Effects of urotensin II on functional activity of late endothelial progenitor cells

Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10(-10), 10(-9), 10(-8), 10(-7) and 10(-6)M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.     

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[³H]glutamate. The apparent binding constant K _d was 135 nM and 393 nM and the maximal binding capacity B_max 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC₅₀ in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [³H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.     

Elevated expression of urotensin II and its receptor in diethylnitrosamine-mediated precancerous lesions in rat liver

Urotensin II (UII) is a somatostatin-like peptide involved in cell proliferation and in tumor biology. To explore the role of liver-derived UII in the pathogenesis of precancerous liver lesions in rat, we investigated the expression of UII and its receptor, UT, in diethylnitrosamine (DEN)-induced precancerous liver lesions and the effects of UII on cell proliferation by hepatic oval cells. Radioimmunoassay, RT-PCR, immunohistochemistry and western blot were used in this study. Compared with untreated controls, rats treated with DEN showed increased UII content by 47.7% in plasma and by 164.9% in liver tissue (all P < 0.01). The expression of UII protein and of both UT mRNA and protein was significantly enhanced in the liver of treated rats. Western blot analysis revealed that the expression of phosphorylated protein kinase C (p-PKC) and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) was increased in the liver of treated animals. Treatment with UII (10⁻¹⁰-10⁻⁶ M) for 24 h significantly increased number of cultured hepatic oval cells (at 10⁻⁹-10⁻⁸ M). However, during the pre-incubation with calphostin C (inhibitor of PKC) or PD98059 (inhibitor of MEK), the proliferation was decreased by 40.1% and 25.4% respectively (both P < 0.05). In DEN-induced precancerous liver lesions, the UII/UT system was up-regulated, which may contribute to the pathogenesis of liver cancer through a PKC- or ERK1/2-dependent pro-mitogenic pathway in an autocrine/paracrine manner.     

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK_1/2 phosphorylation and, to a lesser extent, IP₁ production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.     

Biochemical and functional characterization of alpha-adrenergic receptors in the rabbit vagina

Vascular and non-vascular smooth muscle within the vagina mediate important physiological changes during sexual arousal in women. In this study, we have characterized alpha-adrenergic receptors (AR) in rabbit vagina by assessment of radioligand binding, contractility of isolated tissue strips and genital hemodynamics. [3H]Prazosin and [3H]RX821002 (alpha-1 and alpha-2 AR selective antagonists) bound to rabbit vaginal membrane preparations with high affinity and limited capacity. Competition binding assays using both non-selective and subtype selective ligands for AR (phentolamine, prazosin, delequamine, rauwolscine and UK14304) further confirmed the presence of alpha-1 and alpha-2 AR in vaginal tissue. In organ bath preparations of vaginal tissue strips, norepinephrine-induced contraction was attenuated by alpha-1 and alpha-2 AR antagonists (prazosin, tamsulosin, delequamine and phentolamine). In anesthetized rabbits, intravaginal injection of the alpha-1 AR selective antagonist REC 15/2615 (50 and 100 microg/kg) caused a 2 to 3-fold increase in genital tissue oxyhemoglobin (OHb) concentration. Similar increases in tissue OHb were observed with intravaginal injection of phentolamine (500 microg/kg) or a tri-mixture of vasodilators (PGE1, papaverine, phentolamine). REC 15/2615, phentolamine or the tri-mixture also enhanced the amplitude and/or duration of change in genital tissue OHb after pelvic nerve stimulation. Thus, vaginal tissue expresses functional alpha-1 and alpha-2 AR, which modulate vaginal smooth muscle contractility and genital engorgement.     

Aequorin functional assay for characterization of G-protein-coupled receptors: Implementation with cryopreserved transiently transfected cells

Assay technologies that measure intracellular Ca²⁺ release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca²⁺ sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M₁ receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays.     

Functional expression of metabotropic GABAB receptors in primary cultures of astrocytes from rat cerebral cortex

GABA(B) receptor subunits are widely expressed on neurons throughout the central nervous system (CNS), at both pre- and postsynaptic sites, where they mediate the late and slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. Recently, GABA(B) receptors have been reported to be expressed in astrocytes and microglia in the rat CNS by immunocytochemistry. However, there are few reports available for the functional characterization of GABA(B) receptors on astrocytes. In the present study, we therefore investigated the functional expression and characteristics of GABA(B) receptors in primary cultures of astrocytes from rat cerebral cortex. In the presence of 10 microM GTP, forskolin concentration-dependently increased adenylylcyclase (AC) activity in membranes prepared from rat astrocytes. The selective GABA(B) agonist (R)-baclofen concentration-dependently reduced forskolin-stimulated AC activity in the presence of 10 microM GTP. This effect was reversed by the selective GABA(B) antagonists, CGP-55845 and CGP-54626, and was completely abolished by treatment of astrocytic membranes with pertussis toxin. In addition, RT-PCR, Western blotting, and immunocytochemistry clearly showed that metabotropic GABA(B) receptor isoforms (GABA(B)R1 and GABA(B)R2) are expressed in rat cerebrocortical astrocytes. Taken collectively, these results demonstrate that functionally active metabotropic GABA(B) receptors are expressed in rat cerebrocortical astrocytes.     

