Cytotoxic Effects of the Schweinfurthin Analog 5'-Methylschweinfurthin G in Malignant Plasma Cells.

INTRODUCTION: Multiple myeloma (MM) is a B plasma cell malignancy currently incurable, and novel therapeutics are needed. Evidences regarding the effect of natural compound schweinfurthins suggest that hematological cancers showed growth inhibitory effects to this family of compounds at single nanomolar concentrations. In this study, we evaluated the cytotoxicity of the schweinfurthin synthetic analog 5'-methylschweinfurthin G (MeSG) in MM cell lines, to better understand the validity of this compound as a therapeutic candidate for further studies in MM. METHODS: MeSG toxicity against MM cell lines RPMI-8226, MM.1S, and H-929 was evaluated. Trypan blue exclusion and MTT assays measured cell viability and mitochondrial activity, respectively. Flow cytometry was performed to detect apoptotic mitochondria. Flow cytometry and Western blotting techniques were used to investigate apoptosis and to examine the cell cycle. Western blotting was used to determine AKT activation upon MeSG treatment. RESULTS: We provide evidence that in all MM cells analyzed, MeSG exerts diverse cytotoxic effects. MeSG treatment of MM.1S and H-929, but not in RPMI-8226, causes a loss of mitochondria membrane potential. MeSG causes an arrest in G2/M, especially in RPMI-8226, supported by decreased levels of cyclin-B1 and early increased levels of p21. Finally, there is a diverse response to the MeSG treatment for AKT phosphorylation. MM.1S and H-929 showed a marked decrease in AKT phosphorylation at earlier time points compared to the RPMI-8226 line. CONCLUSIONS: MeSG cytotoxicity has been confirmed in all of 3 cell lines studied. Results suggest an early event of increased reactive oxygen species, and/or involvement of cholesterol homeostasis via decreased AKT activation, both of which are currently under investigation.

Nitrogen Containing Bisphosphonates Impair the Release of Bone Homeostasis Mediators and Matrix Production by Human Primary Pre-Osteoblasts.

Bisphosphonates (BPs) represent the first-line treatment for a wide array of bone disorders. Despite their well-known action on osteoclasts, the effects they induce on osteoblasts are still unclear. In order to shed light on this aspect we evaluated the impact of two nitrogen containing bisphosphonates, Alendronate (ALN) and Zoledronate (ZOL), on human primary pre-osteoblasts. At first, we showed an inhibitory effect on cell viability and alkaline phosphatase activity starting from µM concentrations of both drugs. In addition, an inhibitory trend on mineralized nodules deposition was observed. Then low doses of both ALN and ZOL rapidly increased the release of the pro-inflammatory mediators TNFα and IL-1ß, while increased DKK-1 and Sclerostin, both inhibitors of osteoblastogenesis. Finally, ALN and 10-7M ZOL decreased the expression of type I Collagen and Osteopontin, while both drugs slightly stimulated SPARC production. With these results, we would like to suggest a direct inhibitory action on bone-forming cells by nitrogen containing bisphosphonates.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

Affordable Method for Hematocrit Determination in Murine Models.

Hematocrit (Hct) is a powerful tool often used in a clinical setting for the diagnosis of blood conditions such as anemia. It is also used in the research field as a hematological parameter in both human and mouse models. Measuring Hct, however, involves the use of expensive standardized equipment (such as a CritSpin™ Microhematocrit Centrifuge). Here, we describe a novel, simple, and affordable method to determine the Hct in untreated wild-type (WT) mice and phenylhydrazine (PHZ)-induced anemic mice with reasonable accuracy, using a benchtop centrifuge commonly available in laboratories. Hct of murine samples processed with a benchtop centrifuge, when compared to the standardized method CritSpin™, showed comparable results. This approach for determining Hct of murine can prove useful to research laboratories that cannot afford specialized equipment for Hct studies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Affordable Method for Hematocrit Determination in Murine Models Basic Protocol 2: Murine Sample Validation Support Protocol: Phenylhydrazine-induced anemia in wild-type (WT) mice.

Marginal Bone Maintenance and Different Prosthetic Emergence Angles: A 3-Year Retrospective Study.

