Role of calcium channels in the protective effect of hydrogen sulfide in rat cardiomyoblasts.

BACKGROUND: Hydrogen sulfide contributes to the reduction of oxidative stress-related injury in cardiomyocytes but the underlying mechanism is still unclear. AIMS: Here we investigated the role of voltage-operated calcium channels (VOCCs) as mediators of the beneficial effect of H2S against oxidative stress in cultured rat cardiomyoblasts (H9c2). METHODS: Intracellular calcium signals were measured by fluorimetric live cell imaging and cell viability by colorimetric assay. RESULTS: Treatment with H2S donor (NaHS 10 µM) or Nifedipine (10 µM) decreased resting intracellular calcium concentration [Ca]i, suggesting that L-type VOCCs are negatively modulated by H2S. In the presence of Nifedipine H2S was still able to lower [Ca]i, while co-incubation with Nifedipine and Ni(2+) 100 µM completely prevented H2S-dependent [Ca]i decrease, suggesting that both L-type and T-type VOCCs are inhibited by H2S. In addition, in the same experimental conditions, H2S triggered a slow increase of [Ca]i whose molecular nature remains to be clarified. Pretreatment of H9c2 with NaHS (10 µM) significantly prevented cell death induced by H2O2. This effect was mimicked by pretreatment with L-Type calcium channel inhibitor Nifedipine (10 µM). CONCLUSIONS: The data provide the first evidence that H2S protects rat cardiomyoblasts against oxidative challenge through the inhibition of L-type calcium channels.

Post-ischemic early acidosis in cardiac postconditioning modifies the activity of antioxidant enzymes, reduces nitration, and favors protein S-nitrosylation.

Postconditioning (PostC) modifies the early post-ischemic pH, redox environment, and activity of enzymes. We hypothesized that early acidosis in PostC may affect superoxide dismutase (SOD) and catalase (CAT) activities, may reduce 3-nitrotyrosine (3-NT) protein levels, and may increase S-nitrosylated (SNO) protein levels, thus deploying its protective effects. To verify this hypothesis, we studied the early (7(th) min) and late (120(th) min) phases of reperfusion (a) endogenous SOD and CAT activities and (b) 3-NT protein levels and SNO protein levels. Isolated rat hearts underwent 30-min ischemia/120-min reperfusion (I/R) or PostC (5 cycles of 10-s I/R at the beginning of 120-min reperfusion) either with or without exogenous CAT or SOD infused during the initial 3 min of reperfusion. The effects of early reperfusion with acid buffer (AB, pH 6.8) on endogenous antioxidant enzymes were also tested. Pressure, infarct size, and lactate dehydrogenase release were also measured. At the 7(th) min, PostC induced a significant decrease in SOD activity with no major change both in Mn and Cu/Zn SOD levels and in CAT activity and level. PostC also reduced 3-NT and increased SNO levels. Exogenous SOD, but not CAT, abolished PostC cardioprotection. In late reperfusion (120-min), I/R increased SOD activity but decreased CAT activity and Cu/Zn SOD levels; these effects were reversed by PostC; 3-NT was not affected, but SNO was increased by PostC. AB reproduced PostC effects on antioxidant enzymes. The conclusions are as follows: PostC downregulates endogenous SOD and preserves CAT activity, thus increasing SNO and reducing 3-NT levels. These effects are triggered by early post-ischemic acidosis. Yet acidosis-induced SOD downregulation may limit denitrosylation, thus contributing to PostC triggering. Hence, exogenous SOD, but not CAT, interferes with PostC triggering. Prolonged SOD downregulation and SNO increase may contribute to PostC and AB beneficial effects.

Physiological and pharmacological features of the novel gasotransmitter: hydrogen sulfide.

