Molecular physiology of aquaporins in plants.

In plants, membrane channels of the major intrinsic protein (MIP) super-family exhibit a high diversity with, for instance, 35 homologues in the model species Arabidopsis thaliana. As has been found in other organisms, plant MIPs function as membrane channels permeable to water (aquaporins) and in some cases to small nonelectrolytes. The aim of the present article is to integrate into plant physiology what has been recently learned about the molecular and functional properties of aquaporins in plants. Exhaustive compilation of data in the literature shows that the numerous aquaporin isoforms of plants have specific expression patterns throughout plant development and in response to environmental stimuli. The diversity of aquaporin homologues in plants can also be explained in part by their presence in multiple subcellular compartments. In recent years, there have been numerous reports that describe the activity of water channels in purified membrane vesicles, in isolated organelles or protoplasts, and in intact plant cells or even tissues. Altogether, these data suggest that the transport of water and solutes across plant membranes concerns many facets of plant physiology. Because of the high degree of compartmentation of plant cells, aquaporins may play a critical role in cell osmoregulation. Water uptake in roots represents a typical process in which to investigate the role of aquaporins in transcellular water transport, and the mechanisms and regulations involved are discussed.

The splanchnic removal of cortisol from plasma of anaesthetized adult guinea-pigs.

To evaluate the role of the liver in cortisol catabolism the extraction ratio of both cortisol and cortisone by the organs of the splanchnic area was estimated in guinea-pigs anaesthetized with pentobarbitone. The [3H]cortisol and [3H]cortisone concentrations were measured in portal and sus hepatic venous plasma during a constant infusion of [3H]cortisol or [3H]cortisone. The extraction ratio of cortisol was estimated to be 10-14% in the splanchnic area and the viscera, while in the liver it had a small negative value suggesting that the liver had produced as much or more cortisol than it had taken up. All the cortisone (95%) formed from cortisol in the viscera was eliminated from the plasma compartment by the liver. Some 75-80% of the infused cortisone was converted to cortisol; rather less of the infused cortisol was converted to cortisone (32%). Using estimates of plasma flow derived from sham-operated animals, the uptake of cortisol by the various organs was calculated. The splanchnic area extracted 41% of the infused cortisol from the plasma: 25-27% as cortisol and 13-16% as cortisone. The liver appeared to take up cortisone preferentially. The conversion of cortisone into cortisol within the liver seems to be important in limiting the amount of cortisol removed from the plasma by the splanchnic area. The liver is also important in inactivating the steroids although other sites are probably also involved.

Aldosterone regulates salivary sodium secretion in cattle.

Intravenous infusion of aldosterone (10 microgram/kg body wt per h for 5 h) in four 2-month-old calves decreased salivary and urinary sodium (Na+) concentration and increased salivary potassium (K+) concentration without modifying salivary flow or urinary K+ concentration. Intravenous angiotensin II infusion (0.3 microgram/kg body wt per min for 1 h) in four Na+-replete 16-month-old bulls decreased salivary Na+ concentration and increased that of K+. It also increased plasma cortisol and plasma aldosterone concentrations, and decreased plasma renin activity (PRA). In four 16-month-old bulls Na+ deficiency (induced by chronic cannulation of the right parotid duct and loss of saliva for 5 days) had similar effects to those observed following aldosterone infusion in calves: a decrease in salivary Na+/K+ ratio. This decrease was associated with an increase in PRA and an increase in plasma aldosterone concentration. In these animals a close positive relationship was observed between PRA and plasma aldosterone concentration (r = 0.91; n = 20; P less than 0.01). Thus in cattle, during Na+ deficiency, the effect of aldosterone on parotid glands participates in the regulation of Na+ metabolism.

Interactions of amiodarone with model membranes and amiodarone-photoinduced peroxidation of lipids.

