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Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Ministry for Education, University and Research Introduction The majority of elderly patients with heart failure has a preserved ejection fraction (HFpEF) that constitutes a syndrome characterized by frequent hospitalizations and high mortality. Despite the growing social burden of HFpEF, the comprehension of its pathophysiology is incomplete, and treatment remains largely undefined. Aging itself may contribute independently to deterioration of diastolic function. Methods 18-month old female Fischer 344 rats were treated with oral administration of either sacubitril/valsartan (60 mg/kg/die, 1:1 ratio) or valsartan alone (30 mg/kg/die) for 12 weeks. Tail-cuff method was used to monitor blood pressure weekly. Echocardiography and left ventricle catheterization were employed to assess systolic and diastolic function, at baseline, and before sacrifice. Cardiac tissue was used for molecular biology and histochemistry assays. Results Tail-cuff analysis indicated a comparable decrease in blood pressure between treatments. Hypertrophy also showed a significant reduction with both treatments. On the contrary, myocardial function analysis demonstrated that no treatment was efficacy on diastolic dysfunction. The lack of improvement of cardiac function could be attributed to the inability of the treatments to counteract the accumulation of fibrotic tissue in the left ventricle, which, in turn, is attributable to the failure to reduce the inflammatory process and oxidative stress, and to the inability to modulate angiotensin II pathway. Conclusion Our results evidenced that both sacubitril/valsartan or valsartan treatment was able to improve diastolic function and pro-fibrotic remodeling, partly due to a lack of effect on classical and non-classical pathways of angiotensin II.

Atrial fibrillation (AF) is the most frequent arrhythmia and is associated with substantial morbidity and mortality. Pathophysiological aspects consist in the activation of pro-fibrotic signaling and Ca2+ handling abnormalities at atrial level. Structural and electrical remodeling creates a substrate for AF by triggering conduction abnormalities and cardiac arrhythmias. The care of AF patients focuses predominantly on anticoagulation, symptoms control and the management of risk factors and comorbidities. The goal of AF therapy points to restore sinus rhythm, re-establish atrioventricular synchrony and improve atrial contribution to the stroke volume. New layer of information to better comprehend AF pathophysiology, and identify targets for novel pharmacological interventions consists of the epigenetic phenomena including, among others, DNA methylation, histone modifications and noncoding RNAs. Moreover, the benefits of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in diabetic and non-diabetic patients at cardiovascular risk as well as emerging evidence on the ability of SGLT2i to modify epigenetic signature in cardiovascular diseases provide a solid background to investigate a possible role of this drug class in the onset and progression of AF. In this review, following a summary of pathophysiology and management, epigenetic mechanisms in AF and the potential of sodium-glucose SGLT2i in AF patients are discussed.

Orthodontics is a branch of dentistry that aims at the resolution of dental malocclusions. The specialist carries out the treatment using intraoral or extraoral orthodontic appliances that require forces of a given load level to obtain a tooth movement in a certain direction in dental arches. Orthodontic tooth movement is dependent on efficient remodeling of periodontal ligament and alveolar bone, correlated with several biological and mechanical responses of the tissues surrounding the teeth. A periodontal ligament placed under pressure will result in bone resorption whereas a periodontal ligament under tension results in bone formation. In the primary stage of the application of orthodontic forces, an acute inflammation occurs in periodontium. Several proinflammatory cytokines are produced by immune-competent cells migrating by means of dilated capillaries. In this paper we summarize, also through the utilization of animal models, the role of some of these molecules, namely, interleukin-1βand vascular endothelial growth factor, that are some proliferation markers of osteoclasts and osteoblasts, and the macrophage colony stimulating factor.

