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Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

Sylwia Dabrowska,1 Andrea Del Fattore,2 Elzbieta Karnas,3,4 Malgorzata Frontczak-Baniewicz,5 Hanna Kozlowska,6 Maurizio Muraca,7 Miroslaw Janowski,1,8 Barbara Lukomska1 1NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 2Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy; 3Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 4Malopolska Centre of Biotechnology, Krakow, Poland; 5Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 6Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 7Department of Women’s and Children’s Health, University of Padua, Padua, Italy; 8Russel H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs.Methods: The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B™) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles.Results: The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs.Conclusion: PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI. Keywords: mesenchymal stem cells, extracellular vesicles, cell tracking, fluorescent dye, iron oxide, MRI

Tests of microfiltration efficiency used for the pretreatment of backwash water from sand filters were conducted at two water treatment plants treating surface water and infiltration water. Microfiltration efficiency was evaluated for three membrane modules: two with polymeric membranes and one with a ceramic membrane. This study showed that the contaminants that limit the reuse of backwash water from both plants by returning them to the water treatment line are mostly microorganisms, including pathogenic species ( Clostridium perfringens ). Additionally, in the case of backwash water from infiltration water treatment, iron and manganese compounds also had to be removed before its recirculation to the water treatment system. Unexpectedly, organic carbon concentrations in both types of backwash water were similar to those present in intake waters. Microfiltration provided for the removal of organic matter, ranging from 19.9% to 44.5% and from 7.2% to 53.9% for backwash water from the treatments of surface water and infiltration water, respectively. Furthermore, the efficiency of the iron removal from backwash water from infiltration water treatment was sufficient to ensure good intake water quality. On the other hand, manganese concentrations in the backwash water, from infiltration water treatment, pretreated using the microfiltration process exceeded the levels found in the intake water and were, therefore, an additional limiting factor for the reuse of the backwash water. In both types of backwash water, the number of microorganisms, including Clostridium perfringens (a pathogenic one), was a limiting parameter for backwash water reuse without pretreatment. The results of the present study showed the possibility for using microfiltration for the pretreatment of backwash water, regardless of its origin but not as the sole process. More complex technological systems are needed before recirculating backwash water into the water treatment system. The polyvinylidene fluoride (PVDF) membrane proved to be the most effective for DOC and microorganism removal from backwash water.

Cannabidiol (CBD) is a non-intoxicating compound of Cannabis with anti-fibrotic properties. Pulmonary hypertension (PH) is a disease that can lead to right ventricular (RV) failure and premature death. There is evidence that CBD reduces monocrotaline (MCT)-induced PH, including reducing right ventricular systolic pressure (RVSP), vasorelaxant effect on pulmonary arteries, and decreasing expression of profibrotic markers in the lungs. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg daily for 21 days) on profibrotic parameters in the RVs of MCT-induced PH rats. In MCT-induced PH, we found an increase in profibrotic parameters and parameters related to RV dysfunction, i.e. plasma pro-B-type natriuretic peptide (NT-proBNP), cardiomyocyte width, interstitial and perivascular fibrosis area, amount of fibroblasts and fibronectin, as well as overexpression of the transforming growth of factor β1 (TGF-β1), galectin-3 (Gal-3), suppressor of mothers against decapentaplegic 2 (SMAD2), phosphorylated SMAD2 (pSMAD2) and alpha-smooth muscle actin (α-SMA). In contrast, vascular endothelial cadherin (VE-cadherin) levels were decreased in the RVs of MCT-induced PH rats. Administration of CBD reduced the amount of plasma NT-proBNP, the width of cardiomyocytes, the amount of fibrosis area, fibronectin and fibroblast expression, as well as decreased the expression of TGF-β1, Gal-3, SMAD2, pSMAD2, and increased the level of VE-cadherin. Overall, CBD has been found to have the anti-fibrotic potential in MCT-induced PH. As such, CBD may act as an adjuvant therapy for PH, however, further detailed investigations are recommended to confirm our promising results., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The enhancement of the endocannabinoid tone might have a beneficial influence on hypertension. Polypharmacology proposes multi-target-directed ligands (MTDLs) as potential therapeutic agents for the treatment of complex diseases. In the present paper, we studied JZL195, a dual inhibitor of the two major endocannabinoid-degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Hemodynamic parameters were assessed in conscious animals via radiotelemetry and tail-cuff methods and then evaluated by the area under the curve (AUC). Single administration of JZL195 induced dose-dependent weak hypotensive and bradycardic responses in SHR but not in WKY. Similarly, its chronic application revealed only a slight hypotensive potential which, however, effectively prevented the progression of hypertension and did not undergo tolerance. In addition, multiple JZL195 administrations slightly decreased heart rate only in WKY and prevented the gradual weight gain in both groups. JZL195 did not affect organ weights, blood glucose level, rectal temperature and plasma oxidative stress markers. In conclusion, chronic dual FAAH/MAGL inhibition prevents the progression of hypertension in SHR without affecting some basal functions of the body. In addition, our study clearly proves the suitability of AUC for the evaluation of weak blood pressure changes.