The potent antiarrhythmic drug, amiodarone (AMIO) exhibits phototoxicity, which is thought to be related to its interaction with biological membranes. We report here a spectroscopic study of the interactions of this drug with phosphatidylglycerol (PG) and phosphatidylcholine (PC) liposomes used as membrane model systems. A linear increase in absorbance at 300 nm was observed with increasing addition of AMIO to dimyristoyl-DL-PC (DMPC) liposomes over all the drugs-lipid molar ratio (Ri)s tested. In contrast, in the dimyristoyl-DL-PG (DMPG) liposomes, there was a dramatic increase in absorbance at values of Ri above unity. Light scattering by DMPG liposomes at 350 nm increased with increasing AMIO concentration up to a Ri = 1, and then decreased with increasing drug concentration. Such changes were not observed with the DMPC liposomes. Moreover, addition of AMIO changed the fluorescence polarization rate of 1,6-diphenyl 1,3,5-hexatriene embedded in these liposomes. It reduced the rate below the phase transition temperature (Tt) of the lipid, but increased it above this temperature. These effects on the lipidic phases observed at low Ri were more pronounced on the DMPG than on the DMPC liposomes. The strong interactions of AMIO with phospholipids, especially the acidic ones, were confirmed by liposome size determinations. All these data strongly suggest that the drug was incorporated in the core of the lipid bilayers. Such a penetration would favor a drug-photoinduced peroxidation of lipids. Indeed, UV irradiation of AMIO-DOPG mixtures led to the disappearance of the unsaturated fatty acids of phospholipids, checked by gas chromatography measurements, which was correlated with the amount of oxygen consumed. This showed that AMIO did photosensitize phospholipid peroxidation.

The genomic organization and DNA sequence of the mouse vas deferens androgen-regulated protein gene.

The gene for mouse vas deferens protein (MVDP) is expressed, under androgenic control, exclusively in the epithelial cells of the deferent duct. As a first step in correlating cell-specific and hormonal regulations with the structure of the gene, the complete sequence of the MVDP gene (11 kb) and 0.5 kb of the 5' flanking region have been determined. The size range for the 10 exons is 78 to 168 bp, whereas that of introns is 292 to 2833 bp. A major site of transcription is located on an A residue 46 nucleotides upstream from the A of the ATG initiation codon. A TATA (CATAA) box, a CAAT box, a GC-rich motif and a (5'-TGTTCT-3') element that closely resembles the consensus sequence of the androgen response elements are present in the 5' flanking region of the MVDP gene.

The roles of P-glycoprotein and intracellular metabolism in the intestinal absorption of methadone: in vitro studies using the rat everted intestinal sac.

Methadone is used as a treatment for opiate detoxification in methadone maintenance programs. Intra- and inter-patient variations in methadone bioavailability have been observed after oral methadone treatment and this makes it difficult to predict a dosing regimen. Intestinal absorption and metabolism could explain these variations. The in vitro gut sac model was used to study the intestinal absorption of methadone, and it confirmed that methadone is a substrate for P-glycoprotein. The transport of methadone was increased in presence of P-gp inhibitors verapamil and quinidine. The appearance of a major metabolite of methadone, 2-ethylidene-1, 5-dimethyl-3, 3-diphenyl pyrrolidine (EDDP) in the gut sac contents also demonstrated the existence of intestinal metabolism of methadone.

Measurement of the rate of secretion, peripheral metabolism and interconversion of cortisol and cortisone in adult conscious male guinea-pigs.

Metabolism of the cortisol (F) and the cortisone (E) was studied by continuous infusion of [14C]-F and [3H]-E in adult conscious male guinea-pigs. Parameters, calculated from the specific activities of F and E, are expressed in mumoles/24 h : production rate of F (PRF) = 8 +/- 1 and E (PRE) = 4.9 +/- 0.7 ; secretion rate of F (QF) = 7.1 +/- 0.9 and E (QE) = 0.9 +/- 0.4 ; rate of irreversible metabolism of F (rF) = 6.2 +/- 0.9 and E (rE) = 1.8 +/- 0.3 ; percentage of transfer of F into E = 59 +/- 4% and E into F = 80 +/- 3%. These results demonstrate that adrenal gland in the adult male guinea-pig secretes essentially cortisol and little or no cortisone. Practically all the pool of E is derived from transformation of F into E ; the major part of E production is reconverted into F.

Evidence for a cold-induced aldosterone stimulation in the rat.