Glutamate synthase (NADH-GOGAT) is one of several proteins whose expression is either enhanced in, or specific to developing legume root nodules induced by (Brady)Rhizobium infection. This enzyme catalyzes the second committed step in the assimilation of symbiotically fixed nitrogen; the transamidation of 2-oxoglutarate by glutamine to yield two moles of glutamate. We have previously purifed alfalfa root nodule NADH-GOGAT and raised antibodies against this protein. To investigate the regulation of the gene encoding this enzyme, we have isolated a full length cDNA clone for NADH-GOGAT from an alfalfa nodule cDNA library. This 7.5 kb cDNA includes the sequence encoding the mature 210 kd protein (the amino terminus of which has been sequenced), as well as an apparent transit peptide. Northern blots indicate that mRNA levels are developmentally regulated in the nodule, showing a similar pattern to that of previously characterized genes encoding aspartate aminotransferase and phosphoenoylpyruvate carboxylase.

The present study has been carried out to determine if glycine, an allosteric modulator of NMDA receptor, is involved in the vascular effect induced by the activation of the CNS NMDA receptors.Icv NMDA (from 0.01 to 1µg/rat in the 3rd ventricle) caused a significant increase in arterial blood pressure in conscious freely moving rats. Moreover, the hypertension was associated with behavioural modifications (jumping, rearing, teething and running). Glycine pretreatment (1 and 10µg/raticv), significantly increased the NMDA hypertension. Glycine alone did not cause any arterial blood pressure modification while it induced a slight sedation. HA-966 (an antagonist of the glycine site on NMDA receptor) administration (1-10µg/raticv 5 min before glycine) significantly antagonized the glycine effects on NMDA hypertension.Alone HA-966 neither modified arterial blood pressure nor antagonized NMDA hypertension. In conclusion, our investigations confirm NMDA receptor involvement in cardiovascular function and they demonstrate thatin vivo glycine positively modulates NMDA receptors.

AIM: Target controlled infusion intravenous anesthesia is a growing phenomenon. Nowadays, many anesthesiologists feel the need to monitor depth of anesthesia during total intravenous anesthesia, even though it is not a standard technique worldwide. Spectral Entropy (SE) is a relatively new depth of anesthesia index. The aim of this study was to investigate whether predicted site-effect propofol concentrations, A-line Autoregressive Index (AAI) and SE values are useful for predicting loss of verbal contact (LVC) and loss of consciousness (LOC) during steady-state conditions. METHODS: Forty-four patients scheduled for elective major abdominal surgery were recruited. All patients were unpremedicated. A target controlled infusion of propofol was administered using Schnider's pharmacokinetic model. The initial propofol infusion provided a site-effect concentration of 1.0 mcg mL-1, and was increased stepwise by 1.0 mcg mL-1 every 4 minutes until the concentration reached 6.0 mcg mL-1. A 4 minute interval was chosen to assure that steady state site-effect concentrations were obtained. AAI, SE and propofol site-effect concentrations were recorded when LVC occurred and also when LOC occurred. Population values for predicted site-effect concentrations at the clinical endpoints were estimated and correlated with AAI and SE values. RESULTS: In our study for LOC the effect-site concentration to include 90% of patients was 5.85 ?mcg mL-1 (5.70-5.90) and 3.4 mcg mL-1 (3.24-3.60) for LVC. In this study, 90% of patients lost verbal contact at an AAI value of 68 (64.6-71.4) and an SE value of 68.2 (66.2-70.2). LOC occurred in 90% of patients at an AAI value of 39.2 (37.2-41.1) and an SE value of 40.2 (38.1-41.3). CONCLUSIONS: LOC and LVC occur within a defined range of predicted site-effect concentrations. More emphasis should be given to site-effect concentrations. SE and AAI have similar values at different endpoints and similar correlation with Ceprop. AAI and SE are both useful tools in predicting both LVC and LOC.