Cannabidiol (CBD) is a safe and well-tolerated plant-derived drug with anti-proliferative properties. Pulmonary hypertension (PH) is a rapidly progressive and still incurable disease. CBD diminishes monocrotaline (MCT)-induced PH, including reduced right ventricular systolic pressure, pulmonary vascular hypertrophy, and right ventricular remodeling. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg once daily for 21 days) on selected remodeling parameters in the lung of MCT-induced PH rats. In MCT-induced PH, we found an increase in profibrotic parameters, e.g., transforming growth factor β1 (TGF-β1), galectin-3 (Gal-3), procollagen I, collagen I, C-propeptide, matrix metalloproteinase 9 (MMP-9) and an increased number of mast cells. In our study, we observed that the TGF-β1, Gal-3, procollagen I, collagen I, C-propeptide, and mast cell levels in lung tissue were decreased after CBD administration to MCT-treated rats. In summary, CBD treatment has an anti-proliferative effect on MCT-induced PH. Given the beneficial multidirectional effects of CBD on PH, we believe that CBD can be used as an adjuvant PH therapy, but this argument needs to be confirmed by clinical trials., Competing Interests: Conflict of Interest Statement The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Cannabidiol (CBD) is a plant-derived compound with antioxidant and anti-inflammatory properties. Pulmonary hypertension (PH) is still an incurable disease. CBD has been suggested to ameliorate monocrotaline (MCT)-induced PH, including reduction in right ventricular systolic pressure (RVSP), a vasorelaxant effect on pulmonary arteries and a decrease in the white blood cell count. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg daily for 21 days) on the parameters of oxidative stress and inflammation in the lungs of rats with MCT-induced PH. In MCT-induced PH, we found a decrease in total antioxidant capacity (TAC) and glutathione level (GSH), an increase in inflammatory parameters, e.g., tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), nuclear factor kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1), and cluster of differentiation 68 (CD68), and the overexpression of cannabinoid receptors type 1 and 2 (CB 1 -Rs, CB 2 -Rs). Administration of CBD increased TAC and GSH concentrations, glutathione reductase (GSR) activity, and decreased CB 1 -Rs expression and levels of inflammatory mediators such as TNF-α, IL -1β, NF-κB, MCP-1 and CD68. In conclusion, CBD has antioxidant and anti-inflammatory effects in MCT-induced PH. CBD may act as an adjuvant therapy for PH, but further detailed preclinical and clinical studies are recommended to confirm our promising results.

GPR18 receptor protein was detected in the heart and vasculature and appears to play a functional role in the cardiovascular system. We investigated the effects of the new GPR18 agonists PSB-MZ-1415 and PSB-MZ-1440 and the new GPR18 antagonist PSB-CB-27 on isolated human pulmonary arteries (hPAs) and compared their effects with the previously proposed, but unconfirmed, GPR18 ligands NAGly, Abn-CBD (agonists) and O-1918 (antagonist). GPR18 expression in hPAs was shown at the mRNA level. PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD fully relaxed endothelium-intact hPAs precontracted with the thromboxane A 2 analog U46619. PSB-CB-27 shifted the concentration-response curves (CRCs) of PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD to the right; O-1918 caused rightward shifts of the CRCs of PSB-MZ-1415 and NAGly. Endothelium removal diminished the potency and the maximum effect of PSB-MZ-1415. The potency of PSB-MZ-1415 or NAGly was reduced in male patients, smokers and patients with hypercholesterolemia. In conclusion, the novel GPR18 agonists, PSB-MZ-1415 and PSB-MZ-1440, relax hPAs and the effect is inhibited by the new GPR18 antagonist PSB-CB-27. GPR18, which appears to exhibit lower activity in hPAs from male, smoking or hypercholesterolemic patients, may become a new target for the treatment of pulmonary arterial hypertension.

