Vitamin A active metabolite, all-trans retinoic acid, induces spinal cord sensitization. I. Effects after oral administration.

BACKGROUND AND PURPOSE: Retinoic acid is an active metabolite of vitamin A involved in the modulation of the inflammatory and nociceptive responses. The aim of the present study was to analyze the properties of spinal cord neuronal responses of male Wistar rats treated with all-trans retinoic acid (ATRA) p.o. in the normal situation and under carrageenan-induced inflammation. We also studied the expression and distribution of cyclooxygenases (COX) in the spinal cord. EXPERIMENTAL APPROACH: Properties of spinal cord neurons were studied by means of the single motor unit technique. The expression of COX enzymes in the spinal cord was assessed by Western blot analysis and immunohistochemistry. KEY RESULTS: Intensity thresholds for mechanical and electrical stimulation (C-fibers) were significantly lower in animals treated with ATRA than vehicle, either in normal rats or in rats with inflammation. The size of cutaneous receptive fields was also larger in animals treated with ATRA in the normal and inflammatory conditions. The expression of COX-2 enzyme, but not COX-1, was significantly higher in animals treated with ATRA. COX-2 labeling was observed in dorsal horn cells and in ventral horn motoneurons. CONCLUSIONS AND IMPLICATIONS: In conclusion, the oral treatment with ATRA in rats induces a sensitization-like effect on spinal cord neuronal responses similar to that observed in animals with inflammation and might explain the enhancement of allodynia and hyperalgesia observed in previously published behavioral experiments. The mechanism of action involves an over-expression of COX-2, but not COX-1, in dorsal and ventral horn areas of the lumbar spinal cord.

Vitamin A active metabolite, all-trans retinoic acid, induces spinal cord sensitization. II. Effects after intrathecal administration.

BACKGROUND AND PURPOSE: In our previous study (see accompanying paper) we observed that all-trans retinoic acid (ATRA) p.o. induces changes in spinal cord neuronal responses similar to those observed in inflammation-induced sensitization. In the present study we assessed the it. effects of ATRA, and its mechanisms of action. EXPERIMENTAL APPROACH: The effects of all drugs were studied after it. administration in nociceptive withdrawal reflexes using behavioural tests in awake male Wistar rats. KEY RESULTS: The administration of ATRA in normal rats induced a dose-dependent enhancement of nociceptive responses to noxious mechanical and thermal stimulation, as well as responses to innocuous stimulation. The intensity of the responses was similar to that observed in non-treated animals after carrageenan-induced inflammation. The effect induced by ATRA was fully prevented by the previous administration of the retinoic acid receptor (RAR) pan-antagonist LE540 but not by the retinoid X receptor (RXR) pan-antagonist HX531, suggesting a selective action on spinal cord RARs. The COX inhibitor dexketoprofen and the interleukin-1 receptor antagonist IL-1ra inhibited ATRA effect. The results indicate that COX and interleukin-1 are involved in the effects of ATRA in the spinal cord, similar to that seen in inflammation. CONCLUSIONS AND IMPLICATIONS: In conclusion, ATRA induces changes in the spinal cord similar to those observed in inflammation. The sensitization-like effect induced by ATRA was mediated by RARs and associated with a modulation of COX-2 and interleukin-1 activities. ATRA might be involved in the mechanisms underlying the initiation and/or maintenance of sensitization in the spinal cord.

[All-trans retinoic acid induces apoptosis in human mesangial cells: involvement of stress activated p38 kinase]. / El ácido retinoico todo-trans induce apoptosis en células mesangiales humanas: implicación de la quinasa activada por estrés p38.

All-trans retinoic acid (AR-t) is used for treating acute promyelocytic leukemia and renal cell carcinoma and it also has therapeutic value in several animal models of renal disease. Among its renal targets, mesangial cells have been widely studied: they have both retinoic acid receptors (RAR) and retinoid X receptors (RXR) and the cell growth is inhibited when human mesangial cells are incubated with 1-10 microM AR-t. Although his effect has been related with the antiproliferative action of AR-t, there are no studies on the involvement of apoptosis in AR-t induced cell growth when higher concentrations of retinoid are used. Our studies show that 25 microM AR-t triggers mesangial cell apoptosis assessed by light and fluorescence microscopy (Giemsa stain and acridine orange stain, respectively), DNA electrophoresis, flow cytometry (annexin-V) and immunocytochemistry (TUNEL). AR-t induced apoptosis was not inhibited by preincubation with the RXR pan-antagonist HX531 nor with the RAR pan-antagonist AGN 193109, this suggesting RAR and RXIR are not involved in AR-t induced cell death. Previous results of our group showed that ERK (extracellular regulated kinase) and INK (c-Jun kinase), two members of the MAP (mitogen activated protein) kinase family, are involved in non apoptotic effects of AR-t on mesangial cells. Therefore we focussed on the stress activated p38 kinase, the third member of the MAPK family, to investigate its involvement in AR-t induced apoptosis. The results confirmed a role of p38 since: 1) preincubation with B5203589, a p38 inhibitor, inhibited ARA induced apoptosis; 2) incubation with AR-t induced p38 phosphorilation after few minutes and p38 remained phosphorilated for at least 8 hours and 3) AR-t induced p38 phosphorilation was inhibited by SB203589. These data suggest that AR-t might have toxic side effects on the kidney but also suggest that AR-t could be an useful inhibitor of pathological mesangial cell expansion.