Il ricorso al mifepristone (RU486), in combinazione con analoghi delle prostaglandine, nell’induzione dell’aborto chimico richiede una specifica riflessione in merito ai principali aspetti farmacologici e tossicologici. L’attuale dialettica bioetica e biopolitica, spesso ideologizzata, impone ancor più una trattazione rigorosa basata sulle evidenze scientifiche, a chiarimento soprattutto dei meccanismi d’azione e degli eventi avversi. Questi ultimi a volte anche sottodimensionati o minimizzati. Premessa l’iniquità dell’aborto volontario, l’articolo si prefigge anche lo scopo di chiarire come, in ordine ai dati della più recente bibliografia, il ricorso all’RU486 rappresenti un significativo rischio per la salute delle donne. Una particolare attenzione è riservata alla chiarificazione etiopatogenetica delle emorragie e delle sepsi, dalle quali si sono riscontrati anche diversi decessi. Nell’articolo sono riportati, inoltre, i più attuali sviluppi della ricerca con RU486 sia per il trattamento sperimentale di patologie – ginecologiche e non – che per l’uso della molecola per la contraccezione ormonale e la “contraccezione di emergenza”. ---------- The use of mifepristone (RU486), in combination with prostaglandin analogues, in the chemical abortion, requires a specific reflection on the main aspects in pharmacology and toxicology. The current debate in bioethics and biopolitical, often ideological, imposes a more rigorous treatment based on scientific evidences, especially clarification of the mechanisms of action and severe adverse events. The latter sometimes underestimated or minimized. Declared beforehand the iniquity of volunteer abortion, this article aims also to clarify how, in order to the data from the most recent literature, the use of RU486 represents a significant risk to women’s health. Particular attention is given to etiopathogenetic clarification of bleeding and sepsis, which have also seen several deaths. The article reportes, moreover, the most current developments in the research for RU486 treatment of experimental conditions – gynaecological and not – and for the use of the molecule in the hormonal contraception and the “emergency contraception”.

Il ricorso al mifepristone (RU486), in combinazione con analoghi delle prostaglandine, nell’induzione dell’aborto chimico richiede una specifica riflessione in merito ai principali aspetti farmacologici e tossicologici. L’attuale dialettica bioetica e biopolitica, spesso ideologizzata, impone ancor piu una trattazione rigorosa basata sulle evidenze scientifiche, a chiarimento soprattutto dei meccanismi d’azione e degli eventi avversi. Questi ultimi a volte anche sottodimensionati o minimizzati. Premessa l’iniquita dell’aborto volontario, l’articolo si prefigge anche lo scopo di chiarire come, in ordine ai dati della piu recente bibliografia, il ricorso all’RU486 rappresenti un significativo rischio per la salute delle donne. Una particolare attenzione e riservata alla chiarificazione etiopatogenetica delle emorragie e delle sepsi, dalle quali si sono riscontrati anche diversi decessi. Nell’articolo sono riportati, inoltre, i piu attuali sviluppi della ricerca con RU486 sia per il trattamento sperimentale di patologie – ginecologiche e non – che per l’uso della molecola per la contraccezione ormonale e la “contraccezione di emergenza”. ---------- The use of mifepristone (RU486), in combination with prostaglandin analogues, in the chemical abortion, requires a specific reflection on the main aspects in pharmacology and toxicology. The current debate in bioethics and biopolitical, often ideological, imposes a more rigorous treatment based on scientific evidences, especially clarification of the mechanisms of action and severe adverse events. The latter sometimes underestimated or minimized. Declared beforehand the iniquity of volunteer abortion, this article aims also to clarify how, in order to the data from the most recent literature, the use of RU486 represents a significant risk to women’s health. Particular attention is given to etiopathogenetic clarification of bleeding and sepsis, which have also seen several deaths. The article reportes, moreover, the most current developments in the research for RU486 treatment of experimental conditions – gynaecological and not – and for the use of the molecule in the hormonal contraception and the “emergency contraception”.