The aim of the present retrospective study was to assess marginal bone changes around implants restored with different prosthetic emergence profile angles. Patients were treated with implants supporting fixed dentures and were followed for 3 years. Marginal bone levels (MBL) measured at the prosthesis installation (t0) and at the 3-year follow-up visit (t1) were considered. The MBL change from t0 to t1 was investigated. Two groups were considered: Group 1 for restorations with an angle between implant axis and prosthetic emergence profile >30°, and Group 2 for those with an angle ≤30°, respectively. Moreover, peri-implant soft tissue parameters, such as the modified bleeding index (MBI) and plaque index (PI) were assessed. Seventy-four patients were included in the analysis and a total of 312 implants were examined. The mean EA in groups 1 and 2 was 45 ± 4 and 22 ± 7 degrees, respectively. The mean marginal bone level change (MBL change) of 0.06 ± 0.09 mm and 0.06 ± 0.10 mm were, respectively, in groups 1 and 2. The difference in the MBL change between the two groups was not statistically significant (p = 0.969). The MBL change does not seem to be influenced by the emergence angle for implants with a stable internal conical connection and platform-switching of the abutment diameter.

Combined simvastatin-manidipine protect against ischemia-reperfusion injury in isolated hearts from normocholesterolemic rats.

This study investigated whether oral simvastatin and manidipine interact in protecting the perfused rat heart from ischemia-reperfusion damage. Simvastatin (0.3 to 3 mg/kg) and manidipine (1 to 10 mg/kg) were given orally singly or together to normocholesterolemic rats once a day for seven consecutive days. At the end of treatment, systolic blood pressure and heart rate were measured in conscious rats, and the lipid profile and other biochemical markers, such as thromboxane B(2), nitrite/nitrates and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)) were determined in the plasma. Hearts were then isolated, perfused with Krebs-Henseleit, and subjected to low flow ischemia-reperfusion injury. Post-ischemic recovery of left ventricular function was measured as left ventricular developed pressure and left ventricular end-diastolic pressure. Creatine kinase, lactate dehydrogenase, tumor necrosis factor-alpha and 6-keto-PGF(1alpha) were measured in the heart effluents. In conscious animals, simvastatin alone increased plasma 6-keto-PGF(1alpha) release while manidipine alone reduced systolic blood pressure with a slight sympathetic reflex increase in heart rate, and increased plasma nitrite/nitrates. The combined treatment produced the same effects, but significantly more marked, and accompanied by a significant reduction of thromboxane B(2). Combined treatment was also significantly more effective than the single drugs in protecting the hearts from ischemia-reperfusion injury, with inhibition of creatine kinase, lactate dehydrogenase and tumor necrosis factor-alpha, and enhancement of 6-keto-PGF(1alpha) during reperfusion. These data show that simvastatin and manidipine interact positively in protecting the rat heart from ischemia-reperfusion injury, possibly through increased prostaglandin and nitric oxide formation by the vascular endothelial cells.

Impact of Dental Implant Surface Modifications on Adhesion and Proliferation of Primary Human Gingival Keratinocytes and Progenitor Cells.

The success of dental implants depends mainly on osseointegration and gingival sealing. Therefore, early attachment and spreading of epithelial cells might be critical for a positive outcome. Research in dental implant materials has primarily focused on surface roughness, defined by the average roughness (Ra) index, as it promotes the process of osseointegration. This study explored its influence on soft tissue attachment by looking mainly at adhesion, proliferation, and spreading of primary human cells belonging to the epithelial lineage. Characterized human gingival keratinocytes, gingival and epithelial progenitor cells were seeded on machined (S1; Ra = 0.3 to 0.6 µm), Ti-Unite (S2; Ra = 1.2 µm), and SLA (S3; Ra = 2 µm) implants. Cell adhesion with early proliferation and spreading were evaluated by combining a biochemical vitality test with imaging analyses. Findings showed that adhesion was significantly higher on S1 (36% ± 2%) and S2 (44% ± 7%) than on S3 (23% ± 6%), while early proliferation was slightly improved on S1. The resulting data, obtained through an innovative and easily reproducible in vitro method, suggest that implant surface roughness affects epithelial cell adhesion and proliferation.

