Beneficial Role of Low-Intensity Laser Irradiation on Neural ß-tubulin III Protein Expression in Human Bone Marrow Multipotent Mesenchymal Stromal Cells.

The purpose of the present study was to evaluate the neural protein expression pattern of human multipotent mesenchymal stromal cells (hMSCs) treated with forskolin (free-form/FF). The study investigated forskolin's capacity to enhance intracellular levels of cyclic adenosine monophosphate (cAMP) by activating adenylate cyclase and probably by inducing neuron-like cells in vitro. In addition, because nanotechnology is a growing field of tissue engineering, we also assessed the action of a new system called the nanostructured-forskolin (NF) to examine the improvement of drug delivery. Afterwards, the cells were submitted to low-level laser irradiation to evaluate possible photobiostimulatory effects. Investigations using the immunofluorescence by confocal microscopy and Western blot methods revealed the expression of the neuronal marker ß-tubulin III. Fluorescence intensity quantification analysis using INCell Analyzer System for ß-tubulin III was used to examine significant differences. The results showed that after low-level laser irradiation exposure, there was a tendency to increase the ß-tubulin III expression in all groups, as expected in the photobiostimulation process. Notably, this process induced for irradiation was more pronounced in irradiated nanoforskolin cells (INF) compared to non-irradiated free-forskolin control cells (NFFC). However, there was also an increase in ß-tubulin III protein expression in the groups: irradiated nanocontrol cells (INC) compared to non-irradiated free-forskolin control cells (NFF) and after treatment with non-irradiated free-forskolin (NFF) and non-irradiated nanoforskolin (NNFC). We concluded that the methods using low-level laser irradiation and/or nanoparticles showed an up-regulation of neural-protein expression in hMSCs that could be used to facilitate cellular therapy protocols in the near future.

Functional ß2-adrenoceptors in rat left atria: effect of foot-shock stress.

Altered sensitivity to the chronotropic effect of catecholamines and a reduction in the ß1/ß2-adrenoceptor ratio have previously been reported in right atria of stressed rats, human failing heart, and aging. In this report, we investigated whether left atrial inotropism was affected by foot-shock stress. Male rats were submitted to 3 foot-shock sessions and the left atrial inotropic response, adenylyl cyclase activity, and ß-adrenoceptor expression were investigated. Left atria of stressed rats were supersensitive to isoprenaline when compared with control rats and this effect was abolished by ICI118,551, a selective ß2-receptor antagonist. Schild plot slopes for the antagonism between CGP20712A (a selective ß1-receptor antagonist) and isoprenaline differed from unity in atria of stressed but not control rats. Atrial sensitivity to norepinephrine, as well as basal and forskolin- or isoprenaline-stimulated adenylyl cyclase activities were not altered by stress. The effect of isoprenaline on adenylyl cyclase stimulation was partially blocked by ICI118,551 in atrial membranes of stressed rats. These findings indicate that foot-shock stress equally affects inotropism and chronotropism and that ß2-adrenoceptor upregulation contributes to the enhanced inotropic response to isoprenaline.

Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification / Acúmulo de lipídios intracitoplasmáticos, desenvolvimento e criotolerância de embriões bovinos produzidos in vitro e tratados com diferentes concentrações de forskolin antes da vitrificação

The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5µM (Forsk 2.5 group), 5.0µM (Forsk 5.0 group) or 10.0µM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 µM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.(AU)

Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5µM (grupo Forsk 2,5), 5,0µM (grupo Forsk 5,0) ou 10,0µM (grupo Forsk 10,0). Os embriões do grupo controle foram cultivados na ausência de forskolin. No dia 7 do cultivo, os blastocistos expandidos foram corados com o corante lipofílico Sudan Black B para a determinação do teor de lípidos intracitoplasmáticos ou foram criopreservados através do protocolo Vitri-Ingá®. Não foi observada diferença significativa (P>0,05) na taxa de produção de blastocistos entre o grupo Controle (44,9%) e os demais tratamentos, todavia observou-se menor produção de embriões (P<0,05) no grupo Forsk 10,0 (38,8%) em comparação com os grupos Forsk 2,5 (50,5%) e Forsk 5,0 (54,7%). A quantidade de lipídeos intracitoplasmáticos do grupo Controle (1,00±0,03) foi semelhante (P>0,05) a dos grupos Forsk 2,5 (0,92±0,03) e Forsk 10,0 (1,06±0,03); no entanto, o acúmulo de lípidos nos blastocistos do grupo Forsk 5.0 (0,82 ± 0,04) foi menor do que no grupo controle (P<0,05). A partir destes resultados, o grupo Forsk 5,0 foi testado quanto à criotolerância e foi observado que a taxa de re-expansão da blastocele 24 horas após o aquecimento foi maior (P<0,05) no grupo Forsk 5,0 (72,2%) quando comparado ao grupo Controle (46,2%). Em conclusão, o pré-tratamento com forskolin na concentração de 5,0 µM durante 24 horas antes da vitrificação foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e, consequentemente, melhorou a criotolerância de embriões bovinos produzidos in vitro.(AU)

Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification / Acúmulo de lipídios intracitoplasmáticos, desenvolvimento e criotolerância de embriões bovinos produzidos in vitro e tratados com diferentes concentrações de forskolin antes da vitrificação

The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5µM (Forsk 2.5 group), 5.0µM (Forsk 5.0 group) or 10.0µM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 µM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.(AU)

Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5µM (grupo Forsk 2,5), 5,0µM (grupo Forsk 5,0) ou 10,0µM (grupo Forsk 10,0). Os embriões do grupo controle foram cultivados na ausência de forskolin. No dia 7 do cultivo, os blastocistos expandidos foram corados com o corante lipofílico Sudan Black B para a determinação do teor de lípidos intracitoplasmáticos ou foram criopreservados através do protocolo Vitri-Ingá®. Não foi observada diferença significativa (P>0,05) na taxa de produção de blastocistos entre o grupo Controle (44,9%) e os demais tratamentos, todavia observou-se menor produção de embriões (P<0,05) no grupo Forsk 10,0 (38,8%) em comparação com os grupos Forsk 2,5 (50,5%) e Forsk 5,0 (54,7%). A quantidade de lipídeos intracitoplasmáticos do grupo Controle (1,00±0,03) foi semelhante (P>0,05) a dos grupos Forsk 2,5 (0,92±0,03) e Forsk 10,0 (1,06±0,03); no entanto, o acúmulo de lípidos nos blastocistos do grupo Forsk 5.0 (0,82 ± 0,04) foi menor do que no grupo controle (P<0,05). A partir destes resultados, o grupo Forsk 5,0 foi testado quanto à criotolerância e foi observado que a taxa de re-expansão da blastocele 24 horas após o aquecimento foi maior (P<0,05) no grupo Forsk 5,0 (72,2%) quando comparado ao grupo Controle (46,2%). Em conclusão, o pré-tratamento com forskolin na concentração de 5,0 µM durante 24 horas antes da vitrificação foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e, consequentemente, melhorou a criotolerância de embriões bovinos produzidos in vitro.(AU)

Fast transdifferentiation of human Wharton's jelly mesenchymal stem cells into neurospheres and nerve-like cells.

BACKGROUND: The human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs) represent a tool for cell-based therapies and regenerative medicine. hWJ-MSCs form neurospheres (NSs) within 3-7 days. No data is available to establish the neuro-phenotypic markers and time of formation of nerve-like (NLCs) and glial cells from NSs derived from hWJ-MSCs. NEW METHOD: hWJ-MSCs were incubated with Fast-N-Spheres medium for 24 and 72h. The new formed NSs were in turn incubated with forskolin in neurogenic NeuroForsk medium for 1-7days. RESULTS: hWJ-MSCs cultured with Fast-N-Spheres medium trans-differentiated into NSs in just 24h compared to 72h for hWJ-MSCs cultured with classic growth factor medium. The NSs generated from the Fast-N-Spheres medium expressed reduced levels SOX2, OCT4 and NANOG, as markers of pluripotency compared to undifferentiated hWJ-MSCs. The formed NSs exposed to NeuroForsk medium differentiated into NLCs in 4days as evidenced by high levels of protein expression of the neuronal markers, and no expression of the glial marker GFAP. COMPARISON WITH EXISTING METHOD(S): Currently, the formation and harvest of NSs is expensive and time consuming. Published protocols require 3-7days to form NSs from whole human umbilical cord MSCs. We report for the first time, to our knowledge, the differentiation of NSs-derived from hWJ-MSCs into NLCs. CONCLUSIONS: The fastest method to obtain NSs and NLCs from hWJ-MSCs takes only five days using the two-step incubation media Fast-N-Spheres and NeuroForsk.

Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification

ABSTRACT: The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5M (Forsk 2.5 group), 5.0M (Forsk 5.0 group) or 10.0M (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P 0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P 0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P 0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 M for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.

RESUMO: Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5M (grupo Forsk 2,5), 5,0M (grupo Forsk 5,0) ou 10,0M (grupo Forsk 10,0). Os embriões do grupo controle foram cultivados na ausência de forskolin. No dia 7 do cultivo, os blastocistos expandidos foram corados com o corante lipofílico Sudan Black B para a determinação do teor de lípidos intracitoplasmáticos ou foram criopreservados através do protocolo Vitri-Ingá®. Não foi observada diferença significativa (P>0,05) na taxa de produção de blastocistos entre o grupo Controle (44,9%) e os demais tratamentos, todavia observou-se menor produção de embriões (P 0,05) no grupo Forsk 10,0 (38,8%) em comparação com os grupos Forsk 2,5 (50,5%) e Forsk 5,0 (54,7%). A quantidade de lipídeos intracitoplasmáticos do grupo Controle (1,00±0,03) foi semelhante (P>0,05) a dos grupos Forsk 2,5 (0,92±0,03) e Forsk 10,0 (1,06±0,03); no entanto, o acúmulo de lípidos nos blastocistos do grupo Forsk 5.0 (0,82 ± 0,04) foi menor do que no grupo controle (P 0,05). A partir destes resultados, o grupo Forsk 5,0 foi testado quanto à criotolerância e foi observado que a taxa de re-expansão da blastocele 24 horas após o aquecimento foi maior (P 0,05) no grupo Forsk 5,0 (72,2%) quando comparado ao grupo Controle (46,2%). Em conclusão, o pré-tratamento com forskolin na concentração de 5,0 M durante 24 horas antes da vitrificação foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e, consequentemente, melhorou a criotolerância de embriões bovinos produzidos in vitro.

The Labdane Ent-3-Acetoxy-Labda-8(17), 13-Dien-15-Oic Decreases Blood Pressure In Hypertensive Rats / O Labdano Ácido Ent-3-Acetóxi-Labda-8(17), 13-Dieno-15-Óico Reduz Pressão Arterial Em Ratos Hipertensos

Abstract Background: Labdane-type diterpenes induce lower blood pressure via relaxation of vascular smooth muscle; however, there are no studies describing the effects of labdanes in hypertensive rats. Objective: The present study was designed to investigate the cardiovascular actions of the labdane-type diterpene ent-3-acetoxy-labda-8(17), 13-dien-15-oic acid (labda-15-oic acid) in two-kidney 1 clip (2K-1C) renal hypertension. Methods: Vascular reactivity experiments were performed in aortic rings isolated from 2K-1C and normotensive (2K) male Wistar rats. Nitrate/nitrite (NOx) measurement was performed in aortas by colorimetric assay. Blood pressure measurements were performed in conscious rats. Results: Labda-15-oic acid (0.1-300 µmol/l) and forskolin (0.1 nmol/l - 1 µmol/l) relaxed endothelium-intact and endothelium-denuded aortas from both 2K-1C and 2K rats. Labda-15-oic acid was more effective at inducing relaxation in endothelium-intact aortas from 2K pre-contracted with phenylephrine when compared to the endothelium-denuded ones. Forskolin was more potent than labda-15-oic acid at inducing vascular relaxation in arteries from both 2K and 2K-1C rats. Labda-15-oic acid-induced increase in NOx levels was lower in arteries from 2K-1C rats when compared to 2K rats. Intravenous administration of labda-15-oic acid (0.3-3 mg/kg) or forskolin (0.1-1 mg/kg) induced hypotension in conscious 2K-1C and 2K rats. Conclusion: The present findings show that labda-15-oic acid induces vascular relaxation and hypotension in hypertensive rats.