The expression profiles of genes involved in cell proliferation, differentiation and programmed death were investigated in carotids of spontaneously hypertensive rats (SHR) treated with a model of surgical injury that mimics events occurring during arterial grafts, endarterectomy and organ transplantation. The mRNA level of the c-myc, angiotensin II receptor 1 (AT1), Rb/p105, Rb2/p130, Bcl-2 and Bax-α genes was assessed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique at different times up to 48 h after injury, while the morphological changes were evaluated 30 days after injury. The proliferation marker c-myc increases almost immediately, peaks after 4 h and returns to basal levels after 24 h; the AT1 receptor mRNA reaches its maximal level 48 h after injury. The level of cell cycle exit markers Rb/p105 and Rb2/p130 gradually decreases after injury. The apoptosis marker Bcl-2/Bax-α ratio shows a significant reduction only 4 h after injury, resuming the initial value after 24 and 48 h. Morphological analysis reveals that surgical injury in SHR induces adventitial and medial constrictive remodeling changes rather than intima proliferation as in balloon angioplasty. Both molecular and histological data show substantial differences with respect to normotensive rats.

A group of 24 imidazo[1,2-c]pyrimidines was taken into consideration in order to study their relationships between chemical structure and antiinflammatory activity. Some of these compounds were synthesized by us in the past, the others have been synthesized and characterized to complete the series. Antiinflammatory, analgesic, antipyretic and gastric ulcerogenic activities of such compounds were compared with those of indomethacin and discussed in terms of the effects exerted by the presence on the heterocyclic system of various substituents and an acidic or nonacidic moiety.

Partial phosphorothioate (PS) antisense oligodeoxynucleotides (ODNs) targeted against rat AT1 receptor mRNA have been used to control blood pressure in normotensive (WKY) and spontaneously hypertensive (SHR) rats. Molecules were injected intracerebroventricularly (i.c.v., right lateral ventricle) in freely moving animals. The antisense ODN lowered the mean arterial pressure (MAP) 24 hours (-43 mmHg+/-10) and 48 hours (-30 mmHg+/-13) after injection, while the control ODN molecule had no significant effects. The observed decrease of blood pressure was due to a specific inhibition of AT1 receptor gene expression, since the level of its mRNA, monitored by reverse transcription (RT)- polymerase chain reaction (PCR), was significantly reduced by antisense molecule (-40%), compared to sense one. In normotensive rats no effect on MAP have been observed, while AT1 receptor gene expression is reduced (-40%) by antisense treatment. It is known that SHRs have an enhanced basal activity of the central renin-angiotensin system that induces an increase in central sympathetic outflow. Instead in WKY rats the central sympathetic outflow is not conditioned by the enhanced activity of brain renin-angiotensin system. Therefore in normotensive rats although partial PS ODN reduces the AT1 mRNA level this will not result in a modification of the sympathetic outflow and no change in MAP level would be observed.

Somatostatin is a hormone produced by the hypothalamus, and is widely diffused outside the CNS. It inhibits the secretion of many glands. Recently it has been shown that somatostatin has been proposed in the treatment of phlogistic diseases of the joints by intra-articular administration. The authors show the results of a study conducted in 16 patients (athletes) with arthrosynovitis or tendinitis of the knee or of the ankle, in which somatostatin was administered (250 mg/treatment, 1 treatment/week). Somatostatin significantly reduced pain, improved movement function and decreased the effects of pain on daily activities. The treatment was very well tolerated locally and generally. In conclusion, somatostatin may be considered useful in the treatment of articular and tendineous phlogistic diseases, almost in athletes.

One of the most important factors that determine the therapeutic limits of doxorubicin (DXR), an anthracycline antibiotic, appears in acute and late cardiotoxicity. The aim of our investigations was to evaluate the effects of S-adenosylmethionine (SAMe), an important precursor of the organic sulfated compounds and a fundamental agent in physiologic transmethylation, on DXR-induced cardiotoxicity. For that purpose, normotensive Wistar rats were treated with DXR associated with or without SAMe. Pretreatment with SAMe significantly reduced DXR-induced electrocardiographic and morphologic changes, lethality, and body weight. The protective effect of SAMe may partly be due to its scavenger ability against free radicals and superoxides. In addition to its action as a fundamental precursor of the sulfated compounds, the principal agent in physiological transmethylations, and an increase of glutathione (GSH) synthesis.

