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Biomedical subjects

R D Blakely

Publications and source records attributed to R D Blakely.

At least 37 records · Page 2Linked to original sources
PubMed2000

Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter.

Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585-602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine- and dopamine-containing neurons. Intracerebroventricular anti-dopamine beta-hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP-25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states.

Animals↗
PubMed2000

Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency.

BACKGROUND: Orthostatic intolerance is a syndrome characterized by lightheadedness, fatigue, altered mentation, and syncope and associated with postural tachycardia and plasma norepinephrine concentrations that are disproportionately high in relation to sympathetic outflow. We tested the hypothesis that impaired functioning of the norepinephrine transporter contributes to the pathophysiologic mechanism of orthostatic intolerance. METHODS: In a patient with orthostatic intolerance and her relatives, we measured postural blood pressure, heart rate, plasma catecholamines, and systemic norepinephrine spillover and clearance, and we sequenced the norepinephrine-transporter gene and evaluated its function. RESULTS: The patient had a high mean plasma norepinephrine concentration while standing, as compared with the mean (+/-SD) concentration in normal subjects (923 vs. 439+/-129 pg per milliliter [5.46 vs. 2.59+/-0.76 nmol per liter]), reduced systemic norepinephrine clearance (1.56 vs. 2.42+/-0.71 liters per minute), impairment in the increase in the plasma norepinephrine concentration after the administration of tyramine (12 vs. 56+/-63 pg per milliliter [0.07 vs. 0.33+/-0.37 pmol per liter]), and a disproportionate increase in the concentration of plasma norepinephrine relative to that of dihydroxyphenylglycol. Analysis of the norepinephrine-transporter gene revealed that the proband was heterozygous for a mutation in exon 9 (encoding a change from guanine to cytosine at position 237) that resulted in more than a 98 percent loss of function as compared with that of the wild-type gene. Impairment of synaptic norepinephrine clearance may result in a syndrome characterized by excessive sympathetic activation in response to physiologic stimuli. The mutant allele in the proband's family segregated with the postural heart rate and abnormal plasma catecholamine homeostasis. CONCLUSIONS: Genetic or acquired deficits in norepinephrine inactivation may underlie hyperadrenergic states that lead to orthostatic intolerance.

Adult↗
PubMed2000

Biogenic amine transporters: regulation in flux.

Following vesicular release, the biogenic amine neurotransmitters dopamine, norepinephrine and serotonin are actively cleared from extracellular spaces by presynaptic transporters. These transporters interact with multiple psychoactive agents including cocaine, amphetamines and antidepressants. Recent findings indicate that amine reuptake is likely to be a tightly regulated component of synaptic plasticity rather than a constitutive determinant of transmitter clearance. Protein kinase C activation and transporter phosphorylation have been linked to regulatory protein trafficking, and both phosphorylation and trafficking may be influenced by transporter ligands. Recognition that transmitters, antagonists and second messengers can modify the intrinsic activity, surface expression or protein levels of amine transporters raises new questions about the fundamental nature of drug actions in vivo. The theory that dysregulation of transporters may contribute to disease states is supported by the recent discovery that a coding mutation in the human norepinephrine transporter contributes to orthostatic intolerance.

Biogenic Monoamines↗
PubMed2000

Modified structure of the human serotonin transporter promoter.

Recently, several studies have reported an association between anxiety traits, affective disorders and autism and alleles of a functional promoter polymorphism (5HTT-LPR) in the human serotonin transporter (5HTT, SERT).1-3 The mechanistic basis for allelic differences in transporter transcription are presently unknown. To explore this issue, we cloned the human 5HTT promoter region from a PAC genomic library and now describe an unreported 381-bp insert between the polymorphic region and the transcription start site. We verified the presence of this novel sequence by Southern hybridization of genomic digests and PCR amplifications from multiple unrelated individuals. Sequence analysis of the novel region reveals a number of canonical transcription factor binding sites (eg AP1, Elk1, NFkappaB) that may be important in controlling the response of the 5HTT gene to regulatory factors. PCR studies of genomic templates reveal a low level of amplification of a deleted template matching the size of the originally reported 5HTT promoter. This deleted template is absent from PAC amplifications, suggesting that the human 5HTT promoter may exhibit in vivo instability. Molecular Psychiatry (2000) 5, 110-115.

Alleles↗
PubMed2000

Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells.

