Design of Mixed Medicinal Plants, Rich in Polyphenols, Vitamins B, and Palmitoylethanolamide-Based Supplement to Help Reduce Nerve Pain: A Preclinical Study.

Neuropathy affects 7-10% of the general population and is caused by a lesion or disease of the somatosensory system. The limitations of current therapies highlight the necessity of a new innovative approach to treating neuropathic pain (NP) based on the close correlation between oxidative stress, inflammatory process, and antioxidant action. The advantageous outcomes of a novel combination composed of Hop extract, Propolis, Ginkgo Biloba, Vitamin B, and palmitoylethanolamide (PEA) used as a treatment was evaluated in this study. To assess the absorption and biodistribution of the combination, its bioavailability was first examined in a 3D intestinal barrier model that replicated intestinal absorption. Further, a 3D nerve tissue model was developed to study the biological impacts of the combination during the essential pathways involved in NP. Our findings show that the combination could cross the intestinal barrier and reach the peripheral nervous system, where it modulates the oxidative stress, inflammation levels, and myelination mechanism (increased NRG, MPZ, ERB, and p75 levels) under Schwann cells damaging. This study proves the effectiveness of Ginkgo Biloba, Propolis, Hop extract, Vitamin B, and PEA in avoiding nerve damage and suggests a potential alternative nutraceutical treatment for NP and neuropathies.

The Combined Antioxidant Effects of N-Acetylcysteine, Vitamin D3, and Glutathione from the Intestinal-Neuronal In Vitro Model.

Chronic oxidative stress has been consistently linked to age-related diseases, conditions, and degenerative syndromes. Specifically, the brain is the organ that significantly contributes to declining quality of life in ageing. Since the body cannot completely counteract the detrimental effects of oxidative stress, nutraceuticals' antioxidant properties have received significant attention in recent years. This study assesses the potential health benefits of a novel combination of glutathione, vitamin D3, and N-acetylcysteine. To examine the combination's absorption and biodistribution and confirm that it has no harmful effects, the bioavailability of the mixture was first evaluated in a 3D model that mimicked the intestinal barrier. Further analyses on the blood-brain barrier was conducted to determine the antioxidant effects of the combination in the nervous system. The results show that the combination reaches the target and successfully crosses the blood-brain and intestinal barriers, demonstrating enhanced advantages on the neurological system, such as a reduction (about 10.5%) in inflammation and enhancement in cell myelination (about 20.4%) and brain tropism (about 18.1%) compared to the control. The results support the cooperative effect of N-acetylcysteine, vitamin D3, and glutathione to achieve multiple health benefits, outlining the possibility of an alternative nutraceutical approach.

Developing New Cyclodextrin-Based Nanosponges Complexes to Improve Vitamin D Absorption in an In Vitro Study.

Vitamin D plays an important role in numerous cellular functions due to the ability to bind the Vitamin D receptor (VDR), which is present in different tissues. Several human diseases depend on low vitamin D3 (human isoform) serum level, and supplementation is necessary. However, vitamin D3 has poor bioavailability, and several strategies are tested to increase its absorption. In this work, the complexation of vitamin D3 in Cyclodextrin-based nanosponge (CD-NS, in particular, ßNS-CDI 1:4) was carried out to study the possible enhancement of bioactivity. The ßNS-CDI 1:4 was synthesized by mechanochemistry, and the complex was confirmed using FTIR-ATR and TGA. TGA demonstrated higher thermostability of the complexed form. Subsequently, in vitro experiments were performed to evaluate the biological activity of Vitamin D3 complexed in the nanosponges on intestinal cells and assess its bioavailability without cytotoxic effect. The Vitamin D3 complexes enhance cellular activity at the intestinal level and improve its bioavailability. In conclusion, this study demonstrates for the first time the ability of CD-NS complexes to improve the chemical and biological function of Vitamin D3.

Ovotransferrin Supplementation Improves the Iron Absorption: An In Vitro Gastro-Intestinal Model.

Transferrins constitute the most important iron regulation system in vertebrates and some invertebrates. Soluble transferrins, such as bovine lactoferrin and hen egg white ovotransferrin, are glycoproteins with a very similar structure with lobes that complex with iron. In this in vitro study, a comparison of bovine lactoferrin and ovotransferrin was undertaken to confirm the comparability of biological effects. An in vitro gastric barrier model using gastric epithelial cells GTL-16 and an in vitro intestinal barrier model using CaCo-2 cells was employed to evaluate iron absorption and barrier integrity. An analysis of the molecular pathways involving DMT-1 (divalent metal transporter 1), ferritin and ferroportin was also carried out. These in vitro data demonstrate the activity of both 15% saturated and 100% saturated ovotransferrin on the iron regulation system. Compared with the commercial bovine lactoferrin, both 15% saturated and 100% saturated ovotransferrin were found to act in a more physiological manner. Based on these data, it is possible to hypothesise that ovotransferrin may be an excellent candidate for iron supplementation in humans; in particular, 15% saturated ovotransferrin is the overall best performing product. In vivo studies should be performed to confirm this in vitro data.

A New Palmitoylethanolamide Form Combined with Antioxidant Molecules to Improve Its Effectivess on Neuronal Aging.

Palmitoylethanolamide is a nutraceutical compound naturally produced in many plants and animal source foods, but the natural form is poorly water-soluble. It has demonstrated an anti-inflammatory role as a neuroprotective mediator, acting on several molecular targets of the central nervous system involved on brain aging process. In healthy adults, palmitoylethanolamide is an endogenous PPAR-α (peroxisome proliferator-activated receptor α) agonist through which it performs anti-inflammatory activity and provides its effects by activating the cannabinoid receptor. The different formulations of palmitoylethanolamide (micronized palmitoylethanolamide, FM-LipoMatrix® palmitoylethanolamide and FM-LipoMatrix® palmitoylethanolamide plus lipoic acid and vitamin D3) were analyzed starting from intestinal barrier, to verify their bioavailability, to in primary astrocytes in which cell viability, reactive oxygen species (ROS) and nitric oxide (NO) production, NFKB activity, MAPK, p53 and PPARα activities were investigated. Additionally, cannabinoid and estrogen receptors were analyzed using the western blot technique. The combination of palmitoylethanolamide in FM-LipoMatrix®, lipoic acid and vitamin D3 shows better absorption predicting an improvement on plasma concentration; this formulation also shows a reduction in ROS and NO production and the data show the interaction of palmitoylethanolamide with cannabinoids and estrogen receptors inhibiting neuroinflammatory markers. All these data support the hypothesis of a new potential strategy to restore brain function and slow down brain aging in humans.

Role of Combined Lipoic Acid and Vitamin D3 on Astrocytes as a Way to Prevent Brain Ageing by Induced Oxidative Stress and Iron Accumulation.

Brain ageing is a complex multifactorial process characterized by gradual and continuous loss of neuronal functions. It is hypothesized that at the basis of brain ageing as well as age-related diseases, there is an impairment of the antioxidant defense system leading to an increase of oxidative stress. In this study, two different biological aspects involved in brain ageing and neurodegeneration have been investigated: oxidative stress and iron accumulation damage. In primary mouse astrocytes, the stimulation with 50 µM lipoic acid (LA) and 100 nM vitamin D (vitD) was first investigated in a time-course study to determine the dosages to be used in combination and then in a permeability test using an in vitro blood-brain barrier. In a second set of experiments, the role of oxidative stress was investigated pretreating astrocytes with 200 µM H2O2 for 30 min. The ability of vitD and LA alone and combined together to prevent or repair the damage caused by oxidative stress was investigated after 24 h of stimulation by the MTT test, mitochondrial membrane potential measurement, and Western blot analysis. To induce neurodegeneration, cells were pretreated with 300 µM catalytic iron for 6 days and then treated with vitD and LA alone and combined for additional 6 days to investigate the protection exerted by combination, analyzing viability, ROS production, iron concentration, and activation of intracellular pathways. In our study, the combination of LA and vitD showed beneficial effects on viability of astrocytes, since the substances are able to cross the brain barrier. In addition, combined LA and vitD attenuated the H2O2-induced apoptosis through the mitochondrial-mediated pathway. The combination was also able to counteract the adverse conditions caused by iron, preventing its accumulation. All these data support the hypothesis of the synergistic and cooperative activity exerted by LA and vitD in astrocytes indicating a possible new strategy to slow down ageing.

Cooperative Effects of Q10, Vitamin D3, and L-Arginine on Cardiac and Endothelial Cells.

This work demonstrates the cooperative effect of Q10, vitamin D3, and L-arginine on both cardiac and endothelial cells. The effects of Q10, L-arginine, and vitamin D3 alone or combined on cell viability, nitric oxide, and reactive oxygen species productions in endothelial and cardiac cells were studied. Moreover, the involvement of PI3K/Akt and ERK/MAPK pathways leading to eNOS activation as well as the involvement of vitamin D receptor were also investigated. The same agents were tested in an animal model to verify vasodilation, nitric oxide, and reactive oxygen species production. The data obtained in this work demonstrate for the first time the beneficial and cooperative effect of stimulation with Q10, L-arginine, and vitamin D3. Indeed, in cardiac and endothelial cells, Q10, L-arginine, and vitamin D3 combined were able to induce a nitric oxide production higher than the that induced by the 3 substances alone. The effects on vasodilation induced by cooperative stimulation have been confirmed in an in vivo model as well. The use of a combination of Q10, L-arginine, and vitamin D to counteract increased free radical production could be a potential method to reduce myocardial injury or the effects of aging on the heart.

Iron Absorption from Three Commercially Available Supplements in Gastrointestinal Cell Lines.

This study compares the absorption characteristics of two iron-based dietary supplements and their biocompatibility to bisglycinate iron, a common chelated iron form. The Caco-2 cell line-a model of human intestinal absorption-and GTL-16 cell line-a model of gastric epithelial cells-were used to perform the experiments; in the first experiments, the kinetics of absorption have been evaluated analyzing the divalent metal transporter 1 (DMT1) expression. Three different iron combinations containing 50 µM iron (named Fisioeme®, Sideral® and bisglycinate) were used for different stimulation times (1-24 h). After this, the effects of the three iron formulations were assessed in both a short and a long time, in order to understand the extrusion mechanisms. The effects of the three different formulations were also analyzed at the end of stimulation period immediately after iron removal, and after some time in order to clarify whether the mechanisms were irreversibly activated. Findings obtained in this study demonstrate that Fisioeme® was able to maintain a significant beneficial effect on cell viability compared to control, to Sideral®, and to iron bisglycinate. This observation indicates that Fisioeme® formulation is the most suitable for gastric and intestinal epithelial cells.

Biological effects of combined resveratrol and vitamin D3 on ovarian tissue.

BACKGROUND: Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural antioxidant polyphenol able to exert a wide range of biological effect on several tissues. Despite its important beneficial properties, it has a low water solubility, which limits its therapeutic applications in humans. Resveratrol also acts as a phytoestrogen that modulates estrogen receptor (ER)-mediated transcription. In addition, it has been shown that ovarian tissues benefit greatly from vitamin D3, which exerts its beneficial effects through VDR receptors. The aim was to evaluate the cooperative effects of resveratrol combined with vitamin D3 on ovarian cells and tissues and some other organs as well. Moreover, the modulation of specific intracellular pathways involving ER and VDR receptors has been studied. METHODS: The experiments were performed both in vitro and in vivo, to analyze cell viability, radical oxygen species production, signal transductions through Western Blot, and resveratrol quantification by HPLC. RESULTS: Cell viability, radical oxygen species production, and intracellular pathways have been studied on CHO-K1 cells. Also, the relative mechanism activated following oral intake in female Wistar rats as animal model was investigated, evaluating bioavailability, biodistribution and signal transduction in heart, kidney, liver and ovarian tissues. Both in in vitro and in vivo experiments, resveratrol exerts more evident effects when administered in combination with vitD in ovarian cells, showing a common biphasic cooperative effect: The role of vitamin D3 in maintaining and supporting the biological activity of resveratrol has been clearly observed. Moreover, resveratrol plus vitamin D3 blood concentrations showed a biphasic absorption rate. CONCLUSIONS: Such results could be used as a fundamental data for the development of new therapies for gynecological conditions, such as hot-flashes.

Role of vitamin D3 combined to alginates in preventing acid and oxidative injury in cultured gastric epithelial cells.

BACKGROUND: Gastric diseases are a worldwide problem in modern society, as reported in the USA, in the range of 0.5-2 episodes/year/person and an incidence of 5-100 episodes/1000/week according to seasons and age. There is convincing evidence that oxidative stress is involved in the pathogenesis of acute gastric injury. Acid secreted from gastric parietal cells determines mucosal injuries which in turn cause inflammation and oxidative stress. Consequent inflammation produces free radicals by mitochondria thus causing lipid peroxidation, oxidative and acidic stress, which can lead to cell apoptosis. Vitamin D3, the active form of vitamin D, may counteract intracellular cell death and improve epithelial regeneration. METHODS: This study was planned to assess whether vitamin D3 is a protective factor against acid injury and oxidative stress in gastric epithelial cells. Primary epithelial cells and GTL-16 cells have been used to test the effects of Grisù® alone or in combination with vitamin D3 during oxidative stress or high acid exposition measuring cell viability, ROS production, cellular adhesion time along with apoptotic, autophagic and survival pathways. The combined effect of Grisù® and vitamin D3 was found more effective in counteracting the negative consequences of oxidative stress and acidity conditions than some other gastroprotective agents, such as Maalox® or Gaviscon®. RESULTS: In case of oxidative stress or acidity condition the stimulation with Grisù® alone caused an improvement of cell viability and a reduction of ROS production on epithelial gastric cells. In addition, the adhesion time of the cells was improved. All these effects were increased by the presence of vitamin D3. Similar data were also observed in primary gastric epithelial cells confirming the results obtained in GTL-16 cells. CONCLUSIONS: These results suggest that Grisù® in combination with vitamin D3 may exert a gastroprotective effect to maintain or restore the integrity of gastric epithelium through an antioxidant pathway, inhibiting apoptosis and activating survival kinases. Moreover, the combination of Grisù® and vitamin D3 improves cell viability and decreases ROS production compared to other gastroprotective agents combined with vitamin D3. All these data were validated using primary cells isolated from gastric tissue.

Protective effects of 1α,25-Dihydroxyvitamin D3 on cultured neural cells exposed to catalytic iron.

Recent studies have postulated a role for vitamin D and its receptor on cerebral function, and anti-inflammatory, immunomodulatory and neuroprotective effects have been described; vitamin D can inhibit proinflammatory cytokines and nitric oxide synthesis during various neurodegenerative insults, and may be considered as a potential drug for the treatment of these disorders. In addition, iron is crucial for neuronal development and neurotransmitter production in the brain, but its accumulation as catalytic form (Fe(3+)) impairs brain function and causes the dysregulation of iron metabolism leading to tissue damage due to the formation of toxic free radicals (ROS). This research was planned to study the role of vitamin D to prevent iron damage in neuroblastoma BE(2)M17 cells. Mechanisms involved in neurodegeneration, including cell viability, ROS production, and the most common intracellular pathways were studied. Pretreatment with calcitriol (the active form of vitamin D) reduced cellular injury induced by exposure to catalytic iron.

Protective effects of vitamin D3 on fimbrial cells exposed to catalytic iron damage.

BACKGROUND: Recently, vitamin D3 (1alpha, 25-dihydroxyvitamin D) has shown its capability to take part in many extraskeletal functions and its serum levels have been related to patient survival rate and malignancy of many types of neoplasms, including ovarian cancers. Catalytic iron is a free circulating form of iron that is able to generate reactive oxygen species and consequently to promote a number of cellular and tissutal dysfunctions including tumorigenesis. In fertile women an important source of catalytic iron is derived from retrograde menstruation. Epithelial secretory cells from fimbriae of fallopian tubes are greatly exposed to catalytic iron derived from menstrual reflux and so represent the site of origin for most serous ovarian cancers. The aim of this study was to assess whether vitamin D3 can play a role in counteracting catalytic iron-induced oxidative stress in cells from fimbriae of fallopian tubes. METHODS: The cells, isolated from women undergoing isteroannessiectomy, were treated with catalytic iron 50-75-100 mM and vitamin D3 at a concentration ranging from 0.01 to 10 nM to study cell viability, radical oxygen species production, p53, pan-Ras, Ki67 and c-Myc protein expressions through Western Blot, and immunocytochemistry or immunofluorescence analysis. RESULTS: The pre-treatment with vitamin D3 1 nM showed its beneficial effects that consists in a significant decrease in ROS production. In addition a novel finding is represented by the demonstration that pre-treatment with vitamin D3 is also able to significantly counteract tumoral biomarkers activation, such as p53, pan-Ras, Ki67 and c-Myc, and consequently the catalytic iron-induced cellular injury. CONCLUSIONS: This study demonstrates for the first time that vitamin D3 plays an important role in preventing catalytic iron-dependent oxidative stress in cultured fimbrial cells. These results support the hypothesis that vitamin D3 could counteract carcinogenic changes induced by catalytic iron.

Essential Oil from Berries of Lebanese Juniperus excelsa M. Bieb Displays Similar Antibacterial Activity to Chlorhexidine but Higher Cytocompatibility with Human Oral Primary Cells.

Chlorhexidine (CHX), one of the most effective drugs administered for periodontal treatment, presents collateral effects including toxicity when used for prolonged periods; here, we have evaluated the bactericidal potency and the cytocompatibility of Juniperus excelsa M. Bieb essential oil (EO) in comparison with 0.05% CHX. The EO was extracted from berries by hydrodistillation and components identified by gas chromatography and mass spectrometry. Bacterial inhibition halo analysis, quantitative cell viability 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenyl amino) carbonyl]-2H-tetrazolium hydroxide assay (XTT), and colony forming unit (CFU) count were evaluated against the two biofilm formers Aggregatibacter actinomycetemcomitans and Streptococcus mutans. Finally, cytocompatibility was assessed with human primary gingival fibroblasts (HGF) and mucosal keratinocytes (HK). The resulting EO was mainly composed of monoterpene hydrocarbons and oxygenated monoterpenes. An inhibition halo test demonstrated that both bacteria were sensitive to the EO; XTT analysis and CFU counts confirmed that 10-fold-diluted EO determined a statistically significant (p < 0.05) reduction in bacteria count and viability towards both biofilm and planktonic forms in a comparable manner to those obtained with CHX. Moreover, EO displayed higher cytocompatibility than CHX (p < 0.05). In conclusion, EO exhibited bactericidal activity similar to CHX, but a superior cytocompatibility, making it a promising antiseptic alternative to CHX.

Molecular characterization of allergens in raw and processed kiwifruit.

BACKGROUND: The prevalence of allergy to kiwifruit is increasing in Europe since the last two decades. Different proteins have been identified as kiwifruit allergens; even though with geographic differences, Act d 1, a cysteine protease protein of 30 kDa, and Act d 2, a thaumatin-like protein of 24 kDa, are normally considered the most important. The aim of this study was (i) to identify at molecular level the sensitization pattern in a group of well-characterized patients allergic to kiwifruit and (ii) to assess the role of technological treatments on kiwifruit allergenic potential. METHODS: The differences in the pattern of antigenicity between fresh and processed kiwifruit were evaluated by both immunoelectrophoretic techniques and clinical tests. RESULTS: In the group of patients included in this study, three proteins were identified as major allergens in fresh kiwifruit, as the specific sensitization was present in ≥50% of the subjects. These proteins corresponded to actinidin (Act d 1), pectin methyl aldolase (Act d 6), and thaumatin-like protein (Act d 2). Kiwellin (Act d 5) and proteins of Bet v 1 family (Act d 8/act d 11) were also recognized as minor allergens. Immunoreactivity was totally eliminated by industrial treatments used for the production of kiwifruit strained derivative. CONCLUSIONS: In this group of allergic children, the technological treatments used in the production of kiwifruit strained product reduced drastically the allergenic potential of kiwifruit.

Fimbrial cells exposure to catalytic iron mimics carcinogenic changes.

OBJECTIVE: Recent evidence strongly suggests that the fallopian tube is a site of origin of ovarian cancer. Although histological data show iron deposition in the fallopian tubes, its role remains unclear. To establish whether catalytic iron has a possible role in ovarian carcinogenesis, we isolated human fimbrial secretory epithelial cells (FSECs). METHODS: Fimbrial secretory epithelial cells, isolated from women undergoing isteroannessiectomy, were treated with different doses of catalytic iron (0.05-100 mM) to study cell viability; NO production; p53, Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc protein expressions through Western blot analysis; and immunocytochemistry or immunofluorescence. RESULTS: In FSECs treated with catalytic iron for up to 6 days, we observed an increase in cell viability, NO production, and p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc activations (P < 0.05) in a dose-dependent and time-dependent manner. These same results were also observed in FSECs maintained for respectively 2 and 4 weeks in the absence of catalytic iron after 6 days of stimulation. CONCLUSIONS: Our model aimed at studying the main nongenetic risk factor for ovarian cancer, providing an alternative interpretation for the role of menstruation in increasing risk of this pathology. This in vitro model mimics several features of the precursor lesions and opens new scenarios for further investigations regarding the correlation between damages produced by repeated retrograde menstruation carcinogenic stimuli.

Diversity of oat varieties in eliciting the early inflammatory events in celiac disease.

PURPOSE: Celiac disease (CD) is an autoimmune enteropathy, triggered by dietary gluten. The only treatment is a strict gluten-free diet. Oats are included in the list of gluten-free ingredients by European Regulation, but the safety of oats in CD is still a matter of debate. The present study examined the capability of different oat cultivars of activating the gliadin-induced transglutaminase-2 (TG2)-dependent events in some in vitro models of CD. In addition, we compared this capability with the electrophoresis pattern of peptic-tryptic digests of the proteins of the oat cultivars. METHODS: K562(S) cells agglutination, transepithelial electrical resistance of T84-cell monolayers, intracellular levels of TG2 and phosphorylated form of protein 42-44 in T84 cells were the early gliadin-dependent events studied. RESULTS: The results showed that the Nave oat cultivar elicited these events, whereas Irina and Potenza varieties did not. The ability of a cultivar to activate the above-described events was associated with the electrophoretic pattern of oat proteins and their reactivity to anti-gliadin antibodies. CONCLUSION: We found significant differences among oat cultivars in eliciting the TG2-mediated events of CD inflammation. Therefore, the safety of an oat cultivar in CD might be screened in vitro by means of biochemical and biological assays, before starting a clinical trial to definitely assess its safety.

Correlation between catechin content and NF-κB inhibition by infusions of green and black tea.