The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 during inflammation. However, the roles of TLT-1 in hemostasis and inflammation are not well understood. We are developing the mouse model of TLT-1 to mechanistically test clinical associations of TLT-1 in health and disease. To facilitate our studies, monoclonal murine TLT-1 (mTLT-1) antibodies were produced by the immunization of a rabbit using the negatively charged region of the mTLT-1 extracellular domain 122PPVPGPREGEEAEDEK139. In the present study, we demonstrate that two selected clones, 4.6 and 4.8, are suitable for the detection of mTLT-1 by western blot, immunoprecipitation, immunofluorescent staining, flow cytometry and inhibit platelet aggregation in aggregometry assays. In addition, we found that the topical administration of clone 4.8 delayed the wound healing process in an experimental burn model. These results suggest that TLT-1 plays an important role in wound healing and because both clones specifically detect mTLT-1, they are suitable to further develop TLT-1 based models of inflammation and hemostasis in vivo.

Asymmetric dimethylarginine (ADMA) induces vascular endothelium impairment and aggravates post-ischemic ventricular dysfunction in rats.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide (NO) inhibitor recognized as an independent risk factor for endothelial dysfunction and coronary heart diseases. This study investigated whether ADMA (10 mg/kg day for 14 days) affected endothelial function and aggravated post-ischemic ventricular dysfunction in the perfused rat heart. Systolic blood pressure and heart rate, plasma levels of ADMA and nitrite/nitrate were measured in vehicle- and ADMA-treated rats. Perfused hearts were submitted to global ischemia-reperfusion and vascular endothelial dysfunction was examined with angiotensin II in coronary vessels and aortic rings. Endothelial NO synthase (eNOS) and angiotensin-converting enzyme (ACE) mRNA expression in aortic and cardiac tissues were measured. ADMA-treated rats had higher systolic blood pressure (1.3-fold, P<0.01) and slower heart rate (16%, P<0.05) than controls. Plasma ADMA rose (1.9-fold, P<0.01) and nitrite/nitrate concentration decreased 59% (P<0.001). Ventricular contraction (stiffness) increased significantly, with worsening of post-ischemic ventricular dysfunction. In preparations from ADMA-treated rats the coronary vasculature's response to angiotensin II was almost doubled (P<0.01) and the maximal vasorelaxant effect of acetylcholine in aortic rings was significantly lower than in preparations from vehicle-treated rats. In cardiac and aortic tissues eNOS mRNA and ACE mRNA levels were similar in controls and ADMA-treated rats. The increased plasma levels of ADMA presumably cause endothelial dysfunction because of a deficiency in NO production, which also appears involved in the aggravation of myocardial ischemia-reperfusion injury.

Angiotensin II type 1 receptor antagonism improves endothelial vasodilator function in L-NAME-induced hypertensive rats by a kinin-dependent mechanism.

OBJECTIVE: This study was designed to investigate the ability of a chronic blockade of angiotensin II type 1 receptors with losartan to reverse the endothelial dysfunction present in N-nitro-L-arginine methyl ester (L-NAME)-treated hypertensive rats and the possible dependence of this effect on bradykinin B2-receptor activation. METHODS: Rats treated with L-NAME alone (60 mg/kg per day for 8 weeks) or with L-NAME + losartan, L-NAME + icatibant (a bradykinin B2-receptor antagonist) and L-NAME + losartan + icatibant were studied. Losartan, icatibant or losartan + icatibant were co-administered with L-NAME during the last 4 weeks of the experiment. Endothelial nitric oxide synthase gene expression in aortic tissues, plasma nitrite/nitrate concentrations, the relaxant effect of acetylcholine on norepinephrine-precontracted aortic rings and 6-keto-PGF1alpha release from aortic rings were used as markers of the endothelial function. RESULTS: Rats treated with L-NAME alone and L-NAME + icatibant showed, as compared with untreated animals, a clear-cut increase in systolic blood pressure and a decrease of all the markers of endothelial function evaluated. In L-NAME-rats, administration of losartan reduced the systolic blood pressure and restored endothelial nitric oxide synthase gene expression, plasma nitrite/nitrate levels, the relaxant activity of acetylcholine on aortic rings and the generation of 6-keto-PGF1alpha from the aortic tissues. Co-administration of icatibant with losartan blunted the stimulatory effect of losartan on the markers of endothelial function evaluated. CONCLUSION: These results demonstrated that losartan is capable of reversing the endothelial vasodilator dysfunction in L-NAME-induced hypertensive rats, and that the beneficial effect of losartan is mediated by bradykinin B2-receptor activation.