Anandamide (N-arachidonylethanolamide) is an endogenous cannabinoid that mimics the pharmacologic effects of Delta(9)-tetrahydrocannabinol, the major bioactive substance in marijuana. Anandamide appears to be synthesized, released, and inactivated by mechanisms similar to those for other neurotransmitters. Of interest to the present studies are reports that anandamide undergoes carrier-mediated uptake into neuronal or glial cells after release, followed by rapid intracellular degradation by the intracellular fatty acid amidohydrolase. In addition to effects in the brain, anandamide has multiple effects in the periphery, particularly on cells of the immune system that express both a peripheral cannabinoid receptor and amidohydrolase enzyme. We have performed a detailed characterization of anandamide uptake in the cognate mast cell line RBL-2H3 to test the hypothesis that the uptake system in peripheral cells is also carrier-mediated and functionally similar to that observed in the central nervous system. RBL-2H3 cells exhibited robust, saturable transport of [(3)H]anandamide that was both time- and temperature-sensitive. This transport activity was not dependent on extracellular ion gradients for uptake and was inhibited selectively by other fatty acid-derived molecules, anandamide congeners, and the psychoactive cannabinoids such as Delta(9)-tetrahydrocannabinol. We conclude that anandamide transport in the RBL-2H3 cells is carrier-mediated, and uptake in peripheral cells is functionally and pharmacologically identical with that observed in neurons and astrocytes.

Amidohydrolases↗
PubMed1999

Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants.

Many psychotropic drugs interfere with the reuptake of dopamine, norepinephrine, and serotonin. Transport capacity is regulated by kinase-linked pathways, particularly those involving protein kinase C (PKC), resulting in transporter phosphorylation and sequestration. Phosphorylation and sequestration of the serotonin transporter (SERT) were substantially impacted by ligand occupancy. Ligands that can permeate the transporter, such as serotonin or the amphetamines, prevented PKC-dependent SERT phosphorylation. Nontransported SERT antagonists such as cocaine and antidepressants were permissive for SERT phosphorylation but blocked serotonin effects. PKC-dependent SERT sequestration was also blocked by serotonin. These findings reveal activity-dependent modulation of neurotransmitter reuptake and identify previously unknown consequences of amphetamine, cocaine, and antidepressant action.

Antidepressive Agents↗
PubMed1999

Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.

Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.

Adrenergic Uptake Inhibitors↗
PubMed1999

Pharmacological profile of neuroleptics at human monoamine transporters.

Using radioligand binding techniques, we determined the equilibrium dissociation constants (K(D)) for 37 neuroleptics and one metabolite of a neuroleptic (haloperidol metabolite) for the human serotonin, norepinephrine, and dopamine transporters with [3H]imipramine, [3H]nisoxetine, and [3H]WIN35428, respectively. Among neuroleptics, the four most potent compounds at the human serotonin transporter were triflupromazine, fluperlapine, chlorpromazine, and ziprasidone (K(D) 24-39 nM); and at the norepinephrine transporter, chlorpromazine, zotepine, chlorprothixene, and promazine (K(D) 19-25 nM). At the human dopamine transporter, only pimozide (K(D) = 69+/-3) ziprasidone (K(D) = 76+/-5) had notable potency. These data may be useful in predicting therapeutic and adverse effects, including drug interactions of neuroleptics.

Antipsychotic Agents↗
PubMed1999

Subcellular localization of chromogranins, calcium channels, amine carriers, and proteins of the exocytotic machinery in bovine splenic nerve.

Subcellular fractionation of bovine splenic nerves, which consist mainly of sympathetic nerve fibers, has been useful for characterizing cellular organelles en route to the terminal. In the present study we have characterized the subcellular distribution of both secretory and membrane proteins. A newly discovered chromogranin-like protein, NESP55, was found in large dense-core vesicles. The endogenous processing of NESP55 was comparable to that of chromogranins but more limited than that of secretogranin II and chromogranin B. For membrane proteins three major types of distribution were found. The amine carrier VMAT2 was confined to large dense-core vesicles. VAMP or synaptobrevin was present both in large dense-core vesicles and in lighter vesicles, whereas SNAP-25, syntaxin, and two types (N and L) of Ca2+ channels were found in a special population of lighter vesicles but were not present in large dense-core vesicles or at the most in very low concentrations. The plasma membrane norepinephrine transporter was apparently present in a separate type of vesicle, but this requires further study. These results further characterize vesicles en route to the terminal and establish for the first time that peptides involved in exocytosis (syntaxin, SNAP-25, and N- and L-type Ca2+ channels) are apparently transported to the terminal in a special type of vesicle. The exclusive presence of the amine carrier in large dense-core vesicles indicates that the formation of small dense-core vesicles in the terminals requires a reuse of membrane components of large dense-core vesicles.