Hydrogen sulfide (H(2)S) has been known for hundreds of years because of its poisoning effect. Once the basal bio-production became evident its pathophysiological role started to be investigated in depth. H(2)S is a gas that can be formed by the action of two enzymes, cystathionine gamma-lyase and cystathionine beta-synthase, both involved in the metabolism of cysteine. It has several features in common with the other two well known "gasotransmitters" (nitric oxide and carbon monoxide) in the biological systems. These three gasses share some biological targets; however, they also have dissimilarities. For instance, the three gases target heme-proteins and open K(ATP) channels; H(2)S as NO is an antioxidant, but in contrast to the latter molecule, H(2)S does not directly form radicals. In the last years H(2)S has been implicated in several physiological and pathophysiological processes such as long term synaptic potentiation, vasorelaxation, pro- and anti-inflammatory conditions, cardiac inotropism regulation, cardioprotection, and several other physiological mechanisms. We will focus on the biological role of H(2)S as a molecule able to trigger cell signaling. Our attention will be particularly devoted on the effects in cardiovascular system and in cardioprotection. We will also provide available information on H(2)S-donating drugs which have so far been tested in order to conjugate the beneficial effect of H(2)S with other pharmaceutical properties.

Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts.

Postconditioning (PostC) may limit mitochondrial damage and apoptotic signaling. We studied markers of apoptosis and mitochondrial protection in isolated rat hearts, which underwent a) perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hour reperfusion (R), or c) PostC protocol (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia). Markers were studied in cytosolic (CF) and/or mitochondrial (MF) fractions. In CF, while pro-apoptotic factors (cytochrome c and caspase-3) were reduced, the anti-apoptotic markers (Bcl-2 and Pim-1) were increased by PostC, compared to the I/R group. Accordingly, phospho-GSK-3beta and Bcl-2 levels increased in mitochondria of PostC group. Moreover, I/R reduced the level of mitochondrial structural protein (HSP-60) in MF and increased in CF, thus suggesting mitochondrial damage and HSP-60 release in cytosol, which were prevented by PostC. Electron microscopy confirmed that I/R markedly damaged cristae and mitochondrial membranes; damage was markedly reduced by PostC. Finally, total connexin-43 (Cx43) levels were reduced in the CF of the I/R group, whereas phospho-Cx43 level resulted in higher levels in the MF of the I/R group than the Sham group. PostC limited the I/R-induced increase of mitochondrial phospho-Cx43. Data suggest that PostC i) increases the levels of anti-apoptotic markers, including the cardioprotective kinase Pim-1, ii) decreases the pro-apoptotic markers, e.g. cytochrome c, iii) preserves the mitochondrial structure, and iv) limits the migration of phospho-Cx43 to mitochondria.

Effects of a protocol of ischemic postconditioning and/or captopril in hearts of normotensive and hypertensive rats.

Brief periods (a few seconds) of cyclic coronary occlusions applied early in reperfusion induce a cardioprotection against infarct size, called postconditioning (PostC) in which B(2)-bradykinin receptors play a pivotal role. Since angiotensin-converting enzyme (ACE) inhibitors reduce degradation of kinins, we studied the effects of PostC on infarct size and postischemic myocardial dysfunction in both normotensive (WKY) and spontaneously hypertensive rats (SHR) acutely or chronically treated with the ACE inhibitor Captopril. Isolated hearts from SHR and WKY rats were subjected to the following protocols: (a) ischemia for 30- and 120-min reperfusion (I/R); (b) I/R + PostC protocol (5-cycles 10-s I/R); (c) pretreatment with Captopril for 4-weeks before to subject the hearts to I/R with or without PostC maneuvers. Some SHR hearts were treated with Captopril during the 20- or 40-min early reperfusion with or without PostC maneuvers. Cardiac function was assessed in vivo with echocardiography. Left ventricular pressure and infarct size were measured ex vivo. Chronic Captopril significantly reduced left ventricular hypertrophy in SHR, and reduced infarct size in both WKY and SHR hearts. PostC maneuvers significantly reduced infarct size in WKY, but not in SHR hearts. Yet, PostC slightly improved postischemic systolic function in untreated SHR. Captopril given in reperfusion was unable to limit I/R injury in SHR hearts. Data show that PostC protection against infarct size is blunted in SHR and that PostC is unable to add its protective effect to those of chronic Captopril, which per se reduces cardiac hypertrophy and heart susceptibility to I/R insult.