A group of ethyl esters of 2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acids 3 were obtained by reaction in anhydrous ethanol of some substituted 3-aminopyridazines 1 with ethyl 2-benzoyl-2-bromoacetate 2. The acids 4 obtained via alkaline hydrolysis were tested in vivo for their antiinflammatory, analgesic and ulcerogenic actions and in vitro for their ability to inhibit the prostaglandin biosynthesis. Almost all of the new compounds showed high analgesic activity, whereas the activities exhibited in the other tests were sharply lower. These results are discussed in terms of mechanism of action.

The cardiovascular effects of beta-endorphin [beta-LPH-(61-91)] were investigated after systemic (i.v.) and central (third ventricle and right lateral ventricle) administration in urethane-anesthetized rats. beta-Endorphin (10 and 100 micrograms/kg i.v. or 40 micrograms into the third ventricle or into the lateral ventricle) in normotensive animals induced a decrease in blood pressure and bradycardia. The same dose of beta-endorphin by systemic and central administration induced much more pronounced cardiovascular effects in spontaneously hypertensive rats and in rats rendered hypertensive by DOCA administration than in normotensive rats. Naloxone i.v. pretreatment reduced the cardiovascular effects induced by beta-endorphin.

A group of ethyl 6-methylimidazo[2,1-b]thiazole-5-carboxylates and 2-methylimidazo[2,1-b]benzothiazole-3-carboxylates was prepared by reaction of ethyl 2-chloroacetoacetate with some 2-aminothiazoles and 2-aminobenzothiazoles, respectively. Such reactions may sometimes afford a side product which was isolated and characterized. The ethyl esters were then converted into the corresponding acids by hydrolysis. Three of these acids were evaluated for antiinflammatory, analgesic, and antipyretic activities, as well as for ulcerogenic potential.

The cardiovascular effects of porins were evaluated using porins isolated from Salmonella typhimurium SH5014. In dogs porins depress arterial systemic pressure, vasomotor reactivity of norepinephrine and peripheral vagal stimulation. They are capable of modifying the sinocarotidal baroreceptor reactivity. In mice porins increase the cardiotoxic effects of isoprenaline, thyroxine, emetine and of p-nitrophenol. In rats porins increase the arrhythmogenic and lethal effects of BaCl2 and also give rise to renal lesions, probably at the tubular level.

The synthesis of two series of amides and glycinamides starting from N-phenyl benzonorbornen-2-endo-amine, prepared from benzonorbornen-2-one via sodium borohydride reduction of its N-phenyl imine, is described. Some amides showed a remarkable hypotensive activity in rats, whereas amides and glycinamides usually exhibited a moderate infiltration anesthesia in mice. Effects on heart rate in rats and antiarrhythmic activity in mice are also reported.

The synthesis of title compounds by reaction of fenchone nitrimine with 2-aminoethanol and 3-aminopropanol, followed by stereospecific NaBH4 reduction of the resulting imines, Eschweiler-Clarke reductive methylation and esterification with aliphatic and aromatic acyl chlorides is described. Some esters showed an appreciable hypotensive activity in rats. Effects on heart rate and antiarrhythmic activity in rats, as well as infiltration anesthesia in mice, are also reported.