Ethanol impairs monosaccharide uptake and glycosylation in cultured rat astrocytes

Astrocyte and glial-neuron interactions have a critical role in brain development, which is partially mediated by glycoproteins, including adhesion molecules and growth factors. Ethanol affects the synthesis, intracellular transport, subcellular distribution and secretion of these glycoproteins, suggesting alterations in glycosylation. We analyzed the effect of long-term exposure to low doses of ethanol (30 mm) on glycosylation process in growing cultured astrocytes in vitro. Cells were incubated for short (5 min) and long (90 min) periods with several radioactively labeled carbohydrate precursors. The uptake, kinetics and metabolism of these precursors, as well as the radioactivity distribution in protein gels were analyzed. The levels of GLUT1 and mannosidase II were also determined. Ethanol increased the uptake of monosaccharides and the protein levels of GLUT1 but decreased those of mannosidase II. It altered the carbohydrate moiety of proteins and increased cell surface glycoproteins containing terminal non-reduced mannose. These results indicate that ethanol impairs glycosylation in rat astrocytes, thus disrupting brain development.     

Cardiovascular effects of native and non-native urotensin II and urotensin II-related peptide on rat and salmon hearts

Urotensin II (UII) was first discovered in the urophyses of goby fish and later identified in mammals, while urotensin II-related peptide (URP) was recently isolated from rat brain. We studied the effects of UII on isolated heart preparations of Chinook salmon and Sprague–Dawley rats. Native rat UII caused potent and sustained, dose-dependent dilation of the coronary arteries in the rat, whereas non-native UII (human and trout UII) showed attenuated vasodilation. Rat URP dilated rat coronary arteries, with 10-fold less potency compared with rUII. In salmon, native trout UII caused sustained dilation of the coronary arteries, while rat UII and URP caused significant constriction. Nω-nitro-_l-arginine methyl (l-NAME) and indomethacin significantly attenuated the URP and rat UII-induced vasodilation in the rat heart. We conclude that UII is a coronary vasodilator, an action that is species form specific. We also provide the first evidence for cardiac actions of URP, possibly via mechanisms common with UII.     

Characterization of functional urotensin II receptors in human skeletal muscle myoblasts: comparison with angiotensin II receptors

The properties of urotensin II (U-II) receptor (UT receptor) and angiotensin II (ANG II) receptor (AT receptor) in primary human skeletal myoblasts (HSMM) and differentiated skeletal myotubes (HSMMT) were characterized. Radiolabeled U-II and ANG II bound specifically to HSMM with Kd's of 0.31 nM (2311 receptors/cell) and 0.61 nM (18,257 receptors/cell), respectively. The cyclic segment of U-II peptide, CFWKYC, was the minimal sequence required for binding, with the WKY residues essential. Inhibitor studies suggested AT1 is the predominant ANG II receptor. After radioligand binding, under conditions designed to minimize receptor internalization, half the bound U-II was resistant to acid washing suggesting that U-II binds tightly to its receptor in a quasi-irreversible fashion. The AT1 receptor-bound radioligand was completely removed under the same conditions. RT-PCR detected the expression of mRNAs for UT and AT1 receptors. Western blotting showed that U-II and ANG II signaled via ERK1/2 kinase. UT receptor was not lost upon differentiation into myotubes since both mRNA for UT receptor and U-II binding were still present. ANG II receptors were also present as shown by ANG II-induced calcium mobilization.     

Structure-activity relationship study on human urotensin II.

The vasoactive cyclic undecapeptide urotensin-II (U-II) has been identified as an endogenous ligand for the G-protein coupled receptor now referred to as the UT receptor. The U-II/UT receptor system might be relevant for cardiovascular functions. A structure-activity study of human U-II investigating 31 peptides in the rat aorta bioassay is reported. Ala- and D-scan investigations indicated that the sequence Phe6-Trp7-Lys8-Tyr9 is essential for biological activity and that Lys8 and Tyr9 are particularly important. These two residues were substituted with a series of coded and non-coded amino acids. These studies demonstrated that the positive charge of the primary aliphatic amine at position 8 and its relative spatial orientation is crucial for both receptor occupation and activation, while the only chemical requirement at position 9 is the presence of an aromatic moiety. Moreover, this study led to the identification of UT receptor partial agonists (compounds 23 and 24) which can be used as chemical templates for further investigations aimed at the identification of selective antagonists.     

β-adrenergic receptor regulation,through cyclic AMP,of nerve growth factor expression in rat cortical and cerebellar astrocytes

1. Type 1 astrocytes prepared from 3-day rat cortex and cerebellum express the 1.3-kb nerve growth factor (NGF) mRNA and synthesize and release beta-NGF. 2. Isoproterenol (IP), a beta-adrenergic agonist, stimulates NGF mRNA content in cortical astrocytes; this increase is blocked by the beta-adrenergic antagonist propranolol but not the alpha-antagonist phenoxybenzamine. The EC50 for the effect of IP is 5 nM. 3. IP increases astrocyte cyclic AMP as does forskolin, which directly activates adenylate cyclase and also increases NGF mRNA content. Cerebellar astrocytes contain about one-third as much NGF mRNA, which can also be increased by forskolin and cyclic AMP. 4. These results suggest that CNS astrocytes can serve as a source of NGF and that the NGF gene is one of the class of cyclic AMP regulated genes.     