Positive interaction of the beta2-agonist CHF 4226.01 with budesonide in the control of bronchoconstriction induced by acetaldehyde in the guinea-pigs.

Pretreatment of anaesthetized guinea-pigs with either CHF 4226.01 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methylethyl]amino]ethyl] carbostyril hydrochloride), formoterol or budesonide reduced acetaldehyde (AcCHO)-evoked responses in the lungs with a rank order of potency CHF 4226.01 (ED(50) values, from 1.88 to 3.31 pmol) > formoterol (ED(50) values, from 3.03 to 5.51 pmol) >> budesonide (ED(50) values, from 335 to 458 nmol). The duration of action of CHF 4226.01 in antagonizing the airway obstruction elicited by AcCHO was also substantially longer than formoterol (area under the curve) at 10 pmol, 763+/-58 and 480+/-34, respectively; P<0.01). Continuous infusion of a subthreshold dose of AcCHO enhanced the intratracheal pressure (ITP) increases caused by subsequent challenges with substance P (from 9.7+/-0.8 to 27.5+/-1.6 cm H(2)O as a peak, P<0.001). Pretreatment with either CHF 4226.01 or formoterol prevented the sensitizing effect of AcCHO on substance P responses (ED(50) values, 2.85 and 6.11 pmol, respectively; P<0.01). The ED(50) value of budesonide (396 nmol) in preventing AcCHO-evoked ITP increase was reduced when this glucocorticoid was combined with 0.1 pmol CHF 4226.01 (ED(50) 76 nmol; P<0.001). CHF 4226.01/budesonide was two-fold more effective (P<0.01) than the formoterol/budesonide combination. These results suggest that CHF 4226.01/budesonide, by optimizing each other's beneficial potential in the control of pulmonary changes caused by AcCHO in the guinea-pigs, may represent a new fixed combination in asthma.

Angiotensin-converting enzyme inhibition and angiotensin AT1-receptor antagonism equally improve endothelial vasodilator function in L-NAME-induced hypertensive rats.

Male Sprague-Dawley rats given N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water for 8 weeks showed: (1) a clear-cut increase in systolic blood pressure; (2) a consistent decrease of endothelial-cell nitric oxide synthase (eNOS) gene expression in aortic tissue; (3) a marked reduction of plasma nitrite/nitrate concentrations; (4) a reduction of the relaxant activity of acetylcholine (ACh, from 10(-10) to 10(-4) M) on norepinephrine-precontracted aortic rings (reduction by 48+/-5%); (5) a marked decrease (-58%) of the basal release of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) from aortic rings. In L-NAME-treated rats, administration in the last 4 weeks of either the angiotensin-converting enzyme (ACE) inhibitor enalapril (10 mg/kg/day in tap water) or the angiotensin AT(1)-receptor antagonist losartan (10 mg/kg/day in tap water) decreased systolic blood pressure levels, completely restored eNOS mRNA levels in aortic tissue and plasma nitrite/nitrate levels, and allowed a consistent recovery of both the relaxant activity of acetylcholine and the generation of 6-keto-PGF1alpha. Coadministration of icatibant, a bradykinin B(2)-receptor antagonist (200 microg/kg/day), with enalapril blunted the stimulatory effect of the ACE inhibitor on eNOS mRNA expression, circulating levels of nitrite/nitrate, the relaxant activity of ACh and the release of 6-keto-PGF1alpha in L-NAME-treated rats. The generation of 6-keto-PGF1alpha from aortic rings was also decreased in rats coadministered icatibant with losartan. These findings indicate that (1) the ACE inhibitor enalapril and the angiotensin AT(1)-receptor blocker losartan are equally effective to reverse NAME-induced endothelial dysfunction; (2) the beneficial effect of enalapril on the endothelial vasodilator function in L-NAME-treated rats is mediated by bradykinin B(2)-receptor activation; and (3) the enhanced endothelial generation of prostacyclin induced by losartan in L-NAME rats is also mediated by bradykinin B(2)-receptor activation.