El ácido retinoico todo-trans induce apoptosis en células mesangiales humanas: implicación de la quinasa activada por estrés p38 / All-trans retinoic acid induces apoptosis in human mesangial cells: involvement of stress activated p38 kinase

El ácido retinoico todo-trans (AR-t) se utiliza en clínica en el tratamiento de la leucemiapromielocítica aguda y el cáncer renal. También presenta efecto terapéutico endiversas formas de enfermedad renal experimental. Las células mesangiales son una delas dianas farmacológicas de AR-t mejor estudiadas: presentan receptores de ácido retinoico(RAR) y receptores X de retinoides (RXR) y el AR-t, a concentraciones entre 1 y 10µM, inhibe su crecimiento. Este efecto se ha relacionado con la acción antiproliferativadel AR-t, aunque no se ha estudiado la participación de mecanismos apoptóticos cuandose utilizan mayores concentraciones de AR-t. El presente trabajo demuestra que AR-t25 µM induce apoptosis de células mesangiales humanas en cultivo, caracterizada porestudios de microscopía óptica y de fluorescencia (tinciones de Giemsa y naranja deacridina, respectivamente), electroforesis del ADN fragmentado, citometría de flujo(anexina-V/ioduro de propidio) e inmunocitoquímica (TUNEL). Ni HX531 (pan-antagonistaRXR), ni AGN193109 (pan-antagonista RAR) redujeron el grado de muerte celularinducido por el AR-t, lo que sugiere un mecanismo independiente de receptores. Resultadosprevios de nuestro grupo indican que dos de los tres miembros de las quinasasactivadas por mitógenos (MAP), ERK (quinasa regulada por estímulos extracelulares) yJNK (quinasa de c-Jun), están implicados en efectos no apoptóticos del AR-t en célulasmesangiales. Nos centramos, pues, en el potencial pro-apoptótico del tercer miembro,la quinasa activada por estrés p38. Confirmamos su implicación en la apoptosis inducidapor el AR-t porque: 1) su inhibidor farmacológico, SB203580, previno dicha apoptosis2) El AR-t indujo en pocos minutos la fosforilación de p38, manteniéndose fosforiladadurante las 8 horas posteriores; y 3) dicha fosforilación se inhibió por preincubación conSB203580. Estos datos sugieren una posible toxicidad renal del AR-t, pero también suutilidad para controlar la proliferación patológica de células mesangiales

All-trans retinoic acid (AR-t) is used for treating acute promyelocytic leukemia andrenal cell carcinoma and it also has therapeutic value in several animal models of renaldisease. Among its renal targets, mesangial cells have been widely studied: they haveboth retinoic acid receptors (RAR) and retinoid X receptors (RXR) and the cell growthis inhibited when human mesangial cells are incubated with 1-10 µM AR-t. Althoughhis effect has been related with the antiproliferative action of AR-t, there are no studieson the involvement of apoptosis in AR-t induced cell growth when higher concentrationsof retinoid are used. Our studies show that 25 µM AR-t triggers mesangial cellapoptosis assessed by light and fluorescence microscopy (Giemsa stain and acridineorange stain, respectively), DNA electrophoresis, flow cytometry (annexin-V) andimmunocytochemistry (TUNEL). AR-t induced apoptosis was not inhibited by preincubationwith the RXR pan-antagonist HX531 nor with the RAR pan-antagonist AGN193109, this suggesting RAR and RXIR are not involved in AR-t induced cell death.Previous results of our group showed that ERK (extracellular regulated kinase) andJNK (c-Jun kinase), two members of the MAP (mitogen activated protein) kinasefamily, are involved in non apoptotic effects of AR-t on mesangial cells. Therefore wefocussed on the stress activated p38 kinase, the third member of the MAPK family, toinvestigate its involvement in AR-t induced apoptosis. The results confirmed a role ofp38 since: 1) preincubation with SB203589, a p38 inhibitor, inhibited ARA inducedapoptosis; 2) incubation with AR-t induced p38 phosphorilation after few minutesand p38 remained phosphorilated for at least 8 hours and 3) AR-t induced p38 phosphorilationwas inhibited by SB203589. These data suggest that AR-t migth have toxicside effects on the kidney but also suggest that AR-t could be an useful inhibitor of pathologicalmesangial cell expansion

The oral administration of retinoic acid enhances nociceptive withdrawal reflexes in rats with soft-tissue inflammation.

