Testosterone regulates the expression and functional activity of sphingosine‐1‐phosphate receptors in the rat corpus cavernosum

The bioactive lipid sphingosine‐1‐phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein‐coupled receptors. The S1P1 receptor is associated with nitric oxide (NO)‐mediated SM relaxation, while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho‐kinase pathway. This study is to determine testosterone (T) modulating the expression and functional activity of S1P receptors in corpus cavernosum (CC). Adult male Sprague‐Dawley rats were randomly divided into three groups: sham‐operated controls, surgical castration and T supplemented group. Serum S1P levels were detected by high‐performance liquid chromatography. The expression of S1P1‐3 receptors and sphingosine kinases was detected by real‐time RT‐PCR. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurement were also performed. T deprivation significantly decreased ICP rise. Meanwhile, surgical castration induced a significant increase in serum S1P level and the expression of S1P2‐3 receptors by twofold (P < 0.05) but a decrease in the expression of S1P1 receptor. Castration also augmented exogenous phenylephrine (PE), S1P, S1P1,3 receptor agonist FTY720‐P contractility and S1P2‐specific antagonist JTE013 relaxation effect. T supplemented could restore the aforementioned changes. We provide novel data that castration increased serum S1P concentration and up‐regulated the expression of S1P2‐3 receptors in CC. Consistently, agonizing S1P receptors induced CCSM contraction and antagonizing mediated relaxation were augmented. This provides the first clear evidence that S1P system dysregulation may contribute to hypogonadism‐related erectile dysfunction (ED), and S1P receptors may be expected as a potential target for treating ED.     

The effect of the sphingosine‐1‐phosphate analogue FTY720 on atrioventricular nodal tissue

The sphingosine‐1‐phosphate (S1P) receptor modulator, fingolimod (FTY720), has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular (AV) conduction block have been reported in some patients after the first dose. The underlying mechanism of this AV node conduction blockade is still not well‐understood. In this study, we hypothesize that expression of this particular arrhythmia might be related to a direct effect of FTY720 on AV node rather than a parasympathetic mimetic action. We, therefore, investigated the effect of FTY720 on AV nodal, using in vitro rat model preparation, under both basal as well as ischaemia/reperfusion conditions. We first look at the expression pattern of S1P receptors on the AV node using real‐time PCR. Although all three S1P receptor isoforms were expressed in AVN tissues, S1P1 receptor isoform expression level was higher than S1P2 and S1P3. The effect of 25 nM FTY720 on cycle length (CL) was subsequently studied via extracellular potentials recordings. FTY720 caused a mild to moderate prolongation in CL by an average 9% in AVN (n = 10, P < 0.05) preparations. We also show that FTY720 attenuated both ischaemia and reperfusion induced AVN rhythmic disturbance. To our knowledge, these remarkable findings have not been previously reported in the literature, and stress the importance for extensive monitoring period in certain cases, especially in patients taking concurrently AV node blocker agents.     

PKC theta activity maintains normal quantal size in chromaffin cells

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC θ and PKC δ, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC θ and δ were expressed in chromaffin cells, mice deficient for PKC θ, but not for PKC δ, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC θ pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC θ mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC θ, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.     

The role of sphingosine 1‐phosphate and its receptors in cardiovascular diseases

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1‐phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P‐based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT‐1303) and the non‐selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.     

Enhancement by sphingosine 1‐phosphate in vasopressin‐induced phosphoinositide hydrolysis in aortic smooth‐muscle cells: Involvement of p38 MAP kinase

We previously reported that sphingosine 1‐phosphate (S‐1‐P), a sphingomyelin metabolite, activates p44/p42 mitogen‐activated protein (MAP) kinase and p38 MAP kinase in aortic smooth‐muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C‐catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C₂‐ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S‐1‐P, which alone slightly stimulated the IPs formation, dose‐dependently amplified the AVP‐induced formation of IPs. Tumor necrosis factor‐α enhanced the AVP‐induced formation of IPs. However, S‐1‐P did not enhance the formation of IPs by NaF, a heterotrimeric GTP‐binding protein activator. Pertussis toxin inhibited the effect of S‐1‐P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S‐1‐P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S‐1‐P on the formation of IPs by AVP. SB203580 inhibited the AVP‐induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S‐1‐P. These results indicate that S‐1‐P amplifies AVP‐induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth‐muscle cells. J. Cell. Biochem. 80:46–52, 2000. © 2000 Wiley‐Liss, Inc.     

Lysophospholipids regulate excitability and exocytosis in cultured bovine chromaffin cells

Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angiogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca(2+) current was inhibited by S1P and LPA pre-treatment in a concentration-dependent manner with IC(50)s of 0.46 and 0.79 mumol/L, respectively. Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na(+) current was inhibited by S1P, but not by LPA. However, recovery of Na(+) channels from inactivation was slowed by both LPLs. The outward K(+) current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Consistent with the reduction in Ca(2+) current, exocytosis elicited by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells.     

