Myristoylation of cGMP-dependent protein kinase dictates isoform specificity for serotonin transporter regulation

By transporting serotonin (5-HT) into neurons and other cells, serotonin transporter (SERT) modulates the action of 5-HT at cell surface receptors. SERT itself is modulated by several processes, including the cGMP signaling pathway. Activation of SERT by cGMP requires the cGMP-dependent protein kinase (PKG). Here we show that in HeLa cells lacking endogenous PKG, expression of PKGIα or PKGIβ was required for 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) to stimulate SERT phosphorylation and 5-HT influx. Catalytically inactive PKG mutants and wild-type PKGII did not support this stimulation. However, a mutant PKGII (G2A) that was not myristoylated substituted for functional PKGI, suggesting that myristoylation and subsequent membrane association blocked productive interaction with SERT. PKG also influenced SERT expression and localization. PKGI isoforms increased total and cell surface SERT levels, and PKGII decreased cell surface SERT without altering total expression. Remarkably, these changes did not require 8-Br-cGMP or functional kinase activity and were also observed with a SERT mutant resistant to activation by PKG. Both PKGIα and PKGIβ formed detergent-stable complexes with SERT, and this association did not require catalytic activity. The nonmyristoylated PKGII G2A mutant stimulated SERT expression similar to PKGI isoforms. These results suggest multiple mechanisms by which PKG can modulate SERT and demonstrate that the functional difference between PKG isoforms results from myristoylation of PKGII.     

Serotonin transporter phosphorylation modulated by tetanus toxin

Tetanus toxin (TeTx) modifies Na(+)-dependent, high-affinity 5-hydroxytryptamine (5-HT, serotonin) uptake in a synaptosomal-enriched P(2) fraction from rat brain. The effect corresponds to a rapid and non-competitive uptake inhibition, and it is preceded by induction of phospholipase C (PLC) activity and translocation and down-regulation of the classical protein kinase C (PKC-alpha, -beta and -gamma) isoforms. The effects on serotonin transport and on cPKC activation were similar to the effects exhibited by phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Moreover, after treatment with TeTx, an increase in Ser- and Tyr-specific phosphorylation was found. Activation of PKC by both TeTx and TPA results in a loss of transport capacity and serotonin transporter (SERT) phosphorylation, which are abolished by coapplication of the specific PKC inhibitor bisindolylmaleimide-1. Since a specific PLCgamma1 phosphorylation prior to TeTx's inducing SERT phosphorylation was found, the studies suggest that part of the action of TeTx consists of modifying the signal cascade initiated in tyrosine kinase receptors on nerve tissue previous to its cellular internalization, resulting in transporter phosphorylation.     

Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase

The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including “ecstasy” (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N⁶-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-³³P]ATP but not from similarly labeled N⁶-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.     

Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins

Cyclic GMP-dependent protein kinase (PKG) plays an important role in regulating pulmonary vasomotor tone in the perinatal period. In this study, we tested the hypothesis that a change in oxygen tension affects PKG-mediated pulmonary vasodilation. Isolated intrapulmonary arteries and veins of near-term fetal lambs were first incubated for 4 h under hypoxic and normoxic conditions (Po2 of 30 and 140 mmHg, respectively) and then contracted with endothelin-1. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cell membrane-permeable analog of cGMP, induced a greater relaxation in vessels incubated in normoxia than in hypoxia. beta-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-PET-cGMPS), a selective inhibitor of PKG, attenuated relaxation induced by 8-BrcGMP (10-4 and 3 x 10-4 M). In the presence of Rp-8-Br-PET-cGMPS, the differential responses to 8-BrcGMP between hypoxia and normoxia treatment were abolished in veins but not in arteries. cGMP-stimulated PKG activity was present in arteries but not in veins after 4 h of hypoxia. Both vessel types showed significant increase in cGMP-stimulated PKG activity after 4 h of normoxia. PKG protein (Western blot analysis) and PKG mRNA levels (quantitative RT-PCR) were greater in veins but not in arteries after 4-h exposure to normoxia vs. hypoxia. These results demonstrate that oxygen augments cGMP-mediated vasodilation of fetal pulmonary arteries and veins. Furthermore, the effect of oxygen on response of the veins to cGMP is due to an increase in the activity, protein level, and mRNA of PKG.     

p38 MAPK activation elevates serotonin transport activity via a trafficking-independent, protein phosphatase 2A-dependent process

Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.     

