Michel Fay

Transcriptional Regulation of Sodium Transport by Vasopressin in Renal Cells

We have examined whether arginine vasopressin (AVP) can induce a long-term modulation of transepithelial ion transport in addition to its well known short-term effect. In the RCCD1 rat cortical collecting duct cell line, an increase in both short-circuit current and 22Na transport was observed after several hours of 10−8 m AVP treatment (a concentration above the in vivo physiological range). This delayed effect was partially prevented by apical addition of 10−5 m amiloride and was blocked by 10−6 m actinomycin D and 2 × 10−6 m cycloheximide. The amounts of mRNA encoding the α1 (not β1) subunit of Na+/K+-ATPase and the β and γ (not α) subunits of the amiloride-sensitive epithelial Na+ channel were significantly increased by AVP treatment. The increase in mRNA was blocked by actinomycin D, not by amiloride, suggesting a Na+-independent increase in the rate of transcription of these subunits. The translation rates of the α1 subunit of Na+/K+-ATPase and the β and γ subunits of the rat epithelial sodium channel increased significantly, whereas the translation rates of the other subunits remained unchanged. Finally, the number of Na+ channels present in the apical membrane of the cells increased, as demonstrated by enhanced specific [3H]phenamil binding.

Cell-specific expression of three members of the FXYD family along the renal tubule.

Abstract: The gamma subunit of Na/K/ATPase is a small membrane protein that shares homologies with other members of the FXYD family, like phospholemman and CHIF (corticosteroid hormone‐induced factor). Both the gamma subunit and CHIF modulate sodium pump properties. The gamma subunit increases the apparent affinity of the pump for ATP and reduces its apparent affinity for sodium. CHIF, in contrast, augments its apparent affinity for sodium. Gamma subunit expression is essentially restricted to the kidney, with two main splice variants, γa and γb, which differ only at their extracellular N‐termini. We have investigated in detail the cell‐specific expression of the two splice variants of gamma within the kidney and compared it to that of CHIF. While both gamma variants affect catalytic properties of the pump (without detectable difference between a and b forms), their localization along the nephron is partially distinct. Both variants are coexpressed in the proximal tubule and in the medullary part of the thick ascending limb of Henles loop (TAL). In contrast, their expression differs in the downstream tubular segments. Within the renal cortex, the sole gamma a variant was found in macula densa cells and in principal cells of the initial parts of the collecting duct. Gamma b is in the cortical part of the TAL. Outer and inner medullary collecting ducts lack detectable gamma expression. These latter nephron segments express CHIF, and no overlap between gamma and CHIF expression along the nephron was observed. Such distinct cell‐specific expression argues for complementary roles to modulate Na/K/ATPase activity.

Vasopressin stimulates long-term net chloride secretion in cortical collecting duct cells

The classical short‐term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long‐term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long‐term increase in net chloride secretion. In the RCCD1 rat cortical collecting duct cell line, 10−8 M AVP induced, after several hours, an increase in net 36Cl− secretion. This delayed effect of AVP was inhibited by basal addition of 10−4 M bumetanide and apical addition of 10−4 M glibenclamide, suggesting chloride entry at the basal membrane through a Na+/K+/2Cl− and apical secretion through a chloride conductance. An original acute cell permeabilization method was developed to allow for entry of antibodies directed against the regulatory region (R) of the cystic fibrosis transmembrane regulator (CFTR) into the cells. This procedure led to a complete and specific blocking of the long‐term net chloride secretion induced by AVP. Finally, it was observed that CFTR transcripts steady‐state level was significantly increased by AVP treatment. Besides the well‐documented short‐term effect of AVP on chloride transport, these results provide evidence that in RCCD1 cells, AVP induces a delayed increase in transepithelial net chloride secretion that is mediated by a Na+/K+/2Cl− co‐transporter and CFTR.

Lymphocyte glutathione status in relation to their Con A proliferative response

We studied the intracellular total, oxidized and reduced glutathione levels in thymus and spleen rat lymphocytes cultured with or without Con A and 2‐mercaptoethanol (2‐ME). After 48 h culture, the total glutathione level decreased and the oxidized glutathione level increased in the two types of unstimulated and stimulated cells. In the presence of 2‐ME, the tritiated thymidine incorporation increased in splenocytes but not in thymocytes; on the other hand, the two types of stimulated cells increased their total and oxidized glutathione content. The enhancement of the GSSG/GSH + GSSG ratio, irrespective of culture conditions, indicates a severely disturbed redox state of the cells. 2‐ME acts on the glutathione synthesis of stimulated lymphocytes but is unable to maintain a normal redox state of these cells.

In vitro O2-induced depression of T and B lymphocyte activation is reversed by diethyldithiocarbamate (DDC) treatment

In this study, we tried to establish a relationship between the immunopotentiating effects and the antioxidant activity of the immunostimulating compound, diethyldithiocarbamate (DDC). We studied the effects of DDC treatment on enriched T and B murine spleen lymphocytes in an in vivo-ex vivo model of O2-induced immune depression. Female C57B1/6 mice were injected subcutaneously with a single dose of DDC (125 mg.kg-1). Eight days after DDC injection, we evaluated, in vitro, the concanavalin A response of the T cell fraction and the LPS response of the B cell fraction, under standard (air--5% CO2) and hyperoxic (60% O2--5% CO2) culture conditions. The results show that after a lag period, DDC is able to enhance the mitogenic response of T and B murine lymphocytes under standard culture conditions to restore the ConA response and to partially restore the LPS response under hyperoxic conditions. The results of this study suggest that the immunostimulatory effects of DDC could be related to the antioxidant activity of this compound on the lymphoid cellular metabolism. This activity apparently affects both T and B lymphocytes.

