Exercise training restores oxidative stress and nitric oxide synthases in the rostral ventrolateral medulla of renovascular hypertensive rats.

We hypothesize that exercise training (EX) reverses the level of nitric oxide (NO) and oxidative stress into rostral ventrolateral medulla (RVLM) of renovascular hypertensive rats (two kidneys, one clip - 2K1C). Microinjections of L-arginine (5 nmol), L-NAME (10 nmol), or saline (100 nl) were made into RVLM of 2K1C and normotensive (SHAM) rats sedentary (SED) or subjected to swimming for 4 weeks. mRNA expression (by qRT-PCR) of nitric oxide synthases isoforms (nNOS, eNOS, and iNOS), manganese superoxide dismutase (MnSOD), copper and zinc superoxide (Cu/ZnSOD), catalase (CAT), NADPH oxidase subunit p22(phox), concentration of thiobarbituric acid-reactive substances (TBARS), and CAT activity into RVLM were evaluated. The mean arterial pressure was reduced in 2K1C EX compared with that in 2K1C SED rats. L-arginine into RVLM induced hypertensive effect in 2K1C and SHAM SED rats, while L-NAME prevented hypertensive effect only in SHAM-SED. EX reduced hypertensive effect of L-arginine in SHAM and 2K1C rats. mRNA expression of NOS isoforms, p22(phox), and concentration of TBARS were increased while CAT and Cu/ZnSOD expression and CAT activity decreased into RVLM of 2K1C-SED compared with SHAM-SED rats. Additionally, EX reversed mRNA expression of CAT and NOS isoforms, concentration of TBARS, and CAT activity into RVLM of 2K1C-EX rats. These data suggest that the levels of NOS and oxidative stress into RVLM are important to determine the level of hypertension. Furthermore, EX can restore the blood pressure by reversing the levels of NOS and CAT expression, and reducing TBARS concentration into RVLM for the physiological state.

Melanopsin triggers the release of internal calcium stores in response to light.

Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells.

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 micro M). The intracellular Ca2+ chelator BAPTA/AM (1 micro M) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 micro M protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92 percent increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Psoralen activity and binding sites in melanotic and amelanotic human melanoma cells.

The biological activity and specific binding sites of 8-methoxypsoralen (8-MOP) are assayed using two human melanoma cell lines, melanotic SK-Mel 28 and amelanotic C32TG. Long-term (72 hr) treatment with 8-MOP at a concentration of 10(-4)M results in an increase in melanogenesis and a decrease in proliferation, similar in both cell lines. Daily exposure of these cells to ultraviolet A (UVA) irradiation (1.28 mJ/cm(2)) does not enhance the response to the compound. Daily pulse application (30 min daily) of 8-MOP does not promote any response. However, in combination with UVA, 8-MOP pulse treatment becomes as effective as the long-term treatment. A decrease in cell proliferation in the constant presence of 8-MOP is not coupled with apoptosis, since no increase in the number of apoptotic nuclei was observed after the treatment. The flow cytometry indicates that 8-MOP arrests the cells at the G0/G1 phase, irrespective of the presence or absence of UVA light. In view of the lack of epidermal growth factor (EGF) receptors in both cell lines, it is not likely that such an arrest is associated with the down-regulation of EGF receptors by 8-MOP. It is noted that this compound elicits a biphasic cell response, since cell proliferation increases after the first 24-hr treatment, whereas it decreases in the subsequent 48 hr and thereafter. Competition binding assays using 3H-8-MOP disclosed: 1) the specific binding of the compound in both cell lines occurs in the presence or absence of UVA light, and 2) a higher binding rate at low concentrations of the compound is in SK-Mel 28 (72%) rather than C32TG (58%) cells. The competition assays in the presence of UVA suggest a possible occurrence of covalent bindings between psoralen and receptor, as DNA covalent binding accounted to only 3-5% of the total binding in both cell lines.