Henryk I. Trzeciak

Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation

The aim of the present study was to establish whether piracetam (2-pyrrolidon-N-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP) are capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we observed the cells treated with PIR and VINP during and after in vitro simulated hypoxia. Cell viability was determined by Live/Dead Viability/Cytotoxicity Assay Kit, LDH release assay and MTT conversion test. Apoptotic cell death was distinguished by a method of Hoechst 33342 staining underfluorescence microscope and caspase-3 colorimetric assay. In addition the intracellular levels of ATP and phosphocreatine (PCr) were evaluated by bioluminescence method. Moreover, the effect of the drugs on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. PIR (0.01 and 1 mM) and VINP (0.1 and 10 microM) were added to the medium both during 24 h normoxia, 24 h hypoxia or 24 h reoxygenation. Administration of 1 mM PIR or 0.1 microM VINP to the cultures during hypoxia significantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also confirmed by their stimulation of mitochondrial function, the increase of intracellular ATP, and the inhibition of the caspase-3 activity. The prevention of apoptosis was accompanied by the increase in ATP and PCr levels and increase in the proliferation of astrocytes exposed to reoxygenation. The higher concentration of VINP (10 microM) was detrimental in hypoxic conditions. Our experiment proved the significant cytoprotective effect of 1 mM PIR and 0.1 microM VINP on astrocytes in vitro.

Role of astrocytes in pathogenesis of ischemic brain injury

Astrocytes play an important role in the homeostasis of the CNS both in normal conditions and after ischemic injury. The swelling of astrocytes is observed during and several seconds after brain ischemia. Then ischemia stimulates sequential morphological and biochemical changes in glia and induces its proliferation. Reactive astrocytes demonstrate stellate morphology, increased glial fibrillary acidic protein (GFAP) immunoreactivity, increased number of mitochondria as well as elevated enzymatic and non-enzymatic antioxidant activities. Astrocytes can re-uptake and metabolize glutamate and in this way they control its extracellular concentration. The ability of astrocytes to protect neurons against the toxic action of free radicals depends on their specific energy metabolism, high glutathione level, increased antioxidant enzyme activity (catalase, superoxide dismutase, glutathione peroxidase) and overexpression of antiapoptoticbcl-2 gene. Astrocytes produce cytokines (TNF-±, IL-1, IL-6) involved in the initiation and maintaining of immunological response in the CNS. In astrocytes, like in neurones, ischemia induces the expression of immediate early genes:c-fos, c-jun, fos B, jun B, jun D, Krox-24, NGFI-B and others. The protein products of these genes modulate the expression of different proteins, both destructive ones and those involved in the neuroprotective processes.

Effect of neuroleptics on phospholipase A2 activity in the brain of rats.

The effect of neuroleptics on phospholipase A2 (PLA2) activity in rat brain plasma membranes was studied. Chlorpromazine (10 mg/kg), fluphenazine (5 mg/kg), thioridazine (5 mg/kg), trifluoperazine (5 mg/kg), haloperidol (2 mg/kg), and sulpiride (100 mg/kg) were administered to rats intraperitoneally as a single dose or long-term treatment (4 weeks). The PLA2 activity was determined 24, 48, and 72 h after the last injection of a drug. The enzyme activity was decreased after a single or 4-week administration of chlorpromazine, trifluoperazine, haloperidol, and sulpiride. Fluphenazine and thioridazine caused an increase of PLA2 activity in rat brain both after a single dose and long-term administration. For the first time it was shown that neuroleptics cannot only inhibit but also increase, PLA2 activity. Elucidation of this fact requires further studies.

Aniracetam attenuates apoptosis of astrocytes subjected to simulated ischemia in vitro.

The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms.