Functional interactions between native Gs-coupled 5-HT receptors in HEK-293 cells and the heterologously expressed serotonin transporter

In HEK-293 cells, serotonin (5-hydroxytryptamine, 5-HT) was found to induce cAMP production showing pharmacological characteristics consistent with the 5-HT(7) receptor. The presence of 5-HT(7) (and 5-HT(6)) receptor mRNA was confirmed by RT-PCR. Stable HEK-293 cell lines expressing either wild-type or haemagglutinin (HA)-tagged human 5-HT transporter (SERT) were selected and SERT function was confirmed using [3H]5-HT transport. The presence of SERT caused a 10-fold reduction in the potency of 5-HT-induced cAMP production compared to control cells. Downstream signalling by 5-HT(6/7) receptors could be detected as 5-HT-induced protein kinase A activation and phosphorylation of MAP kinase and CREB using phospho-specific antibodies. SERT inhibitors reversed the reduction in potency of 5-HT-induced cAMP production caused by the presence of SERT, resulting in a concentration-dependent left shift in EC(50) values but also a progressive decrease in the maximal response. Thus, when antidepressants were used to block SERT activity, 5-HT receptor signalling was effectively clamped within a mid-range.     

Chemosensory behaviour and ciliary cyclic GMP-dependent protein kinase in Tetrahymena thermophila

The role of protein kinase, in particular cyclic GMP-dependent protein kinase (PKG), in the control of chemotaxis was studied in Tetrahymena thermophila using the membrane-permeable cGMP analogue 8-bromo-cGMP and the NO-generator sodium nitroprusside (SNP) that stimulates cGMP production by activating guanylate cyclase. Stimulation of chemoattraction was observed in the presence of 8-bromo-cGMP and nitroprusside when used in 10–100 μM concentrations in vivo. In vitro stimulation of ciliary membrane PKG activity was observed when using similar concentrations of cGMP or 8-bromo-cGMP to those in the in vivo experiments. In contrast, the protein kinase flavonol inhibitors quercitin and kaempherol block chemoattraction and reduce ciliary membrane PGK activity in vitro. For the inhibition of PKG, the IC-50 s for quercitin and kaempherol are 22 and 19 μM, respectively. The results suggest a modulating function of PKG on adaptory processes in cilia-mediated chemotaxis.

The ciliary membrane-associated PKG was partially characterized. Without added external protein kinase substrate in vitro, an endogenous ciliary membrane kinase activity showed phosphorylation of 55 and 97 kDa Triton-X-100 soluble proteins when analyzed by SDS-PAGE under reducing conditions and with ³²P-γ-ATP as phosphorylation donor. Phosphoamino acid analysis of PKG-phosphorylated proteins showed ³²P-phosphate labeling of serine and threonine residues. Ciliary membrane-associated PKG was further purified by carboxy-methyl-sephadex-column chromatography. The membrane enzyme was Mg²⁺⁺-dependent and had a pH optimum at 6.4. The carboxy-methyl-sephadex-eluted PKG was analyzed by electrophoresis on sodium dodecyl sulphate polyacrylamide gels showing a molecular weight of 70–75 kDa.     