Resumo Fundamento: Diterpenos do tipo labdano induzem uma queda da pressão arterial por meio do relaxamento do músculo liso vascular; todavia, não há estudos que descrevam os efeitos de labdanos em ratos hipertensos. Objetivo: O presente estudo foi desenvolvido para investigar as ações cardiovasculares do labdano ácido ent-3-acetóxi-labda-8(17),13-dieno-15-óico (labda-15-óico) na hipertensão renal dois rins-1 clipe (2R-1C). Métodos: Foram feitos experimentos de reatividade vascular em anéis aórticos isolados de ratos machos 2R-1C e normotensos (2R). A medição de Nitrato/Nitrito (NOx) foi feita nas aortas por meio de ensaio colorimétrico. As medidas de pressão arterial foram feitas em ratos conscientes. Resultados: O ácido labda-15-óico (0,1 - 300 µmol/l) e a forscolina (0,1 nmol/l - 1 µmol/l) relaxaram as aortas com endotélio intacto e as aortas sem endotélio dos ratos 2R-1C e 2R. O labda-15-óico mostrou-se mais eficaz na indução do relaxamento em aortas com endotélio intacto de 2R pré-contraídas com fenilefrina em comparação àquelas sem endotélio. A forscolina mostrou-se mais potente do que o ácido labda-15-óico na indução do relaxamento vascular nas artérias tanto de ratos 2R-1C quanto de ratos 2R. O aumento dos níveis de NOx induzido pelo ácido labda-15-óico foi menor nas artérias de ratos 2R-1C em comparação a ratos 2R. A administração intravenosa de ácido labda-15-óico (0,3-3 mg/kg) ou forscolina (0,1-1 mg/kg) induziu hipertensão em ratos 2R-1C e 2R conscientes. Conclusão: Os presentes resultados mostram que o labda-15-óico induz relaxamento vascular e hipotensão em ratos hipertensos.

In vitro embryos production after oocytes treatment with forskolin.

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Cryosurvival and pregnancy rates after exposure of IVF-derived Bos indicus embryos to forskolin before vitrification.

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 µM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration

INTRODUCTION: Results from our laboratory have demonstrated that intracerebroventricular administration of sildenafil to conscious rats promoted a noticeable increase in both lumbar sympathetic activity and heart rate, with no change in the mean arterial pressure. The intracerebroventricular administration of sildenafil may have produced the hemodynamic effects by activating sympathetic preganglionic neurons in the supraspinal regions and spinal cord. It is well documented that sildenafil increases intracellular cGMP levels by inhibiting phosphodiesterase type 5 and increases cAMP levels by inhibiting other phosphodiesterases. OBJECTIVE: To examine and compare, in conscious rats, the hemodynamic response following the intrathecal administration of sildenafil, 8-bromo-cGMP (an analog of cGMP), forskolin (an activator of adenylate cyclase), or dibutyryl-cAMP (an analog of cAMP) in order to elucidate the possible role of the sympathetic preganglionic neurons in the observed hemodynamic response. RESULTS: The hemodynamic responses observed following intrathecal administration of the studied drugs demonstrated the following: 1) sildenafil increased the mean arterial pressure and heart rate in a dose-dependent manner, 2) increasing doses of 8-bromo-cGMP did not alter the mean arterial pressure and heart rate, 3) forskolin did not affect the mean arterial pressure but did increase the heart rate and 4) dibutyryl-cAMP increased the mean arterial pressure and heart rate, similar to the effect observed following the intrathecal injection of the highest dose of sildenafil. CONCLUSION: Overall, the findings of the current study suggest that the cardiovascular response following the intrathecal administration of sildenafil to conscious rats involves the inhibition of phosphodiesterases other than phosphodiesterase type 5 that increase the cAMP level and the activation of sympathetic preganglionic neurons.