Amines↗
PubMed1999

[The status of serotonin protein -- a serotonin transporter in thrombocytes in patients with somatoform disorders].

The role of serotonin transporter (SERT) protein in the development of somatoform disorders (SD) was investigated. An association study was performed in terms of the evaluation of the level of SERT immunoreactive (IR-SERT) protein using site-specific antibodies directed at SERT C-terminus fragment, poorly conserved among the other cotransporters. The level of the anxious symptomatology was also estimated in the patients with SD. 22 patients, who met DSM-IV criteria for somatoform disorders, and 32 normals were examined. In platelets from normals, IR-SERT protein migrated as a difuse band between 68 and 105 kDa, and a major sharper band at 43 kDa. Almost complete disappearance of platelet 43 kDa IR-SERT protein band was observed in most of the patients with SD. These findings permitted to suggest a possibility of either biosynthetic or processing abnormality of SERTs in the affected population, that might reflect a dysfunction of serotonin neurotransmission in CNS of the patients with SD.

Adult↗
PubMed1999

Orthostatic intolerance: emerging genetic and environmental etiologies.

Orthostatic intolerance (OI) is common after space flight and resembles the disabling idiopathic orthostatic intolerance commonly observed in otherwise healthy young individuals. OI can arise from reduced sympathetic nervous system activity and, paradoxically, also from increased sympathetic nervous system activity. Patients with early manifestations of pure autonomic failure demonstrate an etiology based upon reduced sympathetic nervous system activity. Patients with hyperadrenergia demonstrate an etiology based on increased sympathetic nervous activity. For many years, we have recognized that the microgravity environment induces adaptation in the cardiovascular system and its autonomic control mechanisms that lead to the presence of OI on return to gravity. Understanding the nature of OI in astronauts returning from space as well as in the relevant patient population on earth has been a priority of Vanderbilt's Center for Space Physiology and Medicine in recent years. A major purpose of the autonomic experiment in the Neurolab mission was to identify whether the OI experienced by astronauts on return to earth was best explained by a hypoadrenergic or hyperadrenergic state. To address this question, we analyzed sympathetic nervous system activity inflight by 1) measurement of plasma catecholamines; 2) assessment of peroneal microneurographic sympathetic nerve traffic; and 3) assessment of norepinephrine spillover and clearance during infusion of tritiated norepinephrine. These studies documented a slight (five bursts per minute) increase in muscle sympathetic nerve activity, a 200 pg/ml increase in plasma norepinephrine level, and a 350 ng/min increase in norepinephrine clearance. Plasma norepinephrine and norepinephrine spillover and clearance were also raised on recovery day. These data indicate that enhanced sympathetic activation, rather than reduced sympathetic activation, accompanies the orthostatic intolerance following microgravity.

Autonomic Nervous System Diseases↗
PubMed1998

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

Animals↗
PubMed1998

Patch-clamp and amperometric recordings from norepinephrine transporters: channel activity and voltage-dependent uptake.

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Carrier Proteins↗
PubMed1998

Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins.

Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) mediate antidepressant-sensitive clearance of 5-HT following release. Although we have been aware for decades that SERT-mediated 5-HT clearance can be modulated by exogenous agents including serotonin-selective reuptake inhibitors, amphetamines, and cocaine, we have had little reason to speculate that SERT activity was actively controlled through endogenous pathways. Recent studies indicate that SERTs are likely to be trafficked to specific plasma membrane subdomains to achieve localized clearance of 5-HT, and that the number of SERTs resident in the plasma membrane is controlled through kinase- and phosphatase-linked pathways. In particular, roles for protein kinase C and phosphatase 2A become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter redistribution and loss of functional uptake capacity. Based on our findings, and the studies of others in native tissues and transfected cells, we propose a model whereby SERTs are organized in a macromolecular complex in the plasma membrane that may serve to locate reuptake activity near release sites. Although many elements of this model remain hypothetical, our findings suggest a much more dynamic picture of transporter-mediated 5-HT reuptake than typically described and suggest opportunities both for the development of new SERT regulatory agents and for the identification of regulatory pathways that may be compromised in mental illness.

Animals↗
PubMed1998

High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol.

Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human and Drosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.

Animals↗
PubMed1998

Phosphorylation and regulation of antidepressant-sensitive serotonin transporters.

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking.

Carrier Proteins↗