Kuo-peing Liu

Attenuated 5‐HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase, phosphatidylinositol 3‐kinase, Akt and mitogen‐activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5‐HT)1A receptor antagonist‐binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5‐HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III‐R criteria. Measurements of [35S]GTPγS binding to Gαi/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5‐HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Gαi/o, Gαq/11 or Gαs proteins, or in the activity of cAMP‐dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5‐HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3‐kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5‐triphosphate. Finally, the activation of extracellular signal‐regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist‐stimulated 5‐HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5‐HT1A receptor transduction pathways that may have implications for cell survival.

Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles

Trapping of weak bases was utilized to evaluate stimulus‐induced changes in the internal pH of the secretory vesicles of chromaffin cells and enteric neurons. The internal acidity of chromaffin vesicles was increased by the nicotinic agonist 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP; in vivo and in vitro) and by high K+ (in vitro); and in enteric nerve terminals by exposure to veratridine or a plasmalemmal [Ca2+]o receptor agonist (Gd3+). Stimulation‐induced acidification of chromaffin vesicles was [Ca2+]o‐dependent and blocked by agents that inhibit the vacuolar proton pump (vH+‐ATPase) or flux through Cl− channels. Stimulation also increased the average volume of chromaffin vesicles and the proportion that displayed a clear halo around their dense cores (called active vesicles). Stimulation‐induced increases in internal acidity and size were greatest in active vesicles. Stimulation of chromaffin cells in the presence of a plasma membrane marker revealed that membrane was internalized in endosomes but not in chromaffin vesicles. The stable expression of botulinum toxin E to prevent exocytosis did not affect the stimulation‐induced acidification of the secretory vesicles of mouse neuroblastoma Neuro2A cells. Stimulation‐induced acidification thus occurs independently of exocytosis. The quantal size of secreted catecholamines, measured by amperometry in cultured chromaffin cells, was found to be increased either by prior exposure to L‐DOPA or stimulation by high K+, and decreased by inhibition of vH+‐ATPase or flux through Cl− channels. These observations are consistent with the hypothesis that the content of releasable small molecules in secretory vesicles is increased when the driving force for their uptake is enhanced, either by increasing the transmembrane concentration or pH gradients.

On the Nature of the Interaction Between Serotonin and Serotonin Binding Protein: Effect of Nucleotides, Ions, and Sulfhydryl Reagents

Abstract: Rat brain serotonin binding protein (SBP) was found to have essential ‐S‐S and ‐SH groups. Both reduction of the disulfide bond by dithiothreitol or mercaptoethanol and modification of ‐SH group(s) by Ellman reagent or alkylating agents caused loss of binding capacity. In contrast, formation of a mixed disulfide bond with sodium metabisulfite did not affect the binding capacity. Serotonin in the presence of Fe2+ and phosphate was found to bind to either an ‐SH group or to a site in very close proximity. Addition of serotonin protected ‐SH groups from modification by Ellman reagent and from denaturation of protein upon storage. Lipids that enhance binding of serotonin to SBP also protected ‐SH groups from modification. Nucleotides were found to be strong inhibitors of the binding of serotonin to SBP. The inhibitory effect of nucleotides was due to their chelating properties and not to their ability to phosphorylate the protein or to bind directly to it. Inhibition by nucleotides and other chelators was reversible. Binding capacity was fully restored after removal of the chelator by molecular sieve chromatography and addition of Fe2+. The ionic environment had a marked effect on the binding: intracellular ions such as K+ were found to enhance the binding, and extracellular ions such as Na+ and Ca2+ inhibited the binding. Based on these data and our previous studies, we suggest that SBP is an intracellular protein that acts as a storage protein. Consistent with our data is formation of a complex of SBP‐S‐Fe‐S that in a hydrophobic surrounding could bind up to four molecules of serotonin in coordination bond with Fe2+ and thereby reduce the osmotic pressure within a storage vesicle. Extracellular ionic conditions that favor the dissociation of the complex would free the amine to interact with its receptor or the presynaptic reuptake carrier.

