Regina Alemany

Sphingosine kinase‐mediated Ca2+ signalling by G‐protein‐coupled receptors

Formation of inositol 1,4,5‐trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G‐protein‐coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC–IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine‐1‐phosphate (SPP), is involved in GPCR‐mediated Ca2+ signalling as proposed for platelet‐derived growth factor and FcϵRI antigen receptors. Inhibition of sphingosine kinase by DL‐threo‐dihydrosphingosine and N,N‐dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK‐293 cells without affecting mAChR‐induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK‐293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK‐293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase–SPP pathway in addition to PLC–IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR‐induced Ca2+ signalling.

Regulation and functional roles of sphingosine kinases

Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca2+ homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.

Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors

Recently, a family of G-protein-coupled receptors named endothelial differentiation gene (Edg) receptor family has been identified, which are specifically activated by the two serum lipids, sphingosine-1-phosphate and lysophosphatidic acid. Sphingosine-1-phosphate can also act intracellularly to release Ca2+ from intracellular stores. Since in several cell types, G-protein-coupled lysophosphatidic acid or sphingosine-1-phosphate receptors mobilize Ca2+ in the absence of a measurable phospholipase C stimulation, it was analysed here whether intracellular sphingosine-1-phosphate production was the signalling mechanism used by extracellular sphingosine-1-phosphate for mobilization of stored Ca2+. Sphingosine-1-phosphate and the low affinity sphingosine-1-phosphate receptor agonist, sphingosylphosphorylcholine, induced a rapid, transient and nearly complete pertussis toxin-sensitive Ca2+ mobilization in human embryonic kidney (HEK-293) cells. The G-protein-coupled sphingosine-1-phosphate receptors, Edg-1, Edg-3 and Edg-5, were found to be endogenously expressed in these cells. Most interestingly, sphingosine-1-phosphate and sphingosylphosphorylcholine did not induce a measurable production of inositol-1,4,5-trisphosphate or accumulation of inositol phosphates. Instead, sphingosine-1-phosphate and sphingosylphosphorylcholine induced a rapid and transient increase in production of intracellular sphingosine-1-phosphate with a maximum of about 1.4-fold at 30 s. Stimulation of sphingosine-1-phosphate formation by sphingosine-1-phosphate and sphingosylphosphorylcholine was fully blocked by pertussis toxin, indicating that extracellular sphingosine-1-phosphate via endogenously expressed G(i)-coupled receptors induces a stimulation of intracellular sphingosine-1-phosphate production. As sphingosine-1-phosphate- and sphingosylphosphorylcholine-induced increases in intracellular Ca2+ were blunted by sphingosine kinase inhibitors, this sphingosine-1-phosphate production appears to mediate Ca2+ signalling by extracellular sphingosine-1-phosphate and sphingosylphosphorylcholine in HEK-293 cells.

Role of sphingosine kinase in Ca2+ signalling by epidermal growth factor receptor

Contribution of sphingosine kinase (SPK)‐catalyzed production of sphingosine‐1‐phosphate (SPP), in comparison to phospholipase C (PLC), to Ca2+ signalling by epidermal growth factor (EGF) was studied in two HEK‐293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca2+ by intracellular SPP. In HEK3 cells, EGF increased [Ca2+]i and stimulated both, SPK and PLC. [Ca2+]i increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca2+]i or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca2+ signalling by EGF in HEK‐293 cells.

Alteration of Lipids, G Proteins, and PKC in Cell Membranes of Elderly Hypertensives

Abstract—In this study, we quantified the levels of lipids and signaling proteins in erythrocyte membranes from elderly normotensive and hypertensive subjects. In hypertensive subjects, the cholesterol/phospholipid ratio increased significantly in erythrocyte membranes, owing to the reduction of phospholipid levels concomitant with a rise in the levels of cholesterol. In addition, differences were also found in the amount of fatty acids in both phospholipid and cholesterol esters. Erythrocyte membranes from hypertensive subjects contained higher levels of monounsaturated and lower levels of polyunsaturated fatty acids. On the other hand, signaling proteins such as G proteins and protein kinase C have been implicated in the control of blood pressure. Previous studies have shown that the cellular localization and the activity of these proteins are modulated by the type and the abundance of membrane lipids. For this reason, we assessed the levels of these signaling molecules in the membrane. We found that the levels of membrane-associated (active/preactive) G proteins (G&agr;i, G&agr;o, and G&bgr;) and protein kinase C were significantly reduced in hypertensive subjects. We believe that these alterations could be related to the etiopathology of hypertension in elderly subjects or alternatively may correspond to adaptive compensatory mechanisms.

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.