Our study aimed to examine the endothelium (vascular)-protecting effects of chronic cannabidiol (CBD) administration (10 mg/kg once daily for 2 weeks) in aortas and small mesenteric (G3) arteries isolated from deoxycorticosterone-induced hypertensive (DOCA-salt) rats and spontaneously hypertensive rats (SHR). CBD reduced hypertrophy and improved the endothelium-dependent vasodilation in response to acetylcholine in the aortas and G3 of DOCA-salt rats and SHR. The enhancement of vasorelaxation was prevented by the inhibition of nitric oxide (NO) with L-NAME and/or the inhibition of cyclooxygenase (COX) with indomethacin in the aortas and G3 of DOCA-salt and SHR, respectively. The mechanism of the CBD-mediated improvement of endothelial function in hypertensive vessels depends on the vessel diameter and may be associated with its NO-, the intermediate-conductance calcium-activated potassium channel- or NO-, COX-, the intermediate and the small-conductance calcium-activated potassium channels-dependent effect in aortas and G3, respectively. CBD increased the vascular expression of the cannabinoid CB 1 and CB 2 receptors and aortic levels of endocannabinoids with vasorelaxant properties e.g., anandamide, 2-arachidonoylglycerol and palmitoyl ethanolamide in aortas of DOCA-salt and/or SHR. In conclusion, CBD treatment has vasoprotective effects in hypertensive rats, in a vessel-size- and hypertension-model-independent manner, at least partly via inducing local vascular changes in the endocannabinoid system.

Currently, no treatment can completely cure pulmonary hypertension (PH), which can lead to right ventricular failure and, consequently, death. Therefore, searching for new therapies remains important. Increased resistance in pulmonary circulation is mainly caused by the excessive contraction and proliferation of small pulmonary arteries. Cannabinoids, a group of lipophilic compounds that all interact with cannabinoid receptors, exert a pulmonary vasodilatory effect through several different mechanisms, including mechanisms that depend on vascular endothelium and/or receptor-based mechanisms, and may also have anti-proliferative and anti-inflammatory properties. The vasodilatory effect is important in regulating pulmonary resistance, which can improve patients' quality of life. Moreover, experimental studies on the effects of cannabidiol (plant-derived, non-psychoactive cannabinoid) in animal PH models have shown that cannabidiol reduces right ventricular systolic pressure and excessive remodelling and decreases pulmonary vascular hypertrophy and pulmonary vascular resistance. Due to the potentially beneficial effects of cannabinoids on pulmonary circulation and PH, in this work, we review whether cannabinoids can be used as an adjunctive therapy for PH. However, clinical trials are still needed to recommend the use of cannabinoids in the treatment of PH.

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB 1 receptor (CB 1 R) expression were elevated. The CB 1 R antagonist AM251 diminished the methanandamide-evoked relaxation only in the sMA of SHR and enhanced the vasoconstriction induced by phenylephrine and the thromboxane analog U46619 in sMA in SHR and WKY. In the sMA of SHR, URB597 elevated anandamide levels, improved the endothelium-dependent vasorelaxation to acetylcholine, and in the presence of AM251 reduced the vasoconstriction to phenylephrine and enhanced the vasodilatation to methanandamide, and tended to reduce hypertrophy. In the aortas, URB597 elevated endocannabinoid levels improved the endothelium-dependent vasorelaxation to acetylcholine and decreased CB 1 R expression. Our study showed that hypertension and chronic administration of URB597 caused local, resistance artery-specific beneficial alterations in the vascular endocannabinoid system, which may bring further advantages for therapeutic application of pharmacological inhibition of FAAH.