A structural explanation of the effects of dissociated glucocorticoids on glucocorticoid receptor transactivation.

There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as “dissociated glucocorticoids” (dGCs). The first published dGCs, RU24782 (9α-fluoro-11β-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11β-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.

Glutathione status of rat thymocytes and splenocytes during the early events of their ConA proliferative responses

Glutathione plays an important role in the lymphocyte mitogenic response. We have demonstrated that 2-ME increases the ConA proliferative response of rat splenocytes and in parallel, causes an enhancement of glutathione synthesis in these cells. On the other hand, 2-ME had the same action on the glutathione level of thymocytes during the late phase of their mitogenic response, but it had no effect on the [3H]thymidine uptake of these cells. To clarify this discrepancy and the role of glutathione during the mitogenic response, we studied the glutathione status of thymus cells during the early phase of the ConA-induced proliferative response in the presence or the absence of 2-ME in parallel with that of whole spleen cells and the T cell fraction of splenocytes. During the early events of the mitogenic response, i.e., during the 24th h, we observed a normal 2 GSSG/GSH + 2 GSSG ratio in cultured cells, indicating a normal redox state, and that ConA involved an increased glutathione level in thymocytes but not in whole splenocytes and in splenic T cells. 2-ME had no effect on the glutathione level of stimulated thymocytes during the early phase of the mitogenic response. This phenomenon could be related to an absence of its effect on [3H]thymidine uptake. On the other hand, 2-ME induced an enhancement of the glutathione level and [3H]thymidine uptake in the two types of stimulated splenocytes. This study suggest that thymocytes do not have the same mechanism of glutathione synthesis induction as that which occurs in splenocytes during the ConA proliferative response. This mechanism could be related to the maturation state of the T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione status during the mitogenic response of rat splenocytes. Effects of oxygen concentration: FO2 21% versus FO2 7%

Glutathione is known to be an important parameter for ConA proliferative response of murine splenocytes. We studied the glutathione status of ConA-stimulated rat splenocytes during the early and late phase of the mitogenic response under low (FO2 7%) and standard (FO2 21%) oxygen concentrations. We determined the intracellular total, oxidized and reduced glutathione levels after 6, 12, 24 and 48 h of culture with or without ConA and/or 2-ME, under FO2 7% and 21%. Our results showed that: The 2 GSSG/GSH + 2 GSSG ratio, which indicated the redox state of the cells, remained normal during the early period of culture (0-24 h), irrespective of culture conditions. After 48 h of culture, this ratio increased dramatically under FO2 21% and less under FO2 7%. The maintenance of the redox state seems to be an oxygen concentration-dependent phenomenon. ConA stimulation involved a glutathione consumption during the early stages of culture; under these conditions 2-ME increased the glutathione synthesis, which was higher under FO2 7% than under FO2 21%. On the other hand, the presence of 2-ME involved an increase of tritiated thymidine uptake in stimulated splenocytes, which was significantly higher under FO2 21% than under FO2 7%. Low oxygen tension (FO2 7%) can induce a higher increase of glutathione synthesis, whereas the respective ConA proliferative response is lower than that observed under standard O2 conditions.

High concentrations of oxygen modulate in vitro Con A responses of rat lymphoid cells. Effect of 2-mercaptoethanol

The effects of different normobaric oxygen concentrations (40, 60 and 95%) on the survival and the proliferative response to Con A of rat lymphoid cells were studied. Spleen, thymus and peripheral blood mononuclear cells were tested. We found that oxygen concentrations modulated the proliferative response independently of cell survival. The addition of 2-mercaptoethanol (2-ME) partially prevented the toxic effects of hyperoxia but the population of thymocytes which responded to Con A stimulation appeared to be less sensitive to the protective action of 2-ME. The relationship between oxygen concentrations and the lymphoid proliferative response could be used as a model of oxidant immunodepression for evaluating pharmacological effects of antioxidant compounds.

Protective effect of LPS and poly A:U against immune oxidative injury: Role of thiols released by activated macrophages

Various drugs with or without sulfur groups have been demonstrated to exert antioxidant properties in vivo. In this latter group immunomodulating agents such as the polyribonucleotide Poly(I)-Poly(C) have been shown to protect rats against hyperoxic pulmonary damage. However, the effects of these agents on the protection of immune cells against oxidative injury have not been described. Our work shows that the immunomodulating agent Poly(A)-Poly(U) administered in vivo to mice protected their lymphoid cells against immune oxidative injury by increasing their glutathione content. This antioxidant effect could be related to the Poly(A)-Poly(U) capacity to induce the release of thiol compounds from macrophages. The antioxidant effects of such a non toxic immunomodulating agent could be of interest in protecting lymphoid cells in pathological situations involving oxidative injury.