The effects of phenelzine and other monoamine oxidase inhibitor antidepressants on brain and liver I2 imidazoline‐preferring receptors

1 The binding of [3H]‐idazoxan in the presence of 10−6 m (−)‐adrenaline was used to quantitate I2 imidazoline‐preferring receptors in the rat brain and liver after chronic treatment with various irreversible and reversible monoamine oxidase (MAO) inhibitors. 2 Chronic treatment (7–14 days) with the irreversible MAO inhibitors, phenelzine (1–20 mg kg−1, i.p.), isocarboxazid (10 mg kg−1, i.p.), clorgyline (3 mg kg−1, i.p.) and tranylcypromine (10 mg kg−1, i.p.) markedly decreased (21–71%) the density of I2 imidazoline‐preferring receptors in the rat brain and liver. In contrast, chronic treatment (7 days) with the reversible MAO‐A inhibitors, moclobemide (1 and 10 mg kg−1, i.p.) or chlordimeform (10 mg kg−1, i.p.) or with the reversible MAO‐B inhibitor Ro 16–6491 (1 and 10 mg kg−1, i.p.) did not alter the density of I2 imidazoline‐preferring receptors in the rat brain and liver; except for the higher dose of Ro 16–6491 which only decreased the density of these putative receptors in the liver (38%). 3 In vitro, phenelzine, clorgyline, 3‐phenylpropargylamine, tranylcypromine and chlordimeform displaced the binding of [3H]‐idazoxan to brain and liver I2 imidazoline‐preferring receptors from two distinct binding sites. Phenelzine, 3‐phenylpropargylamine and tranylcypromine displayed moderate affinity (KiH = 0.3–6 μm) for brain and liver I2 imidazoline‐preferring receptors; whereas chlordimeform displayed high affinity (KiH = 6 nm) for these receptors in the two tissues studied, Clorgyline displayed very high affinity for rat brain (KiH = 40 pm) but not for rat liver I2 imidazoline‐preferring receptors (KiH = 169 nm). 4 Preincubation of cortical or liver membranes with phenelzine (10−4 m for 30 min) did not alter the total density of I2 imidazoline‐preferring receptors, indicating that this irreversible MAO inhibitor does not irreversibly bind to I2 imidazoline‐preferring receptors. In contrast, preincubation with 10−6 m clorgyline reduced by 40% the Bmax of [3H]‐idazoxan to brain and liver I2 imidazoline‐preferring receptors. 5 Chronic treatment (7 days) with the inducers of cytochrome P‐450 enzymes phenobarbitone (40 or 80 mg kg−1, i.p.), 3‐methylcholanthrene (20 mg kg−1, i.p.) or 2‐methylimidazole (40 mg kg−1, i.p.) did not alter the binding parameters of [3H]‐idazoxan to brain and liver I2 imidazoline‐preferring receptors. The compound SKF 525A, a potent inhibitor of cytochrome P‐450 enzymes which forms a tight but reversible complex with the haemoprotein, completely displaced with moderate affinity (KiH = 2–10 μm) the specific binding of [3H]‐idazoxan to brain and liver I2 imidazoline‐preferring receptors. Preincubation of total liver homogenates with 3 times 10−4 m phenelzine in the presence of 10−3 m NADH, a treatment that irreversibly inactivates the haeme group of cytochrome P‐450, did not reduce the density of liver I2 imidazoline‐preferring receptors. These results discounted a possible interaction of [3H]‐idazoxan with the haeme group of cytochrome P‐450 enzymes. 6 Together the results indicate that the down‐regulation of I2 imidazoline‐preferring receptors is associated with an irreversible inactivation of MAO (at least in the brain) that is not related either to the affinity of the MAO inhibitors for I2 imidazoline‐preferring receptors or to an irreversible binding to these putative receptors. These findings indicate a novel effect of irreversible MAO inhibitors in the brain and suggest a new target for these compounds that could be of relevance in the treatment of depression, a disease in which an increased density of brain I2 imidazoline‐preferring receptors has been reported.

2-Hydroxyoleic Acid. A New Hypotensive Molecule

Abstract—Recent studies have shown that diets rich in monounsaturated fatty acids (MUFAs) from olive oil, a natural source of oleic acid, have beneficial effects on blood pressure (BP) in hypertensive patients. With this in mind, we investigated whether a synthetic derivative of the MUFA oleic acid, 2-hydroxyoleic acid (2-OHOA), was capable of regulating the BP of Sprague-Dawley rats. Intraperitoneal and oral administration of 2-OHOA to rats induced significant and sustained decreases in BP in a time-dependent manner. Without affecting heart rate, treatments for 7 days provoked reductions in systolic BP of 20 to 26 mm Hg. At the molecular level, the density of G&agr;s, but not G&agr;i2 or G&agr;o, increased in membranes from the hearts and aortas of 2-OHOA–treated rats, whereas in heart membranes, the density of G&agr;q/11 and protein kinase C&agr; proteins was also augmented. These molecular alterations were reflected in the increase in cAMP levels after G&agr;s protein and &bgr;-adrenergic receptor stimulation. On the contrary, inhibitory hormones reduced adenylyl cyclase activity to the same extent in 2-OHOA–treated rats as in vehicle-treated ones. Our results indicate that cardiovascular tissues from 2-OHOA–treated rats exhibited increased cAMP production in response to G&agr;s activation, which might be attributed to enhanced expression of G&agr;s proteins. As a result of this change, a significant reduction in systolic BP was observed. Therefore, BP can be lowered by administration of 2-OHOA, which might represent the first member of a new family of antihypertensive drugs.

