Jose M. Macarulla

Sphingosine‐1‐phosphate stimulates cortisol secretion

We show here for the first time that sphingosine‐1‐phosphate (Sph‐1‐P) stimulates cortisol secretion in zona fasciculata cells of bovine adrenal glands. This effect was dependent upon protein kinase C (PKC) and extracellular Ca2+, and was inhibited by pertussis toxin. Sph‐1‐P activated phospholipase D (PLD) through a pertussis toxin‐sensitive mechanism, also involving extracellular Ca2+ and PKC. Primary alcohols, which attenuate formation of phosphatidic acid (the product of PLD), and cell‐permeable ceramides, which inhibit PLD, blocked Sph‐1‐P‐induced cortisol secretion. In conclusion, Sph‐1‐P stimulates cortisol secretion through a mechanism involving Gi/o protein‐coupled receptors, extracellular Ca2+, PKC and PLD.

Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways

We reported recently that sphingosine-1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes the phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK 1/2), which is an indication of their activation, in these cells. These effects are probably mediated through the interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pretreatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished the phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells were pretreated with LY 294002 and PD 98059 simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to the stimulation of PLD and aldosterone secretion.

Triton X-100 solubilization of mitochondrial inner and outer membranes

Rat liver mitochondrial inner and outer membranes were subjected to the solubilizing effect of the nonionic detergent Triton X-100 under various conditions. After centrifugation, the supernatants (containing the solubilized fraction) and pellets were characterized chemically and/or ultrastructurally. The detergent seems to act by inducing a phase transition from membrane lamellae to mixed protein-lipid-detergent micelles. Different electron-micro-scopy patterns are shown by the inner membranes after treatment with different amounts of surfactant, whereas the corresponding images from outer membranes vary but slightly. Selective solubilization of various components is observed, especially in the case of the inner membrane. Some membrane lipids (e.g., cardiolipin) are totally solubilized at detergent concentrations when others, such as sphyngomyelin, remain in the membrane. Other inner-membrane components (flavins, cytochromes, coenzymeQ) show different solubilization patterns. This allows the selection of conditions for optimal solubilization of a given membrane component with some degree of selectivity. The influence of Triton X-100 on various mitochondrial inner-membrane enzyme activities was studied. The detergent seems to act especially through disruption of the topology of the functional complexes, although the activity of the individual enzymes appears to be preserved. Relatively simple enzyme activities, such as ATPase, are more or less solubilized according to the detergent concentration, whereas the more complex succinate-cytochromec reductase activity practically disappears even at low Triton X-100 concentrations.

Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. Here, we report for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms α and δ and extracellular Ca2+, and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC α and δ and extracellular Ca2+. Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD activity, blocked S1P-stimulated aldosterone secretion. Furthermore, propranolol, chlorpromazine, and sphingosine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupled receptors, extracellular Ca2+, and the PKC isoforms α and δ are all important components in the cascade of events controlling this process.

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

SummaryThe specific binding of [3H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (KD=8.8nm andBmax=1477 fmol/mg protein) and the other one has low affinity and high binding capacity (KD=91nm andBmax=9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (KD=11.2nm andBmax=1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na+,K+-ATPase digitalis receptor. Probably it is of the same nature that the one determinate for [3H]cortisol and [3H]corticosterone in mouse liver plasma membrane. Beta-and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane.

SummaryThe mechanism of steroid uptake by the cell remains controversial. [3H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (Kd = 33 ± 4 nm, Bmax = 32 ± 2 pmol/mg). [3H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.

Angiotensin II-stimulated cortisol secretion is mediated by phospholipase D

Angiotensin II (Ang-II) regulates a variety of cellular functions including cortisol secretion. In the present report, we demonstrate that Ang-II activates phospholipase D (PLD) in zona fasciculata (ZF) cells of bovine adrenal glands, and that this effect is associated to the stimulation of cortisol secretion by this hormone. PLD activation was dependent upon extracellular Ca2+, and was blocked by inhibition of protein kinase C (PKC). Using the reverse transcription-polymerase chain reaction technique, we demonstrated that ZF cells express both PLD-1 and PLD-2 isozymes. Primary alcohols, which attenuate the formation of phosphatidate (the product of PLD), and cell-permeable ceramides, which inhibit PLD potently, blocked Ang-II-stimulated cortisol secretion. Furthermore, propranolol or chlorpromazine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked cortisol secretion. These data suggest that the PLD/PAP pathway plays an important role in the regulation of cortisol secretion by Ang-II in ZF cells.

Effects of nitric oxide on aldosterone synthesis and nitric oxide synthase activity in glomerulosa cells from bovine adrenal gland

This study investigated the effects of two NO-releasing agents, diethylenetriamine-NO (deta-NO) and sodium nitroprusside (SNP), on basal, ACTH-, and angiotensin II (AngII)-stimulated aldosterone production in glomerulosa cells from bovine adrenal gland. NO donors inhibited basal and ACTH- or AngII-stimulated aldosterone synthesis in a concentration-dependent manner. Deta-NO and SNP also provoked a concentration-dependent stimulation of cGMP production. However, cGMP was not responsible for the inhibition of aldosterone secretion, because a cGMP analog did not reproduce the inhibitory effect. Moreover, soluble guanylyl cyclase or protein kinase G inhibitors did not revert the inhibitory effect of NO on aldosterone production. NO donors did not modify ACTH-stimulated cAMP production or AngII-stimulated PLC activity stimulation, but inhibited 22[R] hydroxycholesterol- or pregnenolone-stimulated aldosteronogenesis. NO can be synthesized in bovine glomerulosa cells because nitrite production was determined and characterization of NOS activity was also performed. Nitrite accumulation was not modified in the presence of ACTH, AngII, or other factors used to induce iNOS. NOS activity that showed a Michaelis-Menten kinetic was NADPH- and calcium-dependent and was inhibited by two competitive inhibitors, L-NAME and L-NMMA. These results show that NO inhibits aldosterone production in glomerulosa cells acting on P450scc and other P450-dependent steroidogenic enzymes, and these cells display NOS activity suggesting that NO can be produced by constitutive NOS isozymes.

Steroid hormone specifically binds to rat kidney plasma membrane

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using anin vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 µM corticosterone. Under standardized conditions of pH 7.4 at 2°C and 30 min incubation time, the binding was saturable and showedKd=13±3 nM andBmax=616±34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes

SummaryThe specific binding of [3H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding areKD=4.4nm andBmax=685 fmol/mg protein in presence of 1 μm of corticosterone. With respect to the binding of 4nm [3H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone>corticosterone>progesterone>cortisol >prednisolone>testosterone>20β-hydroxyprogesterone >cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca2+ nor Mg2+ affected the binding of [3H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [3H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.