Enhanced activity of human serotonin transporter variants associated with autism

Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

The Pro-inflammatory Cytokine TNF-α Regulates the Activity and Expression of the Serotonin Transporter (SERT) in Astrocytes

Pro-inflammatory cytokines have been implicated in the precipitation of depression and related disorders, and the antidepressant sensitive serotonin transporter (SERT) may be a major target for immune regulation in these disorders. Here, we focus on astrocytes, a major class of immune competent cells in the brain, to examine the effects of pro-longed treatment with tumor necrosis factor-alpha (TNF-α) on SERT activity. We first established that high-affinity serotonin uptake into C6 glioma cells occurs through a SERT-dependent mechanism. Functional SERT expression is also confirmed for primary astrocytes. In both cell types, exposure to TNF-α resulted in a dose- and time-dependent increase in SERT-mediated 5-HT uptake, which was sustained for at least 48 h post-stimulation. Further analysis in primary astrocytes revealed that TNF-α enhanced the transport capacity (V_max) of SERT-specific 5-HT uptake, suggesting enhanced transporter expression, consistent with our observation of an increase in SERT mRNA levels. We confirmed that in both, primary astrocytes and C6 glioma cells, treatment with TNF-α activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment with the p38 MAPK inhibitor SB203580 attenuated the TNF-α mediated stimulation of 5-HT transport in both, C6 glioma and primary astrocytes. In summary, we show that SERT gene expression and activity in astrocytes is subject to regulation by TNF-α, an effect that is at least in part dependent on p38 MAPK activation.     

Going with the flow: trafficking-dependent and -independent regulation of serotonin transport

Antidepressant-, cocaine- and 3,4-methylenedioxymethamphetamine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) are expressed on presynaptic membranes of 5-HT-secreting neurons to provide efficient uptake of the biogenic amine after release. SERTs also support 5-HT transport across platelet, placental, gastrointestinal and pulmonary membranes and thus play a critical role in central nervous system and peripheral nervous system 5-HT signaling. SERTs are subject to multiple levels of posttranslational regulation that can rapidly alter 5-HT uptake and clearance rates. Specific cell surface receptors are now known to regulate SERT trafficking and/or catalytic function, with pathways activating protein kinase C, protein kinase G and p38 mitogen-activated protein kinase receiving the greatest attention. Remarkably, disease-associated mutations in SERT not only impact basal SERT activity but also selectively impact one or more SERT regulatory pathway(s). In this review, we describe both trafficking-dependent and trafficking-independent modes of SERT regulation and also the suspected roles played in regulation by SERT-associated proteins. Elucidation of the SERT 'regulome' provides important depth to our understanding of the likely origins of 5-HT-associated disorders and may help orient research to develop novel therapeutics.     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Vascular reactivity, 5-HT uptake, and blood pressure in the serotonin transporter knockout rat

The handling of serotonin [5-hydroxytryptamine (5-HT)] depends on the serotonin transporter (SERT). A SERT knockout (KO) rat is a useful model to test the hypothesis that SERT is the primary mechanism for arterial 5-HT uptake and to investigate the impact of SERT removal on blood pressure. Wild-type (WT) and KO rats were used to measure 5-HT content (plasma, raphe, aorta, carotid, and mesenteric artery), aortic isometric contraction, and blood pressure. HPLC supported the lack of circulating 5-HT in plasma (ng/ml plasma, WT, 310 +/- 96; and KO, 1.0 +/- 0.5; P < 0.05). Immunohistochemistry and Western blot analyses validated the presence of the SERT protein in the WT rats and a lesser expression in the KO rat. The aorta isolated from KO rats had a normal contraction to phenylephrine and norepinephrine and a normal relaxation to the endothelium-dependent agonist acetylcholine compared with the aorta from WT. In contrast, the potency of 5-HT was increased in the aorta from KO rats compared with WT rats [-log EC(50) (M); WT, 5.71 +/- 0.08; and KO, 6.7 +/- 0.18] and maximum contraction was reduced [%phenylephrine (10 muM) contraction, WT, 113 +/- 6%; and KO, 52 +/- 12%]. 5-HT uptake was reduced but not abolished in arteries of the KO compared with the WT rats. Diurnal mean arterial blood pressure, heart rate, and locomotor activity level of the KO rats were similar to the WT rats. These data suggest that there are other mechanisms of 5-HT uptake in the arteries of the rat and that although the absence of circulating 5-HT and/or SERT function sensitizes arteries to 5-HT, SERT dysfunction does not impair normal blood pressure.     