Arachidonic acid-induced Ca2+ entry is involved in early steps of tumor angiogenesis.

Growth factor-induced intracellular calcium signals in endothelial cells regulate cytosolic and nuclear events involved in the angiogenic process. Among the intracellular messengers released after proangiogenic stimulation, arachidonic acid (AA) plays a key role and its effects are strictly related to calcium homeostasis and cell proliferation. Here, we studied AA-induced intracellular calcium signals in endothelial cells derived from human breast carcinomas (B-TEC). AA promotes B-TEC proliferation and organization of vessel-like structures in vitro. The effect is directly mediated by the fatty acid without a significant contribution of its metabolites. AA induces Ca(2+)(i) signals in the entire capillary-like structure during the early phases of tubulogenesis in vitro. No such responses are detectable in B-TECs organized in more structured tubules. In B-TECs growing in monolayer, AA induces two different signals: a Ca(2+)(i) increase due to Ca(2+) entry and an inhibition of store-dependent Ca(2+) entry induced by thapsigargin or ATP. An inhibitor of Ca(2+) entry and angiogenesis, carboxyamidotriazole, significantly and specifically decreases AA-induced B-TEC tubulogenesis, as well as AA-induced Ca(2+) signals in B-TECs. We conclude that (a) AA-activated Ca(2+) entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation and tubulogenesis, and it is down-regulated during the reorganization of tumor-derived endothelial cells in capillary-like structures; and (b) inhibition of AA-induced Ca(2+) entry may contribute to the antiangiogenic action of carboxyamidotriazole.

Postconditioning cardioprotection against infarct size and post-ischemic systolic dysfunction is influenced by gender.

Whether cardioprotection by postconditioning (PostC) is gender dependent is not clear. We studied the effect of PostC in terms of both infarct size (IS) and post-ischemic systolic dysfunction (PSD) reduction. Isolated male and female rat hearts were subjected to 10- or 30-min of global ischemia and 120-min of reperfusion, with or without PostC (i.e., 5 cycles of 10-s reperfusion/ischemia immediately after the ischemia). Surprisingly, after 10-min ischemia, IS and PSD were greater in female than male hearts (IS: 21 +/- 2% Vs. 11 +/- 2%; P < 0.01), while PostC attenuated IS and PSD in female hearts only. After 30-min ischemia IS was smaller in female than male hearts (52 +/- 2% Vs. 61 +/- 3%; P < 0.05), whereas PSD was similar in these two groups. PostC reduced IS in both genders, though the effect was smaller (P < 0.05) in females. Yet, PostC reduced PSD in female, but not in male hearts. Contracture development paralleled IS in all groups. To check the effects of buffer perfusion over heart function, additional hearts underwent 150-min buffer perfusion only. Contractile function of these hearts was not significantly affected over time. In conclusion IS, contracture and PSD are differently affected by gender, depending on ischemia duration. Yet, reduction of IS induced by PostC depends on the extension of IS induced by index-ischemia. While in female hearts reduction of PSD paralleled IS reduction, in male it does not occur. Results suggest that improvement of systolic function is mainly due to the anti-necrotic rather than to the anti-stunning effect exerted by PostC.

Hypoxia and hydrogen sulfide differentially affect normal and tumor-derived vascular endothelium.