NCX 4016, a nitric oxide-releasing aspirin, modulates adrenergic vasoconstriction in the perfused rat tail artery.

1. The ability of the nitric oxide (NO)-releasing aspirin, NCX 4016, to control vasoconstrictor responses induced by electrical field stimulation (TNS) or by exogenous norepinephrine (NE) was investigated in perfused rat tail artery with intact endothelium. 2. NCX 4016 (25, 50 and 100 microM) dose-dependently antagonized the vasoconstriction caused by TNS (from 0.5 to 64 Hz) and by NE (from 0.01 to 10 microM). The vasorelaxant activity of NCX 4016 (100 microM) in NE-precontracted arteries was concomitant with a marked increase of tissue cyclic GMP (4.9 fold, P<0.001) and was significantly antagonized by the inhibitors of soluble guanylate cyclase, methylene blue and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one. 3. The effect of NCX 4016 was endothelium NO-independent since, in preparations perfused with N(G)-monomethyl-L-arginine (10 microM), this compound prevented the rise in basal perfusion pressure and reversed the accentuation of vasoconstrictor responses caused by NO synthase inhibition. 4. Aspirin-moiety released by NCX 4016 inhibited the 6-keto-PGF(1alpha) formation without interfering with the vasorelaxant activity of NCX 4016, while aspirin (100 microM) was devoid of any activity against vasoconstriction induced by both TNS and NE in perfused rat tail artery. 5. NCX 4016 moderated adrenergic vasoconstriction in perfused rat tail arteries by a direct donation of NO without involving the relaxant factors such as PGI(2) and NO from endothelial cells. 6. The results obtained with NCX 4016 in perfused rat tail artery bears some therapeutical potential in conditions associated with vascular smooth muscle hyperreactivity to adrenergic stimulation.

Neuronal sensitization and its behavioral correlates in a rat model of neuropathy are prevented by a cyclic analog of orphenadrine.

N-methyl-D-aspartic acid (NMDA) is an agonist at the homonymous receptor implicated in the development of neuronal sensitization and its behavioral correlates. An effective modulation of the NMDA effects, achieved also by uncompetitive antagonists, could contribute to controlling pain symptoms in several neuropathic syndromes. Because nefopam is a known analgesic derivative of orphenadrine and of its congener diphenhydramine, both uncompetitive NMDA receptor antagonists, we tested the effect of nefopam on the developing pain and neuronal anomalies in an animal model of chronic pain with NMDA receptor involvement. A single intraperitoneal injection of nefopam was administered twenty minutes prior to the chronic constriction injury of the sciatic nerve (CCI rats). In the first 10 days, nefopam (30 mg/kg) significantly decreased behavioral signs of neuropathic pain and the stimulus-evoked electrophysiological anomalies in recordings at 14 days, with only slight manifestation afterwards. The dose of 20 mg/kg was ineffective. Nefopam injected after constriction was ineffective. In normal non-operated rats, Nefopam had no effect on the electrophysiological and behavioral parameters. Iontophoretic nefopam (1 mM, 50-80 nA, positive current) in normal rats did not change the spontaneous neuronal activity, but reduced the mean response to noxious stimuli and the concurrent iontophoretic NMDA evoked activity. In CCI rats, iontophoretic nefopam did not significantly modify the spontaneous hyperactivity but reduced significantly both the frequency of the responses to noxious stimuli, and the duration of the afterdischarge. We propose that nefopam exerts a preventive analgesic effect, with a possible role in modulating NMDA receptor-mediated effects in central sensitization.

Nitric oxide-releasing aspirin inhibits vasoconstriction in perfused tail artery of normotensive and spontaneously hypertensive rats.