Vasomotor reactivity has been evaluated in rats exposed perinatally and postnatally to thallium sulphate (1 mg/dl in their drinking water ad libitum). Prenatal and postnatal exposure to thallium did not modify the values of the systolic arterial blood pressure on the 30th and 60th day in pups of normotensive and DOCA-hypertensive rats. The hypertensive responses induced by endosinusal carotid hypotension and by 1-noradrenaline in pups of normotensive and DOCA-hypertensive rats, exposed or not exposed to thallium sulphate, were more intensive on the 60th than on the 30th day. Similar effects were observed for the hypotensive responses induced by 1-isoprenaline and acetylcholine. Prenatal exposure to thallium did not modify hypertensive responses induced by endosinusal carotid hypotension on the 30th and 60th days, but it caused a decrease of hypertensive responses induced by 1-noradrenaline on the 30th and 60th days and hypotensive responses induced by 1-isoprenaline and acetylcholine exclusively on the 60th day. Postnatal exposure to thallium did not modify hypertensive responses induced by endosinusal carotid hypotension and hypotensive responses induced by acetylcholine, but it caused a decrease of hypertensive responses induced by 1-noradrenaline on the 30th and 60th days in pups of normotensive rats and exclusively on the 60th day in pups of DOCA-hypertensive rats. Moreover, postnatal exposure to thallium caused a decrease of the hypotensive response induced by 1-isoprenaline exclusively on the 60th day. Our findings show that prenatal and postnatal exposure to thallium sulphate modifies the rat's developing vascular autonomic nervous system with a reduction of the α, β-adrenergic and muscarinic vasomotor reactivity.

Exposure to drugs during pregnancy can alter functional development of the vascular system. The present investigation was carried out in order to evaluate the effects of prenatal and postnatal exposure to Ca2+-antagonist (diltiazem, verapamil, and nimodipine) drugs on the development of rat vasomotor reactivity. Studies were carried out on pregnant female albino rats exposed from the first day of pregnancy until weaning to diltiazem and verapamil (6 and 24 mg/kg in their drinking water ad libitum) and nimodipine (3 and 12 mg/kg in their food ad libitum). After weaning, pups were exposed until the 60th day of age to the same treatment as their mothers were. Afterwards, pups from the 60th to 90th day of age were fed with a normal diet. In 30-, 60-, and 90-day-old conscious and anaesthetized pups, we evaluated the following: 1) systolic arterial blood pressure; 2) vasomotor responses elicited by various agents: L-noradrenaline (0.1, 1, and 5 micrograms/kg IV), L-isoprenaline (0.01, 0.1, and 1 micrograms/kg IV), and acetylcholine (0.01, 0.1, and 1 micrograms/kg IV) and by sinus-carotid baroreceptor stimulation; and 3) catecholamine, acetylcholinesterase, and adenosinase plasma levels. Prenatal and postnatal exposure to Ca2+-antagonist drugs significantly (P less than .05) decreased the pressor response to sinus-carotid baroreceptor stimulation and to L-noradrenaline and increased the hypotensive responses to L-isoprenaline and acetylcholine. Moreover, this type of treatment, although it induced a significant (P less than .05) decrease of catecholamine plasma levels, did not modify the acetylcholinesterase and adenosinase plasma levels in 30- and 60-day-old rats. On the 90th day of age, the evaluated parameters were not different from those of control rats. Our results showed that exposure to Ca2+ antagonists during pregnancy and the postnatal period may alter the functional development of rat vasomotor reactivity.

A group of seven acidic phenyl derivatives of some fused imidazole systems were subjected to a series of tests in vivo in order to evaluate their pharmacological activity. Antiinflammatory activity was studied by means of the carrageenin-induced rat paw edema. Hot plate test and writhing induced by acetic acid in mice were used to evaluate analgesic activity. Yeast-induced hyperthermia in rats was employed to study antipyretic action. Irritative and ulcerogenic action on the rat gastric mucosa was examined at higher doses. The antiaggregating activity and the inhibition of platelet malondialdehyde production were studied in vitro. All experimental results are discussed from the point of view of structure-activity relationships and mode of action.

Purpose: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance. Experimental Design: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab. Results: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET–dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR–MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells. Conclusions: These results suggest that overexpression of TGF-α through induction of EGFR–MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer. Clin Cancer Res; 19(24); 6751–65. ©2013 AACR.

In this study we examined the effect of polyunsaturated fatty acids (PUFAs), in particular of docosahexaenoic acid (DHA), on calcium homeostasis in isolated adult rat cardiomyocytes exposed to KCl, ET-1 and anoxia. Free [Ca(2+)](i) in rat cardiomyocytes was 135.7 +/- 0.5 nM. Exposure to 50 mM KCl or 100 nM ET-1 resulted in a rise in free [Ca(2+)](i) in freshly isolated cells (465.4 +/- 15.6 nM and 311.3 +/- 12.6 nM, respectively) and in cultured cells (450.8 +/- 14.8 nM and 323.5 +/- 14.8 nM respectively). An acute treatment (20 minutes) with 10 microM DHA significantly reduced the KCl- and ET-1-induced [Ca(2+)](i) increase (300.9 +/- 18.1 nM and 232.08 +/- 11.8 nM, respectively). This reduction was greater after chronic treatment with DHA (72 h; 257.7 +/- 13.08 nM and 192.18 +/- 9.8 nM, respectively). Rat cardiomyocytes exposed to a 20 minute superfusion with anoxic solution, obtained by replacing O(2) with N(2) in gas mixture, showed a massive increase in cytosolic calcium (1200.2 +/- 50.2 nM). Longer exposure to anoxia induced hypercontraction and later death of rat cardiomyocytes. Preincubation with DHA reduced the anoxic effect on [Ca(2+)](i) (498.4 +/- 7.3 nM in acute and 200.2 +/- 12.2 nM in chronic treatment). In anoxic conditions 50 mM KCl and 100 nM ET-1 produced extreme and unmeasurable increases of [Ca(2+)](i.) Preincubation for 20 minutes with DHA reduced this phenomenon (856.1 +/- 20.3 nM and 782.3 +/- 7.6 nM, respectively). This reduction is more evident after a chronic treatment with DHA (257.7 +/- 10.6 nM and 232.2 +/- 12.5 nM, respectively). We conclude that in rat cardiomyocytes KCl, ET-1 and anoxia interfered with intracellular calcium concentrations by either modifying calcium levels or impairing calcium homeostasis. Acute, and especially chronic, DHA administration markedly reduced the damage induced by calcium overload in those cells.

The purposes of this study were to investigate in vivo the effects of two lazaroids,U-74389G (21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-pregna-1,4,9(11)-triene-3,20-dione (2)-2-butenenedionate) and U-83836E (−)-2-[4-(2,6-di-1-pyrrlidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran- 6 -ol, dihydrochloride against the cardiotoxicity induced by doxorubicin in rat and the mechanisms underlying such a toxicity. Doxorubicin (DXR) administered intraperitoneally (5 mg kg 4 times per week for 1 week) induced significant decrease of body weight, ECG alterations and 100% mortality. The lazaroids used in this study did not protect from DXR-induced cardiotoxicity Our results showed that the compound U-74389G delayed, but did not reduce DXR-induced mortality, and did not prevent body weight loss and ECG changes. The compound U-83836E was unable to modify any toxic effects induced by DXR. These data indicate that oxygen free radicals and the subsequent increase in intracellular calcium are only steps of DXR progressive general toxicity that leads to cardiac injury. In conclusion, we propose that the 21-aminosteroids, potent inhibitors of membrane lipid peroxidation, alone are not enough to protect from DXR toxic effects.

Summary A series of methyl-substituted imidazo[1,2- a ]pyrazines 8 bearing a carboxylic acid group on the imidazole ring were synthesized. The structures of new compounds were confirmed by 1 H- and 13 C-NMR spectral data; the correct assignment of carbon resonances was made by means of HETCOR and COLOC experiments. Antiinflammatory, analgesic and ulcerogenic activities in vivo were evaluated and compared with those of antiinflammatory imidazopyrazines ( 2 and 3 ) and indomethacin. The inhibitory action on cyclooxygenase activity was evaluated in vitro. Compounds 8 were found to be less potent than indomethacin in these assays. SARs are discussed.