding stimuli. We found that repeated exposure to CB experience produced anhedonic-like behavior in terms of reduced saccharin intake and α1-adrenoceptor downregulation in piriform and somatosensory cortices, hippocampus, amygdala and discrete thalamic nuclei. We also found a selective decrease of α1B-adrenoceptor binding sites in the cingulate cortex and hippocampus and an increase of hippocampal α1A and α1B receptor, but not of α1D-adrenoceptor, mRNA levels. Moreover, while a significant decrease of antiapoptotic heat shock proteins Hsp72 and Hsp90 was identified in the prefrontal cortex, a parallel increase of antiapoptotic members of Bcl-2 family proteins was found at the hippocampal level. Together, these data provide evidence that the early exposure to CB experience produced enduring downregulation of α1-adrenoceptors in the prefrontal-limbic forebrain/limbic midbrain network, which plays a key role in the processing of olfactory information and reaction to rewarding stimuli. Finally, these data show that CB experience can "prime" the hippocampal circuitry and promote the expression of antiapoptotic factors that can confer potential neuroprotection to subsequent adversity.

[The effects and its mechanism of N-arginine chitosan as transdermal enhancer].

The purpose of this study is to investigate the penetration effects and mechanism of N-arginine chitosan (ACS). This novel transdermal enhancer with a mimetic structure of cell-penetration peptides was synthesized by introducing hydrophilic arginine groups to the amino-group on chitosan's side chain. The structure of ACS was confirmed by FT-IR, 1H NMR and element analysis. In addition, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the protein conformation and the water content of stratum corneum, and the result suggested that ACS can change the orderly arrangement of the molecules in the stratum corneum, making the stack structure of keratin become loose. And ACS can increase the water content of the stratum corneurn. Inverted fluorescence microscope and flow cytometry were used to examine penetration effect of ACS on Hacat cell. The result confirmed that the uptake of ACS was enhanced with increased substitution degree of arginine by 4-8 folds compared to chitosan. In vitro penetration studies on three electrical types of drugs were carried out using three model drugs of negatively charged aspirin, positively charged terazosin and neutral drug isosorbide mononitrate by the method of Franz diffusion cells. The results showed that ACS has obviously penetration of the negatively charged drug aspirin, and certain penetration of neutral drug issorbide mononitrate, but inhibition of positively charged terazosin. In vivo imaging technology research results show that the ACS can significantly enhance the fluorescence intensity of morin, which is the auto-fluorescence anionic drug. These obtained results suggested that ACS, as a promising transdermal enhancer, can change the structure of the keratinocytes and analog penetrating peptides promote absorption, but have certain selectivity for the drug.

Temperature dependence of the interaction of prazosin with lipid Langmuir monolayers.

The influence of temperature on membrane-prazosin interactions was studied. Prazosin, a quinazoline derivative of 2-furoylpiperazine, is a classic antihypertensive and antiarrhythmic drug. A mixed cholesterol/phospholipid monolayer at the water/air interface was employed as a simplified biomembrane model. Brewster angle microscopy (BAM) was used to visualize the monolayer morphology. It was found that prazosin penetrates Langmuir monolayers and modifies the interactions between membrane components, causing monolayer fluidization. An increase in temperature facilitates penetration of prazosin into the monolayers. Prazosin interacts preferentially with phosphatidylcholine and modifies the morphology of the condensed phase domains of DPPC. In the presence of prazosin, monolayers collapse at lower surface pressures. The difference between the collapse pressures of monolayers on water with and without prazosin increases with temperature.

Pharmacokinetics of terazosin enantiomers in healthy Chinese male subjects.

The purpose of this study was to elucidate the pharmacokinetics of terazosin enantiomers in healthy Chinese male subjects. After a single oral dose of 2-mg terazosin, the plasma concentrations of terazosin enantiomers were measured over the course of 48 h in 12 healthy subjects. The plasma concentrations of (+)-(R)-terazosin at all time points were higher than those of (-)-(S)-terazosin. The area under the plasma concentration-time curve (AUC(0-∞) ) and maximum plasma concentration of (+)-(R)-terazosin were significantly greater than those of the (-)-(S)-terazosin (P < 0.01, respectively). The R/S ratio of AUC(0-∞) of terazosin was 1.68. For the first time, it was proven that the pharmacokinetics of terazosin was stereoselective in healthy Chinese male subjects.

Kuguacin J isolated from Momordica charantia leaves inhibits P-glycoprotein (ABCB1)-mediated multidrug resistance.

Multidrug resistance (MDR) is a major factor in the failure of chemotherapy in cancer patients. Resistance to chemotherapy has been correlated to the overexpression of ABC drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. Our previous study showed that bitter melon (Momordica charantia) leaf extract (BMLE) was able to reverse the MDR phenotype by increasing the intracellular accumulation of chemotherapeutic drugs. In the present study, bioguided fractionation was used to identify the active component(s) of BMLE that is able to modulate the function of P-gp and the MDR phenotype in a human cervical carcinoma cell line (KB-V1). We found that kuguacin J, one of the active components in BMLE, increased sensitivity to vinblastine and paclitaxel in KB-V1 cells. A flow cytometry assay indicated that kuguacin J inhibits the transport function of P-gp and thereby significantly increases the accumulation of rhodamine 123 and calcein AM in the cells. These results were confirmed by [³H]-vinblastine transport assay. Kuguacin J significantly increases intracellular [³H]-vinblastine accumulation and decreased the [³H]-vinblastine efflux in the cells. Kuguacin J also inhibited the incorporation of [¹²5I]-iodoarylazidoprazosin into P-gp in a concentration-dependent manner, indicating that kuguacin J directly interacts with the drug-substrate-binding site on P-gp. These results indicate that kuguacin J modulates the function of P-gp by directly interacting at the drug-substrate-binding site, and it appears to be an effective inhibitor of P-gp activity in vitro and thus could be developed as an effective chemosensitizer to treat multidrug-resistant cancers.