Biochemical characterization and immunohistochemical localization of urotensin II in the human brainstem and spinal cord

The human urotensin II (UII) precursor encompasses several potential cleavage sites and thus, processing of pro-UII may generate various forms of mature UII including the peptides of 11 (UII11), 16 (UII16) and 19 (UII19) residues. Until now, the native form of human UII had not been characterized. Here, we show that the major UII peptide occurring in the human spinal cord corresponds to UII11. In contrast, neither the UII16 nor the UII19 forms could be detected. In 50% of the brainstem and in all the spinal cord extracts analysed, a second minor UII-immunoreactive peptide was resolved. Immunohistochemical labelling of the cervical segment of the human spinal cord revealed that the UII-immunoreactive material was confined to a subset of ventral horn motoneurones. These data provide the first evidence that in the human, the UII precursor, expressed in motoneurones, is processed at the tribasic KKR93 cleavage site to generate a mature form of UII of 11 amino acids. The absence of N-terminally elongated forms of UII of 16 and 19 residues indicates that pro-UII is not cleaved at the R85 or K88 monobasic sites. Finally, the minor UII-immunoreactive peptide detected in several tissue extracts might correspond to an extended form of UII resulting from the processing of the UII precursor at the basic RK50 or RK66 doublets.     

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

A novel cross-talk between endothelin and ErbB receptors controlling glutamate transporter expression in astrocytes

The endothelin and epidermal growth factor (EGF) systems are central to the control of reactive brain processes and are thought to partly exert these tasks by endothelin-induced transactivation of the epidermal growth factor receptor (EGFR) Here we show that beyond EGFR transactivation, endothelins prevent the ligand-induced internalization of the EGFR. We unravel that endothelins abrogate internalization of the EGFR by either promoting the formation of "internalization-deficient" EGFR/ErB2-heterodimers or by activating c-Abl kinase, a negative regulator of EGFR internalization. We further provide evidence that this cross-talk is operational in the control of astrocytic glutamate transport. Specifically, we establish that the inhibitory effects exerted by endothelins on basal as well as EGF-induced expression of the major astroglial glutamate transporter subtype, glutamate transporter 1, are a direct consequence of the endothelin-dependent retention of the EGFR at the cell surface. Together our findings unravel a previously unknown cross-talk between endothelin and epidermal growth factor receptors, which may have implications for a variety of pathological conditions.     

Intracellular Na+ inhibits volume‐regulated anion channel in rat cortical astrocytes

Accumulating evidence indicates that increased intracellular Na⁺ concentration ([Na⁺]_i) in astroglial cells is associated with the development of brain edema under ischemic conditions, but the underlying mechanisms are still elusive. Here, we report that in primary cultured rat cortical astrocytes, elevations of [Na⁺]_i reflecting those achieved during ischemia cause a marked decrease in hypotonicity‐evoked current mediated by volume‐regulated anion channel (VRAC). Pharmacological manipulations revealed that VRAC inhibition was not due to the reverse mode of the plasma membrane sodium/calcium exchanger. The negative modulation of VRAC was also observed in an astrocytic cell line lacking the predominant astrocyte water channel aquaporin 4, indicating that [Na⁺]_i effect was not mediated by the regulation of aquaporin 4 activity. The inward rectifier Cl^? current, which is also expressed by cultured astrocytes, was not affected by [Na⁺]_i increase. VRAC depression by high [Na⁺]_i was confirmed in adult astrocytes, suggesting that it was not developmentally regulated. Altogether, these results provide the first evidence that intracellular Na⁺ dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na⁺ accumulation might be a favorable strategy to counteract the development of brain edema.

     

Patterns of synthesis and secretion of sulfated glycosaminoglycans in primary cortical and cerebellar astrocytes in vitro

We determined the amounts of [35S]-glycosaminoglycans (GAGs) found on the intracellular, pericellular and extracellular compartments of primary cultures of astrocytes derived from newborn rat cortex and cerebellum in vitro. Our results show that the greatest portion of newly synthesized GAGs were found in different cellular compartments, depending on the source of the astrocytes. In the cells derived from the cerebellum, the proportion of [35S]-GAGs secreted to the culture medium preponderates over the amount found in the two other compartments, whereas cells derived from the cortex accumulated higher proportions of [35S]-GAGs in the intracellular compartment than in the two other compartments. Cortical and cerebellar glial cells synthesised and secreted heparan sulfate (HS) and chondroitin 4-sulfate (C-4S). HS was predominantly accumulated on the pericellular surface, while C-4S was mostly secreted to the culture medium. Beside the difference on the distribution of total [35S]-GAGs among the three cellular compartments, no difference was observed on the relative proportions of HS and C-4S within each compartment. By defining the source of GAGs, the present study may help to complement and extend information on biosynthesis of these compounds by mammalian glial cells.