The aim of this study was to investigate the capacity of the 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX 4016), a nitric oxide (NO)-releaser derivative of aspirin, to decrease blood pressure in spontaneously hypertensive rats (SHR) and to counteract the adrenergic vasoconstriction in perfused tail artery of these animals. Oral treatment for 10 consecutive days with NCX 4016 (100 micromol/kg) in SHR and their genetic controls Wistar Kyoto (WKY) rats resulted in a reduction of blood pressure in SHR but not in WKY rats. In SHR, the NCX 4016 treatment increased the serum nitrite/nitrate and diminished the serum thromboxane B2, whereas aspirin did not change blood pressure but abolished the serum thromboxane B2. Perfused tail arteries excised from vehicle-treated SHR exhibited a significant impairment of endothelium-dependent vasorelaxant function. These vessels, prepared from SHR or WKY rats treated orally with NCX 4016 (10, 30 and 100 micromol/kg for 7 consecutive days), revealed a dose-dependent decrease in vasoconstriction in response to transmural nerve stimulation and norepinephrine, whereas aspirin was ineffective. Furthermore, in tail arteries of both SHR and WKY rats treated orally with NCX 4016 (100 micromol/kg for 7 consecutive days), the cGMP increased significantly. In conclusion, NCX 4016, by releasing NO and increasing cGMP in vascular tissue, reduces sympathetic-mediated vasoconstriction in resistance vessels and lowers blood pressure in SHR.

Enalapril and quinapril improve endothelial vasodilator function and aortic eNOS gene expression in L-NAME-treated rats.

Endothelial dysfunction ensuing inhibition of nitric oxide synthase (NOS) was investigated in male Sprague-Dawley rats given N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water for 8 weeks. Age-matched rats served as controls. L-NAME-treated rats, as compared to control animals, showed: (1) a clear-cut increase in systolic blood pressure; (2) a consistent decrease of endothelial-cell NOS (eNOS) gene expression in aortic tissue; (3) a reduction of the relaxant activity of acetylcholine (ACh, from 10(-10) to 10(-4) M) on norepinephrine-precontracted aortic rings (reduction by 52+/-5%); (4) a marked decrease (-50%) of the basal release of 6-keto-prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) from aortic rings. In L-NAME-treated rats, administration in the last 2 weeks of either the angiotensin-converting enzyme inhibitor enalapril (1 mg/kg/day) or the cognate drug quinapril (1 mg/kg/day) decreased systolic blood pressure levels, completely restored eNOS mRNA levels in aortic tissue, and allowed a consistent recovery of both the relaxant activity of ACh and the generation of 6-keto-PGF(1 alpha). No difference was present in the ability of the two angiotensin-converting enzyme inhibitors to reverse NAME-induced endothelial dysfunction. These findings indicate that L-NAME-induced hypertension in the rat relies on the marked impairment of the endothelial vasodilator function, with an ensuing contribution by a decreased production of prostacyclin by the endothelial cells. Angiotensin-converting enzyme inhibition by enalapril or quinapril was equally effective in improving endothelial vasodilator function, prostacyclin endothelial production and restoring aortic eNOS mRNA.

Activity of a new hydrogen sulfide-releasing aspirin (ACS14) on pathological cardiovascular alterations induced by glutathione depletion in rats.

We investigated the effects of the hydrogen sulfide (H2S)-releasing derivatives of aspirin (ACS14) and salicylic acid (ACS21) in a rat model of metabolic syndrome induced by glutathione (GSH) depletion, causing hypertension and other pathological cardiovascular alterations. GSH depletion was induced in normal rats by the GSH-synthase inhibitor buthionine sulfoximine (BSO, 30 mmol/L day for seven days in the drinking water). Systolic blood pressure and heart rate were measured daily by the tail-cuff method, and plasma thromboxane B2, 6-keto-prostaglandin F(2α), 8-isoprostane, GSH, insulin and glucose were determined at the end of the seven-day BSO schedule. In addition, ischemia/reperfusion-induced myocardial dysfunction and endothelial dysfunction were assayed on isolated heart and aortic rings, respectively. Unlike aspirin and salicylic acid, ACS14 and ACS21 reduced BSO-induced hypertension, also lowering plasma levels of thromboxane B2, 8-isoprostane and insulin, while GSH remained in the control range. Neither ACS14 nor ACS21 caused gastric lesions. Both restored the endothelial dysfunction observed in aortic rings from BSO-treated rats, and in ischemia/reperfusion experiments they lowered left ventricular end-diastolic pressure, consequently improving the developed pressure and the maximum rise and fall of left ventricular pressure. Together with this improvement of heart mechanics there were reductions in the activity of creatine kinase and lactate dehydrogenase in the cardiac perfusate. This implies that H2S released by both ACS14 and ACS21 was involved in protecting the heart from ischemia/reperfusion, and significantly limited vascular endothelial dysfunction in aortic tissue and the related hypertension.