Atrial natriuretic peptide-initiated cGMP pathways regulate vasodilator-stimulated phosphoprotein phosphorylation and angiogenesis in vascular endothelium

Nitric oxide (NO)- and atrial natriuretic peptide (ANP)-initiated cGMP signaling cascades are important in the maintenance of cardiovascular homeostasis. The molecular signaling mechanisms downstream of cGMP are not well understood, however. We have used small interfering RNA (siRNA) approaches to specifically knock down a series of signaling proteins in bovine aortic endothelial cells, and we have combined biochemical analyses with physiological assays to investigate cGMP-mediated signal transduction pathways. Activation of particulate guanylate cyclase (GC-A) by ANP leads to a substantial, dose-dependent, rapid, and sustained increase in intracellular cGMP. In contrast, stimulation of soluble guanylate cyclase by NO yields only a weak and transient increase in cGMP. ANP-induced cGMP production is selectively suppressed by siRNA-mediated knockdown of GC-A. ANP greatly enhances the phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a major substrate of cGMP-dependent protein kinase (PKG) that significantly influences actin dynamics. Moreover, the ANP-induced phosphorylation of VASP at Ser-239 is accompanied by increased actin stress fiber formation and enhanced endothelial tube formation. siRNA-mediated knockdown of GC-A, VASP, or PKG abolishes ANP-induced VASP Ser-239 phosphorylation, stress fiber formation, and endothelial tube formation. We have demonstrated similar findings in human umbilical vein endothelial cells, where ANP substantially enhances intracellular cGMP content, phosphorylation of VASP at Ser-239, and endothelial tube formation. Taken together, our findings suggest that ANP-mediated cGMP signal transduction pathways regulate PKG phosphorylation of VASP Ser-239 in endothelial cells, resulting in reorganization of the actin cytoskeleton and enhancement of angiogenesis.     

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.     

Serotonin transporter function and pharmacology are sensitive to expression level: evidence for an endogenous regulatory factor

We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires approximately 5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity.     

DASB -in vitro binding characteristics on human recombinant monoamine transporters with regard to its potential as positron emission tomography (PET) tracer

The efficiency of serotonergic signal transduction is controlled by the density of serotonegic synapses and by the activity of the serotonin transporter (SERT), which selectively clears the synaptic cleft of the neurotransmitter. SERT is located in axons, where it is concentrated in varicosities and terminal boutons and thus is an exquisite marker for serotonergic synapses. This finding has been taken advantage of for neuroimaging serotonergic synaptic contact sites. Previous positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies were often carried out using radioligands that bind with high affinity to SERTs in the brainstem but also exhibit high affinity for dopamine and norepinephrine transporters and therefore did not allow quantification of serotonergic innervations in brain regions also containing dopaminergic or noradrenergic terminals. In order to visualize SERT availability more selectively, in recent years new tracers have been developed, one of which is [11C]DASB (N,N-dimethyl-2-2-amino-4-cyanophenylthiobenzylamine). Here, we have performed a detailed pharmacological characterization of unlabelled as well as radioactive DASB on recombinant human monoamine transporter proteins. Our results show that DASB selectively binds to SERT with high affinity (KD = 3.5 nm) to a site distinct from the serotonin (5-HT) recognition/translocation site. 5-HT inhibits DASB binding to SERT with more than one order of magnitude lower affinity than that of DASB binding (IC50 = 82.4 nm). These findings suggest DASB to be a highly selective PET tracer to visualize the density of serotonergic synapses in human brain.