Cannabidiol (CBD) is known for its vasorelaxant (including in the human pulmonary artery), anti-proliferative and anti-inflammatory properties. The aim of our study was to examine the potential preventive effect of chronic CBD administration (10 mg/kg/day for three weeks) on monocrotaline (MCT)-induced pulmonary hypertension (PH) rats. PH was connected with elevation of right ventricular systolic pressure; right ventricle hypertrophy; lung edema; pulmonary artery remodeling; enhancement of the vasoconstrictor and decreasing vasodilatory responses; increases in plasma concentrations of tissue plasminogen activator, plasminogen activator inhibitor type 1 and leukocyte count; and a decrease in blood oxygen saturation. CBD improved all abovementioned changes induced by PH except right ventricle hypertrophy and lung edema. In addition, CBD increased lung levels of some endocannabinoids (anandamide, N -arachidonoyl glycine, linolenoyl ethanolamide, palmitoleoyl ethanolamide and eicosapentaenoyl ethanolamide but not 2-arachidonoylglycerol). CBD did not affect the cardiopulmonary system of control rats or other parameters of blood morphology in PH. Our data suggest that CBD ameliorates MCT-induced PH in rats by improving endothelial efficiency and function, normalization of hemostatic alterations and reduction of enhanced leukocyte count determined in PH. In conclusion, CBD may be a safe, promising therapeutic or adjuvant therapy agent for the treatment of human pulmonary artery hypertension.

The aim of this study was to investigate the hemodynamic effects of SKA-31, an activator of the small ( K Ca 2.x ) and intermediate ( K Ca 3.1 ) conductance calcium-activated potassium channels, and to evaluate its influence on endothelium-derived hyperpolarization (EDH)- K Ca 2.3 / K Ca 3.1 type relaxation in isolated endothelium-intact small mesenteric arteries (sMAs) from spontaneously hypertensive rats (SHRs). Functional in vivo and in vitro experiments were performed on SHRs or their normotensive controls, Wistar-Kyoto rats (WKY). SKA-31 (1, 3 and 10 mg/kg) caused a brief decrease in blood pressure and bradycardia in both SHR and WKY rats. In phenylephrine-pre-constricted sMAs of SHRs, SKA-31 (0.01-10 µM)-mediated relaxation was reduced and SKA-31 potentiated acetylcholine-evoked endothelium-dependent relaxation. Endothelium denudation and inhibition of nitric oxide synthase (eNOS) and cyclooxygenase (COX) by the respective inhibitors l -NAME or indomethacin, attenuated SKA-31-mediated vasorelaxation. The inhibition of K Ca 3.1 , K Ca 2.3 , K IR and Na + /K + -ATPase by TRAM-34, UCL1684, Ba 2+ and ouabain, respectively, reduced the potency and efficacy of the EDH-response evoked by SKA-31. The mRNA expression of eNOS, prostacyclin synthase, K Ca 2.3 , K Ca 3.1 and K IR were decreased, while Na + /K + -ATPase expression was increased. Collectively, SKA-31 promoted hypotension and vasodilatation, potentiated agonist-stimulated vasodilation, and maintained K Ca 2.3 / K Ca 3.1 -EDH-response in sMAs of SHR with downstream signaling that involved K IR and Na + /K + -ATPase channels. In view of the importance of the dysfunction of endothelium-mediated vasodilatation in the mechanism of hypertension, application of activators of K Ca 2.3 / K Ca 3.1 channels such as SKA-31 seem to be a promising avenue in pharmacotherapy of hypertension., Competing Interests: The authors declare that there are no conflicts of interest.