This study was undertaken to determine the secretion of aldosterone by male Long-Evans rats acclimated for six weeks to moderate cold (15 C), in comparison with rats maintained at thermo-neutral temperature (28 C). The following determinations were made: corticosteroids in plasma and adrenals, PRA, and hydromineral balance. Cold acclimation highly increased the plasma and adrenal levels of aldosterone and corticosterone. The cold stimulation of aldosterone was induced neither by the renin-angiotensin system, nor by alterations of hydromineral balance: PRA, plasma sodium and potassium concentrations, blood hematocrit, and hydromineral balance at 15 C and 28 C did not differ. Moreover this stimulation was induced neither by ACTH, nor by any other hypophyseal factors, since plasma aldosterone levels remained high in hypophysectomized rats. This study provides evidence of an aldosterone stimulation which appeared during moderate cold acclimation; the origin of this stimulation must be investigated.

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.

Antioxidant activity of flavonoids: efficiency of singlet oxygen (1 delta g) quenching.

Flavonoids, polyphenolic pigments widely present in plants, have been reported to act as scavengers of various oxidizing species. However, most often an overall antioxidant effect was measured. In this paper we report the results of a systematic study of the reactivity of 13 selected flavonoids (from the flavonol, flavone, flavanone and flavane families) with singlet oxygen (1O2(1 delta g)) in order to establish a structure-activity relationship. The rate constants of the chemical reaction of these flavonoids with 1O2(k r) and their rate constants of 1O2 physical quenching (kq) have been determined by kinetic measurements and near-IR singlet oxygen luminescence. The efficiency of the physical quenching is mainly controlled by the presence of a catechol moiety on ring B, whereas the structure of ring C (particularly the presence of a hydroxyl group activating the double bond) is the main factor determining the efficiency of the chemical reactivity of these compounds with 1O2. The total reactivity factor determining the efficiency of the chemical reactivity of these compounds with 1O2. The total reactivity scale is dominated by kq, which is in general higher than kr. (+)-Catechin is the most efficient quencher of the series.

[Characterization of cDNA and of the gene corresponding to androgen-dependent protein of the vas deferens in mice]. / Caractérisation de l'ADNc et du gène correspondant à une protéine androgéno-dépendante du canal déférent de souris.

A 34.5 kDa abundant protein named MVDP (Mouse Vas Deferens Protein) is produced and secreted by vas deferens epithelial cells from adult mice. Steady-state levels of MVDP and its 1.4 kb mRNA are markedly decreased 30 days after castration. Testosterone treatment for 2 weeks is necessary to completely reverse the effect of castration. A cDNA encoding MVDP has been cloned and entirely sequenced. A protein of 316 amino acids encoded by an open reading frame of 948 nucleotides shows 82% homology with a human placental aldose reductase. A gene corresponding to MVDP cDNA has been recently isolated ans characterized. The gene extends over approximately 11 kb and consists of 10 exons. Its structure is very similar to that of the human aldose reductase gene. The promotor region of MVDP gene contains an androgen responsive element consensus located 97 nucleotides upstream the transcription initiation site.

Pituitary and adrenal responses to ovine corticotropin releasing factor and vasopressin injected into young and adult guinea-pigs.

Comparison of effects of synthetic ovine corticotropin releasing factor (oCRF), Arginine-Vasopressin (AVP) and the combination of both peptides have been tried in adult and 7-days-old guinea-pigs. On plasmas collected 15 min after interscapulary injection, cortisol, aldosterone and ACTH were measured. The different circulating forms of ACTH were isolated by Sephadex G50 column chromatography, with 1% formic acid and measured by radioimmunoassay. Thus, in the guinea-pig plasma, we detected three immunoreactive forms of ACTH: a "big" molecular form (Mr greater than 20000), an "intermediate" (Mr = 9500) and a "little" ACTH form (Mr = 4500) which was eluted in the same fractions as human 1-39 ACTH. In adult guinea-pigs, CRF increased total ACTH and the "intermediate" form and also plasma cortisol concentrations whereas AVP remained without significant effect excepted a rise in cortisol levels. Injected together, CRF and AVP enhanced plasma concentrations of total ACTH, of the three circulating forms and of cortisol. In 7-days-old guinea-pigs, both CRF and AVP increased plasma concentrations of total, of "intermediate" ACTH and of cortisol and aldosterone whereas the combination of both peptides enhanced dramatically plasma concentration of total ACTH suggesting a magnifying effect of AVP on CRF activity still more efficient in young than in adult guinea-pigs.