OBJECTIVE AND DESIGN: To study the involvement of all-trans retinoic acid (ATRA) in the development and maintenance of inflammatory pain. SUBJECTS: Adult male Wistar rats and murine neuro2a and human SH-SY5Y neuroblastoma cells. TREATMENT: Soft-tissue inflammation was induced by the intraplantar administration of 100 microl of carrageenan lambda. The oral treatment with either ATRA or vehicle lasted for seven days and consisted in a dose of 15 mg/kg the first two days and a dose of 10 mg/kg the following five days. Neuroblastoma cells were incubated for 16 h with ATRA. METHODS: Rats were tested twice daily for intensity and evolution of withdrawal reflexes evoked by mechanical and thermal stimulation. The expression of COX enzymes was studied in spinal cords and neuroblastoma cells by western blot. RESULTS: The animals treated with ATRA showed a significantly more intense development of mechanical allodynia (p < 0.01), mechanical hyperalgesia (p < 0.01), thermal hyperalgesia (p < 0.001) and reduction of threshold for mechanical (29 +/- 4 vs. 60 +/- 6 mN, p < 0.001) and thermal stimulation (12 +/- 0.3 vs. 8.4 +/- 0.3 s, p < 0.001) than control animals. Recovery to mechanical baseline data was slower in animals treated with ATRA, the main difference was observed in the test carried out on day 2, p.m. In neuroblastoma cells incubated with ATRA, a concentration-dependent increase in the expression of COX-2 protein was observed. Changes in the expression of COX-1 enzyme were not clear. An increase in COX-2 expression in the lumbar spinal cord was also observed in animals treated with ATRA. CONCLUSIONS: A clear relationship between the oral administration of ATRA and an enhancement of the nociceptive withdrawal reflexes was observed in rats. This relationship was associated with an increment of the expression of the COX-2 enzyme.

A dual effect of somatostatin on the proliferation of cultured rat mesangial cells.

The present experiments were designed to analyze the ability of somatostatin to modulate the proliferation of cultured rat mesangial cells. In the absence of fetal calf serum, somatostatin stimulated cell proliferation in a dose-dependent manner. In contrast, in proliferating cells, somatostatin inhibited cell proliferation, also in a dose-dependent fashion. Zaprinast, a rather specific cyclic GMP phosphodiesterase blocker, inhibited the somatostatin-dependent proliferation in the absence of growth factors. However, it potentiated the inhibitory effect on proliferating cells. These results support a dual role for somatostatin in the regulation of mesangial cell proliferation. The inhibitory effect of the peptide may be mediated by cyclic GMP.

The effects of cholesterol depletion on the sodium pump in human red cells.

Sodium pump function has been studied in human erythrocytes depleted of membrane cholesterol by incubation with phosphatidylcholine liposomes. The cells were sodium loaded by incubation in alkaline sodium phosphate and sodium pump activity was assessed by measurements of ouabain-sensitive 86Rb uptake at 37 degrees C. Cholesterol depletion had a biphasic effect; depletion by 5-25% increased sodium pump activity by a mean of 16.1% (S.D. 3.2%), whereas depletion by 35-50% decreased sodium pump activity by a mean of 14.8% (S.D. 3.8%). Cholesterol depletion had no reproducible effect on the ouabain-insensitive uptake of Rb. These results support the hypothesis that there may be an optimum membrane cholesterol content for sodium pump function.

Changes in the hemostatic system and compensatory responses during alimentary hyperlipidemia.

Compensatory responses tending to prevent a thrombotic state in rats fed a high lipid diet have been investigated. Platelet membranes from these animals had an increased cholesterol content but the membrane fluidity was found to be within values nearly normal. A decrease in phosphatidylethanolamine was noted. These changes may maintain normal platelet sensitivity to aggregating agents. In fact, platelets from hyperlipidemic rats were hypersensitive to thrombin, but not to adenosine diphosphate. In addition, platelets were apparently able to correct, at least in part, the stated hyperactivity of hyperlipidemic plasma to coagulate, as shown by thrombelastographic tests in both platelet-rich plasma and plasma from hyperlipidemic rats. Finally, thrombelastographic features of whole blood from these animals were found to be normal. This suggests an important role of blood cells in compensating plasma hyperactivity to coagulate during hyperlipidemia.