Recent evidence suggests that endocannabinoids acting via cannabinoid CB 1 receptors may modulate vascular responses of various vasoconstrictors in the rodent systemic vasculature. The aim of the study was to investigate whether endocannabinoids modulate the contractile responses evoked by a thromboxane A 2 analog (U46619), angiotensin II (ANG II), serotonin (5-HT), and phenylephrine, which stimulate distinct G q/11 protein-coupled receptors (thromboxane, ANG II type 1, 5-HT 2 , and α 1 -adrenergic receptors) in isolated endothelium-intact human and rat pulmonary arteries (hPAs and rPAs, respectively). The CB 1 receptor antagonist AM251 (1 μM) and diacylglycerol lipase (2-arachidonoylglycerol synthesis enzyme) inhibitor RHC80267 (40 μM) enhanced contractions induced by U46619 in hPAs and rPAs and by ANG II in rPAs in an endothelium-dependent manner. AM251 did not influence vasoconstrictions induced by 5-HT or phenylephrine in rPAs. The monoacylglycerol lipase (2-arachidonoylglycerol degradation enzyme) inhibitor JZL184 (1 μM), but not the fatty acid amide hydrolase (anandamide degradation enzyme) inhibitor URB597 (1 μM), attenuated contractions evoked by U46619 in hPAs and rPAs and ANG II in rPAs. 2-Arachidonoylglycerol concentration-dependently induced relaxation of hPAs, which was inhibited by endothelium denudation or AM251 and enhanced by JZL184. Expression of CB 1 receptors was confirmed in hPAs and rPAs using Western blotting and immunohistochemistry. The present study shows the protective interaction between the endocannabinoid system and vasoconstriction in response to U46619 and ANG II in the human and rat pulmonary circulation. U46619 and ANG II may stimulate rapid endothelial release of endocannabinoids (mainly 2-arachidonoylglycerol), leading to CB 1 receptor-dependent and/or CB 1 receptor-independent vasorelaxation, which in the negative feedback mechanism reduces later agonist-induced vasoconstriction., (Copyright © 2017 the American Physiological Society.)

Background: Whole-cell-based vaccines modified with Hyper-IL-6 (H6) and Hyper-IL-11 (H11) have demonstrated high activity in murine melanoma and renal cancer models., Materials and Methods: H6 and H11 cDNA was transduced into TRAMP cells (TRAMP-H6 and TRAMP-H11). An orthotopic TRAMP model was employed. The efficacy of TRAMP-H6 and TRAMP-H11 in combination with docetaxel was evaluated. Immune cells infiltrating tumors were assessed., Results: Immunization with TRAMP-H6 and TRAMP-H11 vaccines extended OS of mice. Addition of docetaxel to TRAMP-H6 and TRAMP-H11 vaccines further extended OS of the animals. Vaccination with TRAMP-H6 alone and TRAMP-H11 combined with docetaxel augmented tumor infiltration by activated CD8(+) and CD4(+) T-cells and attracted higher number of activated, mature DCs infiltrating tumors. Addition of docetaxel to TRAMP-H6, TRAMP-H11, TRAMP-Adv700 vaccines enhanced the infiltration of the tumor by NK cells., Conclusion: Addition of docetaxel to modified TRAMP vaccines improved clinical benefit of treated mice and enhanced anti-tumor immune response., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: The aim of our study was (1) the pharmacological characterization of EP(3) receptors in human pulmonary arteries and (2) the examination of the potential involvement of these receptors in the regulation of neurogenic tachycardia in pithed rats. L-826266 served as the EP(3) receptor antagonist., Methods: Experiments were performed on isolated human pulmonary arteries and pithed rats., Results: The prostanoid EP(1)/EP(3) receptor agonist sulprostone (1 nM - 100 μM) concentration-dependently contracted isolated human pulmonary arteries (pEC50, 6.88 ± 0.10). The EP(1) receptor antagonist SC 19920 (100 μM) did not affect the vasoconstriction induced by sulprostone, the TP receptor antagonist sulotroban (10 μM) only slightly attenuated the effects elicited by sulprostone >>3 μM, whereas L-826266 (10 μM) shifted its concentration-response curve to the right (apparent pA(2) value 6.18; incubation time 0.5 h). In rings exposed to L-826266 (0.1, 1 or 10 μM) for 3 h, a concentration-dependent inhibitory effect against the sulprostone-induced vasoconstriction was obtained, yielding a Schild plot-based pA(2) value of 7.39. In pithed rats, sulprostone (10 - 1,000 nmol/kg), but not the IP/EP(1) receptor agonist iloprost (1-100 nmol/kg), inhibited the electrically evoked increase in heart rate (HR) dose-dependently, maximally by at least 80%. L-826266 (3 μmol/kg) did not affect basal HR and diastolic blood pressure, but reduced the inhibitory effect of sulprostone 1,000 nmol/kg by about 20%., Conclusion: EP(3) receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibers supplying the heart of pithed rats strongly inhibit the neurogenic tachycardia.