New bradykinin analogs in contraction of rat uterus

In this study, we evaluated 20 of our previously synthesized peptides on isolated rat uterus by Holtons procedure with minor modifications, and compared their activity with that assessed previously by their ability to inhibit vasodepressor response to exogenous bradykinin (BK) in conscious rats. We used [D-Arg(0), Hyp(3), Thi(5, 8), (D-Phe)(7)]BK, the B(2) antagonist of Vavrek and Stewart as a model when designing our analogs. We observed that, in the case of the rat uterus test, the activity of peptides modified by acylation of the N-terminus with various bulky groups depends substantially on the chemical character of the substituent. We also learned that, contrary to previous examples, acylation of the N-terminus of antagonists, which contain a sterically restricted fragment in the C-terminal part, may not improve their antagonistic potencies. Besides an improved characterization of a series BK analogs, our studies have provided new information on the structure-activity relationship, which in turn may be of value in the design of more potent and selective bradykinin antagonists. The results of our studies appear to support the hypothesis of others about the presence of different subtypes of B(2) receptors in rat uterus and blood vessels.

Does Trimetazidine Exert Cytoprotective Activity on Astrocytes Subjected to Hypoxia In Vitro

The aim of the present study was to establish whether trimetazidine (TMZ) is capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we tried to observe the cells treated with TMZ before, during and after hypoxia simulated in vitro. Cell viability was determined by Live/Dead (viability/cytotoxicity) Assay Kit and MTT conversion test. Apoptotic cell death was distinguished by a method using fluorescence microscopy with Hoechst 33342. The effect of the drug on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. TMZ stimulates the proliferation of astrocytes most significant one when the astrocytes are exposed to the drug in normoxia, hypoxia and/or re-oxygenation. Adding TMZ into cultures during re-oxygenation and hypoxial re-oxygenation significantly decreases the number of dead and apoptotic cells. Our experiment has proved that TMZ exerts the most significantly cytoprotective effect on astrocytes in vitro when added during hypoxia and/or re-oxygenation. We may conclude that the protective effect of TMZ depends on the sequence of drug adding and hypoxia/ re-oxygenation onset.

Analogues of arginine vasopressin modified in position 2 or 3 with naphthylalanine : Selective antagonists of oxytocin in-vitro

In this study we describe the synthesis and some pharmacological properties of six new analogues of arginine vasopressin (AVP).

Surprising Pharmacological Activity of Analogues Designed by Substitution of Position 3 in Arginine Vasopressin (AVP) and 8‐D‐Arginine Vasopressin with l‐2‐Naphthylalanine

In an attempt to develop more active and selective analogues of arginine vasopressin (AVP), two peptides have been designed, synthesized and tested for vasopressor (V1‐receptors) and antidiuretic (V2‐receptors) activities. We also estimated the uterotonic and anti‐uterotonic activities of these compounds in‐vitro.

Effects of short- and long-term protriptyline treatment on phospholipid metabolism in rat brain

Abstract: Wistar rats were injected intraperitoneally with 10 mg/kg of protriptyline according to one of the following schedules: a single dose or daily for 4 days (short‐term), or daily for 2 or 13 weeks (long‐term). Total lipid, total phospholipid, and individual phospholipid contents in the brain were determined. Further, the incorporation of 32P into individual phospholipids in vivo and the fatty acid composition of phosphatidylethanolamine in the brains of rats treated with protriptyline for 13 weeks were studied. Three alternative phases of changes of total and individual phospholipid contents in the brain during 13 weeks of experimentation were distinguished. An increase of phospholipid contents after 4 days, a decrease after 2 weeks, and a further increase after 13 weeks of protriptyline administration were found. However, phosphatidylinositol and phosphatidic acid levels after 13 weeks of protriptyline administration were diminished. The decrease of specific radioactivity of phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine and the increase of phosphatidylinositol, phosphatidic acid, and sphingomyelin in rats treated with the drug for a longer period of time were noted. No greater differences in fatty acid composition of phosphatidylethanolamine in the brains of the same group of rats were observed as compared to control. These results indicate that during long‐term treatment with protriptyline the contents of lipids and phospholipids in rat brain are altered. The modification of the biological function of phospholipids in brain cell membranes is suggested.

The Effect of Antidepressants on Phospholipase A2 Activity in the Brain of Rats

Phospholipase A2 (PLA2) cleaves fatty acids from the sn-2 position of phospholipids and plays an important role in phospholipid turnover of cell membranes, which in turn affects membrane fluidity and function. The products of PLA2 action are important factors in signal transduction (1). Moreover, there are suggestions that PLA2 participate in the pathogenesis of depression (2).