Plasma concentrations of adrenocorticotropin-related peptides in lambs injected with ovine corticotropin releasing factor or vasopressin.

Synthetic ovine corticotropin-releasing factor (oCRF) and arginine vasopressin (AVP) were intravenously injected each alone or in combination (each peptide: 1 microgram/kg body weight) in lambs on days 1, 3, 7 and 20 after birth. Plasma samples were collected just before and 10 and 30 min after injection. Plasma concentrations of cortisol and aldosterone were measured. Adrenocorticotropin (ACTH)-related peptides were isolated by Sephadex G50 column chromatography and measured by radioimmunoassay. Three different peaks with an ACTH immunoreactivity were found in lamb plasma: a "big" ACTH molecular form (Mr = 30,000), an "intermediate" (Mr = 8000) and a "little" (Mr = 4500). In 1 and 3 days-old lambs, both CRF and AVP increased preferentially "intermediate" ACTH. In 7 and 20 days-old lambs, an increase in "little" ACTH occurred after CRF whereas "intermediate" ACTH rose after AVP. The rise in plasma levels of different molecular forms of ACTH after stimulation by CRF or AVP could suggest that the biological pathway of ACTH synthesis, storage and release may occur in different intracellular pools or rather in different pituitary cells. Intermediate ACTH stimulated adrenal secretion of cortisol as soon as the first day of postnatal life and increased plasma aldosterone concentration in 7 and 20 day-old lambs. At these stages aldosterone level did not change after a rise in "little" ACTH.

Plasma concentrations of adrenocorticotropin-related peptides after corticotropin-releasing hormone and vasopressin injections in sheep.

Ovine corticotropin-releasing hormone (1 micrograms/kg body weight) and arginine vasopressin (1 micrograms/kg) were injected iv in sheep, both separately and in combination. Plasma were sampled just before and 5, 15 and 30 min after the injection. Adrenocorticotropin-related peptides were isolated by Sephadex G-50 column chromatography and measured by RIA. Cortisol and aldosterone were determined on the same plasma samples. Three molecular forms of immunoreactive ACTH (IR-ACTH) were isolated: 'big' (greater than 20,000 mol wt), 'intermediate' (= 8000 mol wt) and 'little' (= 4500 mol wt). Following CRH injections, the three molecular forms of ACTH were enhanced, particularly the 'little' form, whereas 'intermediate' IR-ACTH was highly and specifically responsive to AVP. After a simultaneous injection of CRH and AVP, additive increases occurred for 'intermediate' and 'little' IR-ACTH. The release of different molecular forms of IR-ACTH after stimulation by CRH or AVP of corticotrope cells suggests that ACTH-related peptides could be stored in different intracellular pools or secreted by different pituitary cells.

Lack of effect of metoclopramide on plasma aldosterone concentrations in young calves.

In five 10-day-old Holstein X Friesian male calves, the intravenous injection of the dopamine blocker metoclopramide (1 mg/kg bwt) had no significant effect on plasma aldosterone concentration. Plasma sodium, potassium, cortisol, corticosterone concentrations and plasma renin activity measured in these animals during 120 min following metoclopramide injection were never significantly different from those simultaneously measured in 5 control calves.

Construction of a directory of tobacco plasma membrane proteins by combined two-dimensional gel electrophoresis and protein sequencing.

The polypeptide pattern of the plasma membrane from tobacco was studied by two-dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in-gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG-IEF, and N-terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG-IEF gels and in-gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant-specific genes.

Sodium intake by pregnant cows and plasma aldosterone and cortisol concentrations in the fetus during late pregnancy.