Role of alpha adrenoceptors and nitric oxide on cardiovascular responses in acute and chronic hypertension

No disponible

The contribution of á-adrenoceptors and nitric oxide (NO) on the alterations of sympathetically mediated cardiovascular responses after acute (AcH) and chronic (ChH) hypertension was evaluated in pithed aortic coarcted hypertensive rats. Pressor and tachycardia response produced by electrical stimulation of preganglionic sympathetic fibers or exogenous noradrenaline (NA) were recorded in the absence and presence of prazosin (á1-antagonist), rauwolscine (á2-antagonist), or N G-nitro-L-arginine methyl ester (L-NAME; an inhibitor of NO synthase). Compared with age-matched sham-operated rats (Nt), the pressor response produced by electrical stimulation or NA was smaller in AcH rats and larger in ChH rats. Prazosin caused a decrease of pressor response elicited by electrical stimulation or NA in all groups. However, this effect was higher in ChH. Rauwolscine produced a similar increase of sympathetically mediated pressor response in Nt and AcH rats. Nevertheless, this antagonist did not affect the sympathetically mediated pressor response in ChH rats. In addition, rauwolscine did not affect the NA-induced pressor response in all groups. The pressor response elicited by L-NAME was larger in all groups compared without L-NAME and in presence of L-arginine. Moreover, L-NAME in the presence of NA increased sympathetically mediated pressor response is in all groups, compared without it or in the presence of L-arginine. Compared with Nt, basally produced NO in aortic rings was increased in AcH but decreased in ChH. Collectively, our data suggest that decreased cardiovascular reactivity in AcH is due to an increase in basally produced NO. In ChH, enhanced cardiovascular response appears to be associated with a decrease in produced NO and an increase in released NA from sympathetic nerves (AU)

Insulin suppresses the expression and function of breast cancer resistance protein in primary cultures of rat brain microvessel endothelial cells.

The aim of this study was to investigate the role of insulin in the regulation of breast cancer resistance protein (BCRP) function and expression using primary cultured rat brain microvessel endothelial cells (rBMECs) as an in vitro model of the blood brain barrier (BBB). The prazosin uptake assay and western blot analysis were used to assess the function and expression of BCRP, respectively. It was noted that the uptake of prazosin by rBMECs was time-, concentration- and temperature-dependent. The BCRP inhibitors novobiocin and imatinib mesylate significantly increased the uptake of prazosin by the cells in a concentration-dependent manner. The cells were also incubated with sera from diabetic rats for 72 h, serving as a diabetic in vitro model. We found that the uptake of prazosin by rBMECs incubated in the diabetic rat sera was 39.8% of that in normal rat sera, and insulin treatment reversed this decrease. Further results showed that insulin down-regulated the function and expression of BCRP in rBMECs in a concentration-dependent manner. Treatment with an antibody against the insulin receptor abolished the down-regulation of BCRP function and expression that was induced by insulin. These results indicate that insulin suppressed the function and expression of BCRPs in rBMEC primary cultures.

Substrate- and species-dependent inhibition of P-glycoprotein-mediated transport: implications for predicting in vivo drug interactions.

P-glycoprotein (P-gp)-based drug interactions are a major concern in the clinic and in preclinical drug development, especially with respect to the intestinal absorption of drugs and distribution of drugs across the blood-brain barrier. Thus, there is significant interest in developing in vitro (e.g., cell culture) and in vivo models (e.g., rodents) to predict such interactions. In order to generate accurate predictions from these models, however, an understanding of the magnitude of substrate- and species-dependent differences in P-gp inhibition is required. We have used a sensitive flow cytometry assay to measure the ability of various drugs to inhibit the initial rate of accumulation of two fluorescent drug analogs (probe substrates), 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -indacene (BODIPY)-verapamil and BODIPY-prazosin, into Lewis lung carcinoma-porcine kidney 1 (LLC-PK1) cells expressing human or rat P-gp. The inhibition of P-gp-mediated efflux of these two fluorescent substrates by several drugs, including quinidine and itraconazole, was found to be substrate- and/or species-dependent. These data suggest that to provide accurate prediction of clinically significant P-gp drug interactions, multiple P-gp substrates will need to be used in both in vitro and in vivo (including human) drug interaction studies. In addition, extrapolation of P-gp-based drug interaction in rodents to humans must be conducted with caution.

Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.

Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17ßG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions.

Glutaminyl cyclase contributes to the formation of focal and diffuse pyroglutamate (pGlu)-Aß deposits in hippocampus via distinct cellular mechanisms.

In the hippocampal formation of Alzheimer's disease (AD) patients, both focal and diffuse deposits of Aß peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aß peptides were identified as a highly pathogenic and seeding Aß peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aß aggregates. First, we demonstrate that both, focal and diffuse pE-Aß deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aß aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aß deposits originating from QC-positive neurons and diffuse pE-Aß deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aß deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aß and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aß deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aß plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons.