A New Method for the Visualization of Living Dopaminergic Neurons and Prospects for Using It to Develop Targeted Drug Delivery to These Cells

This is the first study aiming to develop a method for the long-term visualization of living nigrostriatal dopaminergic neurons using 1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine-BODIPY (GBR-BP), the original fluorescent substance, which is a derivative of GBR-12909, a dopamine uptake inhibitor. This method is based on the authors’ hypothesis about the possibility of specifically internalizing into dopaminergic neurons substances with a high affinity for the dopamine transporter (DAT). Using a culture of mouse embryonic mesencephalic and LUHMES cells (human embryonic mesencephalic cells), as well as slices of the substantia nigra of adult mice, we have obtained evidence that GBR-BP is internalized specifically into dopaminergic neurons in association with DAT via a clathrin-dependent mechanism. Moreover, GBR-BP has been proven to be nontoxic. As we have shown in a primary culture of mouse metencephalon, GBR-BP is also specifically internalized into some noradrenergic and serotonergic neurons, but is not delivered to nonmonoaminergic neurons. Our data hold great promise for visualization of dopaminergic neurons in a mixed cell population to study their functioning, and can also be considered a new approach for the development of targeted drug delivery to dopaminergic neurons in pathology, including Parkinson’s disease

GPR55 Receptor Activation by the N-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca2+, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential

Neuroprotective and cerebrovascular effects of endogenous N-Arachidonoyl-GABA and its putative Cox-2 metabolite – GABA conjugate with Prostaglandin E2

Introduction: The aim of the study was to compare the neuroprotective and cerebrovascular effects of bioactive, endogenous lipid – N-arachidonoyl-GABA (AA-GABA) and GABA conjugate with prostaglandin E2 (PGE2-GABA) by evaluation of a morphological state of rat brain tissue and lipofuscin levels under the condition of permanent focal brain ischemia, as well as cerebral circulation under the condition of global transient ischemia. Materials and methods: The study has been implemented using the models of the left middle cerebral artery occlusion (MCAO) and global transient ischemia of the brain. A morphological examination of the brain tissue, a registration of local blood flow by laser flowmeter, and quantitative measurement of lipofuscin by fluorescence spectroscopy were used. Results and discussion: AA-GABA and the putative COX-2 metabolite PGE2-GABA showed significant neuroprotective and cerebrovascular effects in rat models of global and focal cerebral ischemia. In the MCAO model, AA-GABA and PGE2-GABA at a dose of 2 mg/kg/day administered i.p. for 6 or 12 days led to: 1) significant restoration of neurons and glial cells with intracellular regeneration of cytoplasmic and nuclear structures, 2) decrease in brain tissue edema; 3) attenuated thrombosis and stasis, and 4) absence of large necrotic foci in rat brain tissue. AA-GABA and PGE2-GABA at the same dose prevented excessive accumulation of lipofuscin in both brain hemispheres in rats with MCAO. All the studied compounds increase cerebral blood circulation in rats subjected to global transient ischemia. However, the cerebrovascular effect of PGE2-GABA was superior to the activity of AA-GABA and all other tested compounds. AA-GABA and PGE2-GABA, unlike PGE2 and nimodipine, increase the cerebral blood flow in rats with global transient brain ischemia and have no influence on the intact animals. Apparently, the GABAergic vascular system of the brain is involved in the mechanisms of the neuroprotective action of AA-GABA and PGE2-GABA. Conclusion: For the first time, we demonstrated the ability of AA-GABA and its putative metabolite COX-2 PGE2-GABA to improve cerebral circulation, attenuate structural damage and lipofuscin accumulation during cerebral ischemia. The natural origin of AA-GABA, which possesses neuroprotective and cerebrovascular activity, as well as anti-aggregatory activity, allows considering AA-GABA as one of the endogenous protective factors in ischemic brain lesions. Graphical abstract

GPR55 Receptor Activation by the <i>N</i>-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca2+, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential

The Interaction of the Endocannabinoid Anandamide and Paracannabinoid Lysophosphatidylinositol during Cell Death Induction in Human Breast Cancer Cells

Endocannabinoid anandamide (AEA) and paracannabinoid lysophosphatidylinositol (LPI) play a significant role in cancer cell proliferation regulation. While anandamide inhibits the proliferation of cancer cells, LPI is known as a cancer stimulant. Despite the known endocannabinoid receptor crosstalk and simultaneous presence in the cancer microenvironment of both molecules, their combined activity has never been studied. We evaluated the effect of LPI on the AEA activity in six human breast cancer cell lines of different carcinogenicity (MCF-10A, MCF-7, BT-474, BT-20, SK-BR-3, MDA-MB-231) using resazurin and LDH tests after a 72 h incubation. AEA exerted both anti-proliferative and cytotoxic activity with EC50 in the range from 31 to 80 µM. LPI did not significantly affect the cell viability. Depending on the cell line, the response to the LPI–AEA combination varied from a decrease in AEA cytotoxicity to an increase in it. Based on the inhibitor analysis of the endocannabinoid receptor panel, we showed that for the former effect, an active GPR18 receptor was required and for the latter, an active CB2 receptor. The data obtained for the first time are important for the understanding the manner by which endocannabinoid receptor ligands acting simultaneously can modulate cancer growth at different stages

The Impact of the Angulus Biopsy on the Detection of Staging and the Grading of Chronic Gastritis

There is a generally recognized need for a morphological assessment of the individual risk of developing gastric cancer in a patient with chronic gastritis, according to the OLGA system (Operative Link for Gastritis Assessment). At the same time, the role of assessing the biopsy from the incisura angularis remains controversial. The aim of our study was to assess the value of incisura angularis biopsy in staging gastritis according to the OLGA system by examining the atrophic and inflammatory changes in the antrum, incisura angularis, and body. Materials and Methods: A total of 718 patients (576 women and 142 men) aged 20 to 84 years were examined. Most of the patients were in the age group of 50 to 70 years (54.6%). Depending on the detection of H. pylori and autoimmune gastritis markers, all patients were divided into three groups. The first group included 380 patients with H. pylori gastritis without signs of autoimmune gastritis. The second group consisted of 209 patients with autoimmune gastritis, in whom no infection was detected during the examination, and there were no indications of H. pylori eradication. The third group consisted of 129 patients with chronic gastritis of combined etiology (autoimmune and H. pylori). Endoscopy biopsies were taken according to the updated Sydney System. Histological assessments of the grade and the stage of gastritis were carried out according to the standard OLGA-based protocol. Then, the same assessments were evaluated without taking into account histological changes in the incisura angularis. Results: When assessing the severity of inflammatory changes in the gastric mucosa according to the OLGA system, grade II (72.3%) was most often detected in all groups of patients. A severe degree of activity of chronic gastritis was most often observed in the group of patients with H. pylori gastritis (6.1%). These indicators practically did not change if the assessment did not take the angulus biopsy into account. When assessing the severity of atrophy of the glands in the gastric mucosa in patients of the first group, mild stages of atrophy prevailed. Without taking into account the angulus biopsy, a decrease in the stage of atrophy was observed in 27 cases (7.11%), and in only 4 cases did stage IV change to stage III, while in 23 cases, discrepancies were noted only within groups with a mild stage of atrophy. There were no transitions from stage III to stage II. In the group of patients with autoimmune gastritis, pronounced stages of atrophy prevailed—in more than 77%. Without taking into account the angulus biopsy, a decrease in the stage of atrophy was observed in eight cases (3.83%), and in three (1.4%) patients, stage III was changed to stage II. In the group of patients with combined etiology (autoimmune + H. pylori), severe stages of atrophy also prevailed (70.5%). A decrease in the stage of atrophy without taking into account the angulus biopsy was only observed in three patients (2.32%), of which two cases concerned patients with mild stages of atrophy. Thus, in general, severe stages of atrophy of the gastric mucosa (stages III and IV according to the OLGA staging system) were detected in 313 patients (43.59%). If the assessment of the atrophy stage did not take into account changes in the angulus biopsy, then severe stages of atrophy (III and IV according to OLGA) were detected in 310 patients (43.17%). In total, changes in the assessment of the atrophy stage occurred in 38 patients (5.29%), and this was more often observed in patients with stages I and II of atrophy. Conclusions: Accounting for histological changes in the incisura angularis does not significantly affect the assessment of the grade and stage of chronic gastritis according to the OLGA system, regardless of the etiology of atrophic gastritis