Vascular biology: the role of sphingosine 1‐phosphate in both the resting state and inflammation

The vascular and immune systems of mammals are closely intertwined: the individual components of the immune system must move between various body compartments to perform their function effectively. Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, exerts effects on the two organ systems and influences the interaction between them. In the resting state, the vascular S1P gradient contributes to control of lymphocyte recirculation through the blood, lymphoid tissue and lymphatic vasculature. The high level of S1P in blood helps maintain endothelial barrier integrity. During the inflammatory process, both the level of S1P in different immune compartments and S1P receptor expression on lymphocytes and endothelial cells are modified, resulting in functionally important changes in endothelial cell and lymphocyte behaviour. These include transient arrest of lymphocytes in secondary lymphoid tissue, crucial for generation of adaptive immunity, and subsequent promotion of lymphocyte recruitment to sites of inflammation. This review begins with an outline of the basic biochemistry of S1P. S1P receptor signalling is then discussed, followed by an exploration of the roles of S1P in the vascular and immune systems, with particular focus on the interface between them. The latter part concerns crosstalk between S1P and other signalling pathways, and concludes with a look at therapies targeting the S1P-S1P receptor axis.     

RCAN1 regulates vesicle recycling and quantal release kinetics via effects on calcineurin activity

We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over‐expressing RCAN1 (RCAN1^ox), but not in wild‐type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK‐506, replicates this effect in WT cells but has no additional effect in RCAN1^ox cells. When we chronically expose WT cells to cyclosporine A and FK‐506 we find that catecholamine release per vesicle and pre‐spike foot (PSF) signal parameters are decreased, similar to that in RCAN1^ox cells. Inhibiting calcineurin activity in RCAN1^ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1^ox cells, the smaller vesicle and dense core size we observe in RCAN1^ox cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.     

Sphingosine‐1‐phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti‐myeloperoxidase antibody‐positive IgG

Cumulating evidences suggested an important role of sphingosine‐1‐phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)‐ANCA‐positive IgG‐mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO‐ANCA‐positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane‐bound and soluble ICAM‐1 and VCAM‐1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO‐ANCA‐positive IgG‐induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO‐ANCA‐positive IgG. S1P enhanced MPO‐ANCA‐positive IgG‐induced membrane‐bound and soluble ICAM‐1/VCAM‐1 up‐regulation of GEnCs. Soluble ICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG increased upon pre‐incubation of S1PR1 antagonist, while pre‐incubation of GEnCs with the S1PR1 agonist down‐regulated sICAM‐1 level. Blocking S1PR2‐4 reduced sICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG. Pre‐incubation with S1PR5 agonist could increase sICAM‐1 level in the supernatants of GEnC stimulated by S1P and MPO‐ANCA‐positive IgG. S1P can enhance MPO‐ANCA‐positive IgG‐mediated GEnC activation through S1PR2‐5.     

Taipoxin induces F-actin fragmentation and enhances release of catecholamines in bovine chromaffin cells

Adrenomedullary bovine chromaffin cells were used to study the uptake and cellular effects of the phospholipase type A2 (PLA2) neurotoxin taipoxin in a neuroendocrine model. This toxin entered rapidly inside cultured cells. Within 1 h, taipoxin accumulated on the plasma membrane, independently of calcium presence, and caused fragmentation of the F-actin cytoskeleton. Toxin-induced cell death occurred after 24 h of incubation with the appearance of toxin containing large vesicles. Secretory experiments performed in cell populations showed an increased exocytosis in taipoxin-treated cells stimulated by depolarization or by incubation with the calcium-ionophore A23187. Like F-actin fragmentation, this effect is abolished by replacement of Ca2+ with Sr2+ during toxin incubation. The effect of taipoxin on exocytosis is not enhanced by latrunculin A, a F-actin disassembling drug altering secretion. Secretory studies in single taipoxin-treated cells using amperometry, showed an increase in the number of released vesicles without modification of the kinetic parameters of individual vesicle fusions. Taken together, these results suggest that taipoxin causes F-actin fragmentation and enhances secretion by redistribution of vesicles among secretory pools.     

Key role of the nicotinic receptor in neurotransmitter exocytosis in human chromaffin cells

The whole-cell secretory response evoked by acetylcholine (ACh) in human chromaffin cells was examined using a new protocol based on quickly switching from the voltage-clamp to the current-clamp (CC) configuration of the patch-clamp technique. Our experiments revealed that Ca(2+) entry through the nicotinic receptor at hyperpolarized membrane potentials contributed as much to the exocytosis (100.4 +/- 27.3 fF) evoked by 200 ms pulses of ACh, as Ca(2+) flux through voltage-dependent Ca(2+) channels at depolarized membrane potentials. The nicotinic current triggered a depolarization event with a peak at +49.3 mV and a 'plateau' phase that ended at -23.9 mV, which was blocked by 10 mumol/L mecamylamine. When a long ACh stimulus (15 s) was applied, the nicotinic current at the end of the pulse reached a value of 15.45 +/- 3.6 pA, but the membrane potential depolarization still remained at the 'plateau' stage until withdrawal of the agonist. Perfusion with 200 mumol/L Cd(2+) during the 15 s ACh pulse completely abolished the plasma membrane depolarization at the end of the pulse, indicating that Ca(2+) entry through Ca(2+) channels contributed to the membrane potential depolarization provoked by prolonged ACh pulses. These findings also reflect that voltage-dependent Ca(2+) channels were recruited by the small current flowing through the desensitized nicotinic receptor to maintain the depolarization. Finally, muscarinic receptor activation triggered a delayed exocytotic process after prolonged ACh stimulation, dependent on Ca(2+) mobilization from the endoplasmic reticulum. In summary, we show here that nicotinic and muscarinic receptors contribute to the exocytosis of neurotransmitters in human chromaffin cells, and that the nicotinic receptor plays a key role in several stages of the stimulus-secretion coupling process in these cells.     

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.     

Selective recapture of secretory granule components after full collapse exocytosis in neuroendocrine chromaffin cells

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.     

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.     

Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.