The purpose of this study was to evaluate the influence of maternal dietary Na intake on the plasma concentrations of aldosterone and cortisol in cows and their fetuses during late pregnancy. Seven cows received a diet with normal amounts of Na (25 g Na per cow/d) and seven others an Na-loaded diet (210 g Na per cow/d) during the last 40 d of gestation. Maternal and fetal blood samples were collected regularly during the last month of gestation through jugular vein puncture and cotyledonary artery indwelling catheters. Serum Na and K concentrations and plasma osmolalities increased, but concentrations of aldosterone decreased in maternal and fetal plasma when cows were fed the Na-loaded diet. Diets did not modify concentrations of cortisol in maternal and fetal plasma. Thus, an increase in Na intake by dams influenced concentration of Na and K in fetal plasma and fetal adrenal secretion of aldosterone.

Isolation of the mouse aldose reductase promoter and identification of a tonicity-responsive element.

Aldose reductase (AR; EC 1.1.1.21) is an oxidoreductase that catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step of the polyol pathway. AR is of great interest due to its implication in the etiology of diabetic complications. In renal medullary cells, AR also plays an osmoregulatory role by accumulating sorbitol to maintain the intracellular osmotic balance during antidiuresis. We have previously cloned the AR cDNA from mouse kidney, and we report here the isolation of the mouse AR gene promoter. Transient transfection of chloramphenicol acetyltransferase reporter constructs containing various 5'-flanking regions of the mouse AR gene in CV1 cells led to the identification of a sequence spanning base pairs -1053 to -1040, required for an enhancer activity in hypertonic compared with isotonic cell culture conditions. This sequence is similar to the tonicity-responsive element first characterized in the betaine-gamma-aminobutyric acid transporter promoter.

A thiol protease and an anionic peroxidase are induced by lowering cytokinins during callus growth in Petunia.

We previously identified a group of proteins that increase early in Petunia hybrida calli subcultured on a low-cytokinin medium, unlike the calli subcultured on a high-cytokinin medium. The calli on the low-cytokinin medium do not regenerate (J.-P. Renaudin, C. Tournaire, B, Teyssendier de la Serve [1991] Physiol Plant 82: 48-56). Two of these proteins, P21 and P17, have been identified by peptide sequencing and cloned. P21 is highly homologous to a group of thiol proteases, including barely aleurain, rice oryzain gamma, Arabidopsis SAG2, and mammalian cathepsin H. P17 is highly homologous to a group of anionic peroxidases from potato and tomato. A study of their expression in two P. hybrida lines, PC6 and St40 which differ in their ability to regenerate, showed that the genes for P21 and P17 are differentially expressed depending on the type and the age of the organ, with the highest expression in senescing leaves and in aged calli. The data are in favor of these genes being associated with an early step of senescence, which may be due, in part, to a reduction in total cytokinin. The two Petunia lines are, thus, functionally different concerning the action of cytokinin in two developmental phenomena: in vitro organogenesis and senescence.

Plasma adrenocorticotrophin, cortisol and aldosterone responses to ovine corticotrophin-releasing factor and vasopressin in sheep.

Ovine corticotrophin-releasing factor (oCRF) (1 microgram/kg) and arginine vasopressin (AVP) (1 microgram/kg) were injected iv in sheep, both separately and in combination. Plasma levels of immunoreactive ACTH (IR-ACTH), cortisol, and aldosterone were measured for 3 h after the injections. Mean levels before injections were 8 +/- 4 pmol/l for ACTH, 7 +/- 3 nmol/l for cortisol, and 28 +/- 9 pmol/l for aldosterone. CRF caused a rapid rise in IR-ACTH and a peak level of 125 +/- 52 pmol/l was obtained 15 min after injection. Highest values for cortisol and aldosterone levels were 40 +/- 9 nmol/l and 64 +/- 13 pmol/l, respectively, 30 min after injection. AVP also increased IR-ACTH (maximum level: 202 +/- 77 pmol/l at 5 min) and aldosterone (128 +/- 36 pmol/l at 15 min), whereas the cortisol increase was lower than after CRF. Simultaneous injection of CRF and AVP produced an addition of the IR-ACTH response (295 +/- 82 pmol/l at 15 min), but the changes in cortisol levels were similar to those obtained after CRF alone and those in aldosterone levels resembled those induced by AVP alone. Plasma Na and K, osmolality, and plasma renin activity (PRA) were not modified by either CRF or AVP. It is suggested that the increase in aldosterone levels after CRF could be mediated by ACTH and that after AVP by an IR-ACTH peptide with less effect on cortisol secretion.