BACKGROUND: endothelial cells play a key role in vessels formation both under physiological and pathological conditions. Their behavior is influenced by blood components including gasotransmitters (H2S, NO and CO). Tumor cells are subjected to a cyclic shift between pro-oxidative and hypoxic state and, in this scenario, H2S can be both cytoprotective and detrimental depending on its concentration. H2S effects on tumors onset and development is scarcely studied, particularly concerning tumor angiogenesis. We previously demonstrated that H2S is proangiogenic for tumoral but not for normal endothelium and this may represent a target for antiangiogenic therapeutical strategies. METHODS: in this work, we investigate cell viability, migration and tubulogenesis on human EC derived from two different tumors, breast and renal carcinoma (BTEC and RTEC), compared to normal microvascular endothelium (HMEC) under oxidative stress, hypoxia and treatment with exogenous H2S. RESULTS: all EC types are similarly sensitive to oxidative stress induced by hydrogen peroxide; chemical hypoxia differentially affects endothelial viability, that results unaltered by real hypoxia. H2S neither affects cell viability nor prevents hypoxia and H2O2-induced damage. Endothelial migration is enhanced by hypoxia, while tubulogenesis is inhibited for all EC types. H2S acts differentially on EC migration and tubulogenesis. CONCLUSIONS: these data provide evidence for a great variability of normal and altered endothelium in response to the environmental conditions.

Fasting cycles retard growth of tumors and sensitize a range of cancer cell types to chemotherapy.

Short-term starvation (or fasting) protects normal cells, mice, and potentially humans from the harmful side effects of a variety of chemotherapy drugs. Here, we show that treatment with starvation conditions sensitized yeast cells (Saccharomyces cerevisiae) expressing the oncogene-like RAS2(val19) to oxidative stress and 15 of 17 mammalian cancer cell lines to chemotherapeutic agents. Cycles of starvation were as effective as chemotherapeutic agents in delaying progression of different tumors and increased the effectiveness of these drugs against melanoma, glioma, and breast cancer cells. In mouse models of neuroblastoma, fasting cycles plus chemotherapy drugs--but not either treatment alone--resulted in long-term cancer-free survival. In 4T1 breast cancer cells, short-term starvation resulted in increased phosphorylation of the stress-sensitizing Akt and S6 kinases, increased oxidative stress, caspase-3 cleavage, DNA damage, and apoptosis. These studies suggest that multiple cycles of fasting promote differential stress sensitization in a wide range of tumors and could potentially replace or augment the efficacy of certain chemotherapy drugs in the treatment of various cancers.

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells.

Hydrogen sulfide (H(2)S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H(2)S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca(2+) imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H(2)S donor, activates cytosolic calcium (Ca(c)) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1 nM-200 µM) evoked Ca(c) signals also in "normal" human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1-10 µM). Remarkably H(2)S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5mM, 30 min), an inhibitor of the H(2)S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Ca(c) signals induced by VEGF (20 ng/ml). We conclude that H(2)S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment.

Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs). Sodium hydrosulphide (NaHS), a H2S donor, caused an elevation in [Ca2+]i, which disappeared in absence of extracellular Ca2+. NaHSinduced Ca2+ inflow was sensitive to high doses of Gd3+, but not BTP-2. Inhibition of the reverse-mode of the Na+-Ca2+ exchanger (NCX), with KB-R7943 or upon removal of extracellular Na+, abrogated the Ca2+ response to NaHS. Moreover, NaHS-elicited Ca2+ entry was significantly reduced by TEA and glybenclamide, which hinted at the involvement of ATP-dependent K+ (KATP) channels. Conversely, NaHS-evoked Ca2+ signal was not affected by the reducing agent, dithiothreitol. Acute addition of NaHS hindered both Ca2+ release and Ca2+ entry induced by ATP, a physiological agonist of ECs. Consistently, inhibition of endogenous H2S synthesis with DL-propargylglycine impaired ATP-induced Ca2+ inflow, whereas it did not affect Ca2+ mobilization. These data provide the first evidence that H2S may stimulate Ca2+ influx into ECs by recruiting the reverse-mode of NCX and KATP channels. In addition, they show that such gasotransmitter may modulate the Ca2+ signals elicited by physiological stimuli in intact endothelium.