Background: The area of vascular supply of particular coronary arteries is directly linked to the varying typology of the coronary vasculature. This factor may have a significant influence on the coronary vessel diameters. To date there has been no published research that analyses the relationship between the type of coronary vasculature and the dimensions of the epicardial arteries in the human foetus. There are only a few papers that deal with this issue in the postnatal period of human life., Material and Methods: The study was carried out on a group of 187 human foetuses aged five to seven months of intrauterine life. Prior to examination all foetuses had been conserved in a 9% formaldehyde solution for a minimum of three months. All foetuses had been aborted naturally. None of them had any external signs of malformations or developmental abnormalities. The number of foetuses in the particular age groups was variable. Adachi/Bianchi classification was used to categorize the particular vasculature types: type I -- classic, neither artery is dominating; type II -- dominant right coronary artery; type III -- dominant left coronary artery., Results and Conclusions: The analysis of differences between the artery dimensions in particular types of coronary vasculature revealed that such differences existed between types I and II and also between types II and III.

Background and Purpose: Intravenous injection of the endocannabinoid anandamide induces complex cardiovascular changes via cannabinoid CB(1), CB(2) and vanilloid TRPV1 receptors. Recently, evidence has been accumulating that in vitro, but not in vivo, anandamide relaxes blood vessels, via an as yet unidentified, non-CB(1) vascular cannabinoid receptor, sensitive to O-1918 (1,3-dimethoxy-5-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-benzene). We here examined whether the anandamide-induced hypotension in urethane-anaesthetized rats was also mediated via a non-CB(1) vascular cannabinoid receptor., Experimental Approach: Effects of two antagonists (O-1918 and cannabidiol) of the non-CB(1) vascular cannabinoid receptor on anandamide-induced changes in mean, systolic and diastolic blood pressure (MBP, SBP, DBP), mesenteric (MBF) and renal (RBF) blood flow and heart rate (HR) in urethane-anaesthetized rats was examined., Key Results: In anaesthetized rats, anandamide (1.5-3 micromol.kg(-1)) and its stable analogue methanandamide (0.5 micromol.kg(-1)) caused a delayed and prolonged decrease in MBP, SBP, DBP, MBF and RBF by about 10-30% of the respective basal values without changing HR. In pithed rats, anandamide (3 micromol.kg(-1)) decreased blood pressure by about 15-20% of the basal value without affecting HR, MBF and RBF. All vascular changes were reduced by about 30-70% by cannabidiol and O-1918 (3 micromol.kg(-1), each)., Conclusions and Implications: Non-CB(1) cannabinoid vascular receptors, sensitive to O-1918, contribute to the hypotensive effect of anandamide in anaesthetized rats. Activation of these receptors may be therapeutically important as the endocannabinoid system could be activated as a compensatory mechanism in various forms of hypertension.

The morphometrical and volumetrical changes of the middle cerebral artery (MCA) during the fetal period of development were analyzed by digital-image analysis system (DIAS). Examinations were performed on 304 MCAs from 152 brains of human fetuses ranging from the 12th to 40th weeks of gestation. MCAs were analyzed with respect to its branching from the internal carotid artery and its division into the main cortical branches. No statistically significant differences were found between the mean values of the diameter, length and volume of the left and right M1 segments of the MCAs in all studied age groups.