Roles of extracellular signal‐regulated kinase and Akt signaling in coordinating nuclear transcription factor‐κB‐dependent cell survival after serotonin 1A receptor activation

To investigate the functional consequences of cross‐talk between multiple effectors of serotonin (5‐HT) 1A receptor, we employed transfected Chinese hamster ovary cells. Activation of 5‐HT1A receptor stimulated extracellular signal‐regulated kinase (ERK)1/2, Akt and nuclear transcription factor‐κB (NF‐κB). Stimulation of cells with 5‐HT1A receptor agonist induced a rapid but transient ERK1/2 phosphorylation followed by increased phosphorylation of Akt. Elevated Akt activity in turn suppressed Raf activity and induced a decline in ERK activation. The activation of ERK and Akt downstream of 5‐HT1A receptor was sensitive to inhibitors of Ras, Raf and phosphatidylinositol 3‐kinase (PI3K). Stimulation of 5‐HT1A receptor also resulted in activation of NF‐κB through a decrease in inhibitor of nuclear transcription factor‐κB. In support of the importance of 5‐HT1A receptor signaling for cell survival, inhibition of NF‐κB facilitated caspase 3 activation and cleavage of poly (ADP‐ribose) polymerase, while treatment of cells with agonist inhibited caspase 3, DNA fragmentation and cell death. Both agonist‐dependent NF‐κB activation and cell survival were decreased by Akt Inhibitor II or by overexpression of dominant‐negative Akt. These findings suggest a role for 5‐HT1A receptor signaling in the Ras/Raf‐dependent regulation of multiple intracellular signaling pathways that include ERK and PI3K/Akt. Of these, only PI3K/Akt and NF‐κB activation were required for 5‐HT1A receptor‐dependent cell survival, implying that the relative distribution of signals between competing transduction pathways determines the functional outcome of 5‐HT1A receptor activation.

In vitro autoradiography of serotonin 5-HT2A/2C receptor-activated G protein: Guanosine-5?-(?-[35S]thio)triphosphate binding in rat brain

Agonist activation of G protein‐coupled receptors induces an increase in the binding of guanosine 5′‐(γ‐[35S]thio)triphosphate ([35S]GTPγS); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide‐binding regulatory protein (G protein). The present study uses agonist‐stimulated [35S]GTPγS binding to characterize serotonin 5‐HT2A/2C receptors in rat brain membrane fractions and demonstrate the anatomical localization of the receptors by in vitro autoradiography on slide‐mounted sections. The stimulatory effect of the agonist [1‐(2,5‐dimethoxy‐4‐iodophenyl)]‐2 aminopropane (DOI) is compared to that of serotonin (5‐HT). Autoradiography revealed a similar localization of DOI‐ and 5‐HT‐stimulated binding of [35S]GTPγS in distinct areas of prefrontal and parietal cortex, consistent with previously reported 5‐HT2A receptor distribution. Specific binding was demonstrated in the frontal and parietal cortex, medial prefrontal, and cingular and orbital‐insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5‐HT2A antagonist, and ketanserin, an antagonist of 5‐HT2A/2C receptors, blocked DOI stimulation in all labeled areas, whereas 5‐HT stimulation was only partially blocked (70–80%). A small but significant inhibition was observed with the specific antagonist of 5‐HT2C/2B, SB 206553. This autoradiographic technique provides a useful tool for measuring in situ changes in specific receptor–Gq protein coupling in anatomically discrete brain regions, under physiological and pathological conditions. J. Neurosci. Res. 61:674–685, 2000.

Identification, Purification, and Characterization of Two Forms of Serotonin Binding Protein from Rat Brain

Abstract: Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5‐Hydroxytryptamine (5‐HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4‐B, Affi‐Gel‐Blue, hydroxylapatite, and phosphocellulose. The 45K form of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate‐gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two‐dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5‐HT binding capacity; the enhancement of 5‐HT binding by Fe2+; and inhibition by –SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross‐reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants—56K form showed higher affinity for 5‐HT (KD1= 0.4 nM; KD2= 32 nM), whereas the 45K form showed lower affinity (KD1= 9.7 nM; KD2= 120 nM); (2) ratio of number of 5‐HT binding sites with low affinity to those with high affinity—56K (19:1), 45K (10:1); (3) isoelectric point—the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis. We suggest that a precursor‐product relationship may exist between 45K and 56K SBP and that the two forms may be located within different parts of serotonergic neutrons.