The effects of chronic imidazoline drug treatment on glial fibrillary acidic protein concentrations in rat brain.

1 The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (western blotting) in the rat brain after treatment with various imidazoline drugs and other agents. 2 Chronic (7 days) but not acute (1 day) treatment with the imidazoline drugs, cirazoline (1 mg kg−1, i.p.) and idazoxan (10 mg kg−1, i.p.), but not with the structurally related α2‐adrenoceptor antagonists, RX821002 (2‐methoxy idazoxan) (10 mg kg−1, i.p.) and efaroxan (10 mg kg−1, i.p.), markedly increased (45%) GFAP immunoreactivity in the rat cerebral cortex. Chronic treatment (7 days) with yohimbine (10 mg kg−1, i.p.), a non‐imidazoline α2‐adrenoceptor antagonist, did not significantly modify GFAP immunoreactivity in the cerebral cortex. 3 Chronic treatment (7 days) with cirazoline and idazoxan did not alter the density of brain monoamine oxidase (MAO)‐B sites labelled by [3H]‐Ro 19–6327 (lazabemide), another relevant astroglial marker. Moreover, these imidazoline drug treatments did not modify the levels of α‐tubulin in the cerebral cortex. These negative results reinforced the specificity of the effects of imidazoline drugs on GFAP. 4 Irreversible inactivation of brain α2‐adrenoceptors (and other neurotransmitters receptors) after treatment with an optimal dose of the peptide‐coupling agent EEDQ (1.6 mg kg−1, i.p., for 6–24 h) did not alter GFAP immunoreactivity in the cerebral cortex. These results further disproved the involvement of these receptors on astroglial cells in the tonic control of GFAP levels. 5 The binding of [3H]‐idazoxan in the presence of 10−6 m (–)‐adrenaline was used to quantitate in parallel I2‐imidazoline preferring sites in the rat brain after the same treatments. Chronic treatment (7 days) with cirazoline and idazoxan, but not with RX821002, efaroxan or yohimbine, significantly increased (25%) the density of I2‐sites in the cerebral cortex. The up‐regulation of I2‐sites induced by cirazoline was not observed in the liver, a tissue that also expresses I2‐sites but lacks glial cells. 6 A strong correlation (r = 0.97) was found when the mean percentage changes in GFAP immunoreactivity were related to the mean percentage changes in I2 imidazoline sites after the various drug treatments. 7 Together the results suggest a direct physiological function of glial I2‐imidazoline preferring sites in the regulation of GFAP levels.

The hypotensive drug 2-hydroxyoleic acid modifies the structural properties of model membranes

We studied the interactions of the hypotensive drug, 2-hydroxyoleic acid (2OHOA), with model membranes using the techniques of DSC, 31P NMR and X-ray diffraction. We demonstrate that 2OHOA alters the thermotropic behaviour of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), thereby promoting the formation of hexagonal phases (HII), despite stabilizing the lamellar phase (Lα). The lattice parameters of lamellar and non-lamellar structures were not altered by the presence of 2OHOA. The molecular bases underlying the alterations in membrane structure provoked by 2OHOA were analysed by comparing the effects produced by 2OHOA with the closely related fatty acids (FAs), oleic acid (OA) and elaidic acid (EA). The capacity of C-18 FAs to induce HII-phase formation followed the order OA>2OHOA>EA. Furthermore, while 2OHOA stabilized the Lα phase, OA destabilized it. The net negative charge of 2OHOA at physiological pH (∼7.4) influenced its effect on membrane structure. By analysing the molecular architecture of 2OHOA in DEPE monolayers, interactions between the carboxylate groups of 2OHOA and the amine groups of DEPE were observed, as well as between the 2-hydroxyl group of the FA and the carbonyl oxygen of the phospholipid acyl chain. These structural characteristics provoked an increase in the P-to-N and P-to-P distances of neighbouring phospholipid headgroups in the presence of 2OHOA, with respect to those observed with OA and EA. The higher headgroup area at the lipid–water interface in presence of 2OHOA could account for the differential effect of this drug on the phase behaviour of DEPE membranes.