The aim of our study was to examine whether non beta(1)-/beta(2)-adrenoceptors participate in the relaxation of the human pulmonary artery. For this purpose the vasodilatory effect of the non-conventional partial beta-adrenoceptor agonist cyanopindolol was examined. Cyanopindolol (1-300 microM), studied in the presence of the beta(1)-/beta(2)-adrenoceptor antagonist propranolol, relaxed the human pulmonary artery preconstricted with serotonin 1 microM in a concentration-dependent manner (maximally by about 80%). This effect was diminished by bupranolol 10 microM (an antagonist of beta(1)-beta(3)-adrenoceptors and the low affinity state of the beta(1)-adrenoceptor) and CGP 20712 10 microM (known to antagonize the low-affinity state of the beta(1)-adrenoceptor at high concentrations). In further experiments, the effect of beta-adrenoceptor ligands on the serotonin-induced vasoconstriction was examined. The concentration-response curve for serotonin was not affected by cyanopindolol 30 microM, bupranolol 10 microM and CGP 20712 10 microM but shifted to the right by cyanopindolol 100 and 300 microM; the serotonin 5-HT(2A) receptor antagonist ketanserin 0.3 microM abolished the maximum contraction elicited by serotonin. In conclusion, the present study reveals that the vasodilatory effect of cyanopindolol in the human pulmonary artery consists of two components, i.e. activation of a propranolol-insensitive atypical beta-adrenoceptor and antagonism against 5-HT(2A) receptors.

1 Activation by CGP 12177 and cyanopindolol of the human and rat low-affinity state of beta(1)-adrenoceptors increases frequency and contractile force and hastens relaxation in isolated cardiac tissues, and probably relaxes isolated vessels. In order to identify the positive inotropic, positive lusitropic and vasodilator effects of both agonists also in vivo, we have determined their effects on the left ventricular systolic pressure (LVSP), the rate of intraventricular pressure rise (+dP dt(-1)(max)) and decline (-dP dt(-1)(max)), the diastolic blood pressure (DBP) and the mesenteric blood flow (MBF) in pithed and vagotomized rats. 2 CGP 12177 (0.1-100 nmol kg(-1)) and cyanopindolol (1-1000 nmol kg(-1)) dose-dependently enhanced all cardiac parameters. The nonselective beta-adrenoceptor antagonist bupranolol 10 micromol kg(-1) diminished the CGP 12177 (100 nmol kg(-1))-stimulated increases in LVSP from 26.3+/-8.2 to 13.1+/-1.8 mmHg (P<0.05), +dP dt(-1)(max) from 5287+/-290 to 2439+/-296 mmHg s(-1) (P<0.001) and -dP dt(-1)(max) from -3836+/-301 to -2187+/-443 mmHg s(-1) (P<0.05), respectively. The beta(1)-adrenoceptor antagonist CGP 20712A 10 micromol kg(-1) (known to block the low-affinity state of beta(1)-adrenoceptors at high doses) inhibited increases in +/-dP dt(-1)(max) elicited by the highest dose of CGP 12177. 3 The highest doses of CGP 12177 and cyanopindolol increased DBP by about 10 mmHg and MBF by 1.4+/-0.3 and 0.6+/-0.3 ml min(-1), respectively. The vascular effects of CGP 12177 were not affected by bupranolol and CGP 20712A. 4 In conclusion, activation of the low-affinity state of beta(1)-adrenoceptors by CGP 12177 and cyanopindolol in pithed rats causes a positive inotropic and lusitropic effect. By contrast, the vascular effects of CGP 12177 and cyanopindolol are not mediated by these receptors and have only marginal influence under in vivo conditions.

We examined whether the beta2-adrenoceptor agonists fenoterol and salbutamol, the beta3-adrenoceptor agonists CL 316243 and ZD 2079, and the agonists of the low-affinity state of beta-adrenoceptors, cyanopindolol and CGP 12177 block alpha1-adrenoceptors in that concentration range in which they relax the human pulmonary and rat mesenteric arteries preconstricted with phenylephrine 10 microM and 1 microM, respectively. For quantification of vasodilatation pEC25 values and for the antagonism toward alpha1-adrenoceptors, pA2 values were determined. We found that in the rat mesenteric artery, (1) the pEC25 values of the beta-adrenoceptor ligands resemble their respective pA2 values (difference < or = 0.9 log units), and (2) the order of potencies is the same for both parameters, ie, cyanopindolol approximately fenoterol > CGP 12177 > salbutamol > ZD 2079 > CL 316243. In the human pulmonary artery, (1) the pEC25 values are slightly lower (by 0.6-1.3 log units) than their respective pA2 values, and (2) the rank order of potencies is the same for both parameters. In conclusion, the present study suggests that ligands of beta2-adrenoceptors and of non-beta1-non-beta2-adrenoceptors relax rat and human vessels preconstricted with phenylephrine or norepinephrine mainly through their alpha1-adrenolytic effects. Hence, for the investigation of the role of beta-adrenoceptors in vessels, the constrictor agent should be chosen with great caution.