Positive interaction of the novel beta2-agonist carmoterol and tiotropium bromide in the control of airway changes induced by different challenges in guinea-pigs.

This study evaluated the bronchodilating activity of the beta(2)-agonist carmoterol and the muscarinic M(3)-antagonist tiotropium, given intratracheally alone or in combination in anaesthetized artificially ventilated normal and actively sensitized guinea-pigs. Carmoterol (0.3-100pmol) and tiotropium (10-1000pmol) were superfused (0.01ml/min) for 5min before challenges with acetylcholine (20mug/kg i.v.), histamine (10mug/kg i.v.) or ovalbumin (5mg/kg i.v.). Both compounds given alone were markedly active against all the challenges. Tiotropium resulted more effective towards cholinergic challenge and carmoterol was very potent against histamine and ovalbumin-induced reaction, being effective already at 1pmol. In the presence of tiotropium, the bronchodilating activity of carmoterol was significantly augmented. The ED(50) value of carmoterol on the acetylcholine challenge was reduced by about 10 and 28 times (0.1 and 0.3pmol of tiotropium), that on the histamine one by 4.5 and 13 times (1 and 3pmol of tiotropium) and that on the ovalbumin-induced one by 8 and 25 times (10 and 30pmol of tiotropium). A positive interaction was also evident when other parameters were evaluated. The histamine-induced release of thromboxane B(2) was markedly reduced (56%, P<0.001) by combining completely ineffective doses of the two drugs (0.3 and 3pmol for carmoterol and tiotropium, respectively). In ovalbumin-challenged animals the time to death, amounting in control animals to 7.2+/-0.9min, was dose-dependently prolonged up to achieve complete protection from death with combination of 1 and 30pmol of carmoterol and tiotropium, respectively. The favorable interaction between carmoterol and tiotropium can represent a good option in the control of bronchopulmonary diseases marked by an increase of airway resistances.

Nitric oxide and prostacyclin pathways: an integrated mechanism that limits myocardial infarction progression in anaesthetized rats.

Nitric oxide (NO) and cyclooxygenase-derived prostaglandins, such as prostacyclin (PGI2), are involved in vascular homeostasis. To better understand the reciprocal role of both NO and PGI2 on myocardial infarction in the rat, we have investigated the cardioprotective effect of nitro-naproxen, isosorbide dinitrate (ISDN), L-arginine, defibrotide and naproxen. In this study, male Wistar rats were treated orally once a day for 5 consecutive days with the compounds under investigation and then, under anesthesia, the animals were subjected to acute myocardial ischemia (30 min) and reperfusion (120 min). Systemic blood pressure, left ventricular pressure and related parameters of cardiac mechanics were recorded. Ventricular arrhythmias and infarct size of the left ventricular wall were also evaluated. Furthermore, cardiac myeloperoxidase (MPO) and plasma creatine phosphokinase (CPK) activities were determined. Defibrotide, nitro-naproxen, ISDN and L-arginine all provided a cardioprotection characterized by significant prevention of arrhythmias with high survival rate of the rats. Infarct size restriction was paralleled by reduction of both cardiac MPO and plasma CK. Cardioprotection of nitro-naproxen, ISDN and L-arginine involve nitrites/nitrates and PGI2-increased in the circulation associated to a reduction of thromboxane B2 (TXB2) in the blood. Defibrotide displays a cardioprotection by increasing PGI2 release and by reducing TXB2 in the blood. Naproxen was devoid a lower protecting activity on myocardial infarction, and PGI2 inhibition may have played a critical role in this context. The results suggested that the increase of both NO and PGI2 brings about a cascade of integrated cellular and molecular events which are of paramount importance in prevention of myocardial ischemic insult.