Inhibition of 5-HT1A receptor-dependent cell survival by cAMP/protein kinase A: Role of protein phosphatase 2A and Bax

Serotonergic 5‐HT1A receptor signaling leading to nuclear factor‐κB (NF‐κB) activation appears to be critical for cell survival. Adenylyl cyclase and protein kinase A (AC/PKA) are effectors of the 5‐HT1A receptor that are inhibited by Gαi subunits. Conversely, Gβγi subunits downstream from the 5‐HT1A receptor participate in the activation of extracellular signal‐regulated kinases (ERK1/2), phosphatidylinositol 3‐kinase (PI3K), Akt, and NF‐κB. To model the contribution of pro‐ and antiapoptotic signaling cascades downstream of activated 5‐HT1A receptor in cell survival, Chinese hamster ovarian (CHO) cells were employed that exogenously overexpress 5‐HT1A receptors. Stimulation with the 5‐HT1A receptor agonist 8‐OH‐DPAT and pharmacological agonists of AC induced PKA and protein phosphatase 2A (PP2A) activity, which in turn inhibited: Akt activity, IκBα degradation, nuclear translocation of NF‐κB, and expression of X‐linked inhibitor of apoptosis protein (XIAP/BIRC4). Pharmacological inhibition of PP2A with calyculin A potentiated Akt activity while attenuating ERK1/2 signaling via increased inhibitory phosphorylation of Raf (pSer259). In contrast, increased cAMP levels enhanced Bax translocation to the mitochondria, resulting in the release of cytochrome c, caspase‐3 activation, and apoptosis induction. Our data suggest a central role of cAMP/PKA‐dependent PP2A in shifting the homeostasis of intracellular signaling downstream of activated 5‐HT1A receptor toward cell death in biological systems linked to neuropsychiatric disorders.

A Ca2+‐Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

Abstract: The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5‐hydroxytryptamine (5‐HT, serotonin). A protein kinase activity that copurifies with SBP (SBP‐kinase) was partially characterized and compared with calcium/calmodulin‐dependent protein kinase II (CAM‐PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5‐HT. SBP‐kinase and CAM‐PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+‐dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule‐associated proteins well and did not phosphorylate myosin, phosphorylase b., and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP‐kinase and CAM‐PK II were observed: (1) CAM enhanced CAM‐PK II activity, but had no effect on SBP‐kinase; (2) synapsin I was an excellent substrate for CAM‐PK II, but not for SBP‐kinase; (3) 5‐HT inhibited both the autophosphorylation of SBP‐kinase and the phosphorylation of SBP, but had no effect on CAM‐PK II. These data indicate that SBP‐kinase is different from CAM‐PK II. Phosphopeptide maps of SBP and SBP‐kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5‐HT within neurons.

Regulation of dopamine D1-receptor activation in vivo by protein phosphatase 2B (calcineurin)

Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist‐promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1‐agonist‐stimulated [35S]GTPγS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild‐type mice and D2‐receptor mutants. These studies revealed a decreased agonist‐stimulated G‐protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPγS binding in a concentration‐dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal andD1‐agonist‐stimulated phosphorylation of D1‐receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increasedkinase‐mediated phosphorylation.

Prenatal development of serotonin binding protein in relation to other transmitter-related characteristics of central serotonergic neurons

Serotonin binding protein (SBP) is a neuron-specific protein that binds serotonin (5-HT) with high affinity and is concentrated in synaptic vesicles. 5-HT has been shown to be stored in situ in a macromolecular complex with SBP. We have now investigated the ontogeny of SBP in the rat CNS. The time course of the appearance of SBP was related to the appearance of tryptophan hydroxylase, endogenous 5-HT and monoamine oxidase (MAO; types A and B). Binding of [3H]5-HT by SBP was assayed using molecular sieve chromatography. SBP had appeared by day E16; its activity then rose rapidly and reached adult levels (150 nmol [3H]5-HT/g protein) at days E18-E19. Tryptophan hydroxylase activity was measured by following the accumulation in vivo (30 min) of 5-hydroxytryptophan (5-HTP) in the brains of rat embryos whose mothers were treated with the aromatic L-amino acid decarboxylase inhibitor, NSD-1015, (100 mg/kg; i.p.). Tryptophan hydroxylase activity was first detectable at E15, remained present but at a low level through day E20 and then rapidly increased to reach 75% of the adult level at birth (747 pmol/g brain wet wt.). The development of stores of endogenous 5-HT paralleled the time course of development of tryptophan hydroxylase rather than that of SBP. 5-HT was first detected at E15, remained low until the end of intrauterine life and at birth was 50% of the adult level (2640 pmol/g brain wet wt.). MAO activity was determined in crude mitochondrial fractions by measuring 5-hydroxyindoleacetic acid produced from 5-HT as substrate. This activity was already present prior to day E15 (the activity of type B MAO was higher than that of type A) and reached adult levels at day E20 (55 pmol/mg protein/min; A, B). It is concluded that the potential of neurons to store 5-HT, as measured by the activity of SBP, develops more rapidly than their ability to produce 5-HT. Moreover, although the ratio of its two forms changes, MAO activity appears very early in development.