This study investigates whether infusions of green and black tea inhibit the NF-κB driven transcription in human epithelial gastric AGS cells. Water extracts were prepared from different brands of green and black tea available on the Italian market. Teas with or without caffeine were studied. An industrially prepared freeze-dried water extract of green tea was also tested. Catechin and caffeine contents were measured by HPLC analysis. The decrease in phenol and catechin content three months after the expiry date was also investigated. The NF-κB driven transcription and the free radical scavenger activity were inhibited, and this effect was related to catechin levels. The potency of epigallocatechin 3-gallate in inhibiting NF-κB driven transcription is so great that tea extracts low in epigallocatechin 3-gallate are still highly active. In one decaffeinated sample of green tea, the phenol and catechin content was very low, probably as a consequence of caffeine removal. The decrease in catechin levels after 3 months did not reduce the inhibition of NF-κB driven transcription by tea infusions. This is the first paper reporting the inhibitory effect of NF-κB of commercial green and black infusions at the gastric level, evaluating their stability as well.

Different expression and function of the endocannabinoid system in human epicardial adipose tissue in relation to heart disease.

BACKGROUND: The endocannabinoid system reportedly plays a role in the pathogenesis of cardiovascular diseases. This system is expressed also in adipose tissue, which could thus be involved in cardiac disorders through modulation of metabolically triggered inflammation. The current study aims to determine the relevance of the endocannabinoid system in epicardial adipose tissue in heart disease. METHODS: Expression of the endocannabinoid receptors CB1 and CB2, and of the endocannabinoid-degrading enzyme, fatty acid amidohydrolase, and activation of protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC), endothelial nitric oxide synthase (eNOS) and inducible (i)NOS, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) (a member of the reperfusion-injury salvage kinase pathway), were analyzed by Western blot in patients after coronary artery bypass surgery (ischemics; N = 18) or valve surgery (nonischemics; N = 15) and in preadipocytes isolated from epicardial adipose tissue. RESULTS: In ischemics, the CB1-to-CB2 expression ratio shifted toward CB1 and was accompanied by higher PKA activation. In contrast, in nonischemics, CB2, fatty acid amidohydrolase, PLC and PKC, and ERK1/2 were upregulated. Moreover, NO production and iNOS-to-eNOS ratios were higher in preadipocytes from ischemics. CONCLUSIONS: These results show a different modulation and functioning of the endocannabinoid system in ischemics compared with nonischemics. Hence, while CB2, PLC and PKC, ERK1/2, and eNOS are more strongly expressed in patients without ischemic heart disease, high CB1 and PKA expression is associated with low survival intracellular pathway activation and high iNOS activation in ischemic heart disease patients. The changes in the endocannabinoid system in ischemics may contribute to cardiac dysfunction and therefore represents a potential therapeutic target.

CCK receptors-related signaling involved in nitric oxide production caused by gastrin 17 in porcine coronary endothelial cells.

In anesthetized pigs gastrin-17 increased coronary blood flow through CCK1/CCK2 receptors and ß(2)-adrenoceptors-related nitric oxide (NO) release. Since the intracellular pathway has not been investigated the purpose of this study was to examine in coronary endothelial cells the CCK1/CCK2 receptors-related signaling involved in the effects of gastrin-17 on NO release. Gastrin-17 caused a concentration-dependent increase of NO production (17.3-62.6%; p<0.05), which was augmented by CCK1/CCK2 receptors agonists (p<0.05). The effect of gastrin-17 was amplified by the adenylyl-cyclase activator and ß(2)-adrenoceptors agonist (p<0.05), abolished by cAMP/PKA and ß(2)-adrenoceptors and CCK1/CCK2 receptors blockers, and reduced by PLC/PKC inhibitor. Finally, Western-blot revealed the preferential involvement of PKA vs. PKC as downstream effectors of CCK1/CCK2 receptors activation leading to Akt, ERK, p38 and endothelial NOS (eNOS) phosphorylation. In conclusion, in coronary endothelial cells, gastrin-17 induced eNOS-dependent NO production through CCK1/CCK2 receptors- and ß(2)-adrenoceptors-related pathway. The intracellular signaling involved a preferential PKA pathway over PKC.