S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.

(1) We examined whether beta3- and/or atypical beta-adrenoceptors relax the rat isolated mesenteric artery. (2) Mesenteric arteries precontracted with phenylephrine were relaxed by beta-agonists with the following potencies (pD2): nonselective agonist isoprenaline (6.00)>nonconventional partial agonist cyanopindolol (5.45)>beta2-agonist fenoterol (4.98)>nonconventional partial agonist CGP 12177 (4.19)>beta3-agonist ZD 2079 (3.72). The beta3-agonist CL 316243 1 mm relaxed the vessel only marginally. (3) The concentration-response curves (CRCs) for cyanopindolol, CGP 12177 and ZD 2079 were not affected by the nonselective beta-antagonist propranolol 0.3 microm, the beta2-antagonist ICI 118551 1 microm and by CL 316243 60 microm, but shifted to the right by bupranolol (pA2 5.3-5.7), CGP 20712 (5.4) and SR 59230A (6.5-6.7) (the latter three drugs block atypical and/or beta3-adrenoceptors at high concentrations). (4) The CRC for isoprenaline was shifted to the right by propranolol (pA2 7.0) but, in the presence of propranolol 0.3 microm, not affected by SR 59230A 1 microm. The CRC for fenoterol was shifted to the right by propranolol (pA2 6.9) and ICI 118551 (6.8). (5) Removal of endothelium diminished the vasorelaxant effects of cyanopindolol, CGP 12177 and ZD 2079. (6) Fenoterol and cyanopindolol also relaxed (endothelium-intact) mesenteric arteries precontracted with serotonin. The relaxant effect of cyanopindolol was antagonized by bupranolol to about the same degree as in phenylephrine-contracted vessels. (7) In conclusion, beta2- and atypical beta-adrenoceptors (but not beta3-adrenoceptors) relax the rat mesenteric artery. The atypical beta-adrenoceptor, which is partially located endothelially, may differ from the low-affinity state of the beta1-adrenoceptor.

1. Atypical beta-adrenoceptors resistant to propranolol, but blocked by bupranolol, increase contractile force and/or frequency of the heart in humans and rats. We compared the potencies of the enantiomers of bupranolol and examined the possible effects of seven bupranolol analogues including bevantolol (BEV) at this receptor in pithed and vagotomized rats. 2. CGP 12177, an agonist of the atypical beta-adrenoceptor, increased heart rate dose-dependently. Its dose-response curve was shifted to the right by S-(-)-bupranolol 10 micro mol kg(-1) by a factor of 8.4, but not affected by the same dose of R-(+)-bupranolol. 3. Desmethylbupranolol and compounds BK-21, BK-22, BK-23 and BK-25 also increased heart rate dose-dependently. The beta(1)-adrenoceptor antagonist CGP 20712 given in combination with the beta(2)-adrenoceptor antagonist ICI 118,551 (0.1 micro mol kg(-1) each) reduced the positive chronotropic action of the five bupranolol analogues without affecting that of CGP 12177. The potencies of the bupranolol analogues to increase heart rate were correlated (r=0.91, P<0.05) with their affinities for beta(1)-adrenoceptor binding sites in rat brain cortex membranes labelled with [(3)H]CGP 12177 (in the presence of ICI 118,551). 4. BK-26 and BEV, 10 micro mol kg(-1) each, had only minor effects on heart rate by themselves and did not antagonize the effect of CGP 12177. However, at 1 micro mol kg(-1), they antagonized the increase in heart rate elicited by the beta(1)-adrenoceptor agonist prenalterol. 5. In conclusion, bupranolol is a stereoselective antagonist at the atypical cardiostimulant beta-adrenoceptor. The effects of the bupranolol analogues are related to the activation or blockade of beta(1)-adrenoceptors, but not of atypical beta-adrenoceptors.