Zdenek B. Pristupa

Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H]CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS‐7 cells following modulation of protein kinase C (PKC)‐ and protein kinase A (PKA)‐dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 μM), but not αPDD, reduced the Vmax (∼1 pmol/min/105 cells) for [3H]DA uptake by ∼40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by ∼30%, an effect mimicked by the potent PKA inhibitor Rp‐cAMPS. Activation of PKA‐dependent pathways with Sp‐cAMPS did not significantly modify DA uptake. Neither the Km of [3H]DA uptake (∼200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA‐dependent mechanisms, the estimated density of hDAT as indexed by whole‐cell [3H]CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS‐7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC‐ and PKA‐dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function. Synapse 30:79–87, 1998.

Molecular and functional characterization of a partial cDNA encoding a novel chicken brain melatonin receptor.

An approach based on homology probing was used to clone a partial cDNA encoding a novel melatonin (ML) receptor (MLR) from chicken (Gallus domesticus) brain. Based on available deduced amino‐acid sequence, the chicken MLR (cMLR) displayed greater sequence homology to the frog (Xenopus) MLR than cloned human/mammalian receptors, with overall identities of 73% and 66%, respectively. In order to gain functional expression, a chimeric frog/chicken (flc)MLR was constructed in which the 5′ end of the cMLR, including the N‐terminus, TM1 and part of the first intracellular loop was substituted by fMLR sequence. [125I]Iodo‐ML bound with high affinity (K d of ∼35 pM) to COS‐7 cells transiently expressing the flcMLR in a saturable and guanine nucleotide‐sensitive manner with the following rank order of potency: 2‐iodo‐ML > ML > 6‐Cl‐ML > S20750 > 6‐OH‐ML > S20643 > S20753 > N‐acetyl‐5HT > > 5‐HT. Estimated K i values for these compounds at the flcMLR correlated well to those obtained in native chicken brain membranes. In line with the observed structural similarity to the fMLR, the flcMLR exhibited affinities for ML, 6‐Cl‐ML and 6‐OH‐ML ∼10‐fold lower than mammalian receptors. Functionally, opposing interactions between ML and dopamine receptor signal transduction pathways were observed with ML potently inhibiting dopamine D1A‐receptor‐mediated cAMP accumulation in cells (HEK‐293) transiently co‐expressing these receptors. cMLR mRNAs were found expressed in chicken brain and kidney with trace levels observed in the lung. The availability of cloned vertebrate MLRs distinct at both the amino acid and pharmacological level from their mammalian counterparts may now allow for the identification of those amino‐acid residues and structural motifs that regulate ML‐binding specificity and affinity.

cDNA array reveals increased expression of glucose-dependent insulinotropic polypeptide following chronic clozapine treatment: role in atypical antipsychotic drug-induced adverse metabolic effects

Clozapine is an atypical antipsychotic drug with unique pharmacological and therapeutic properties. Unlike the typical antipsychotic drug, haloperidol, clozapine does not cause extrapyramidal side effects; however, weight gain, dyslipidemia, and type II diabetes are commonly associated with the use of this drug in subjects with schizophrenia. The aim of this study was to profile gene expression in the rat striatum following clozapine treatment. Chronic treatment with clozapine revealed upregulation of several genes including the glucose-dependent insulinotropic polypeptide (GIP) gene by over 200% in the rat striatum. The cDNA array results for the GIP gene were confirmed by real-time RT-PCR as well as by radioimmunoassay. Expression of the GIP gene in the central nervous system is consistent with the results of retinal GIP gene expression as reported by other investigators. Taken together, these findings implicate the possible role of GIP as a neuromodulator in the central nervous system. GIP is an insulinotropic agent with stimulatory effects on insulin synthesis and release from the pancreas. However, changes in brain GIP levels are most likely unrelated to the metabolic adverse effects (dyslipidemia, type II diabetes, weight gain) associated with clozapine treatment. Therefore, we also measured GIP gene expression in the K-cell-rich regions, duodenum and jejunum (small intestine), and plasma GIP levels using radioimmunoassay following chronic treatment with clozapine. GIP mRNA levels in the small intestine and the plasma GIP at the protein level were significantly elevated in clozapine-treated subjects. Furthermore, as observed in humans, chronic clozapine treatment also caused weight gain, and increased levels of insulin, triglycerides and leptin in the plasma. These results suggest that adverse metabolic effects associated with clozapine treatment may be related to its ability to increase intestinal gene expression for GIP.

Dopaminergic Properties and Experimental Anti-Parkinsonian Effects of IPX750 in Rodent Models of Parkinson Disease

With a view toward improving the neural bioavailability of administered dopaminergic compounds, including dopamine, synthetic efforts have been directed toward enhancing the brain bioavailability of these compounds by accessing cellular sugar transport systems with stereoselective dopaminergic drugs. While synthesis and chemistry of the resultant class of compounds has recently been described in US Patent No. 6,548,484, the associated biologic properties have not previously been reported. One member of this new class, IPX-750, is a pro-drug dopamine-gluconamine designed to retain stereospecificity of binding at: glucose transporters (GLUT 1/GLUT 3 and intestinal Na+/glucose co-transporters SGLT1), dopamine transporter (DAT); and, dopaminergic receptors of the D1/D2 families. Designed to be cleavable by tissue amidases, results reported here show that intact IPX-750 pro-drug retains dopaminergic agonist binding and biologic activities both in vitro and in vivo. IPX-750, like dopamine, exhibited predominant D5/D1 binding specificity with lower binding activity at D2. As expected, binding was highly stereo-specific, ie, IPX-760, a benzamide differing in just a hydrogen atom and keto oxygen from IPX-750, bound with 6-fold lower activity at D5. In cell culture, activation resulted from binding of IPX-750 at D1 or D5 in transfected cells was measured by increased intracellular cAMP. Interestingly, considering prior reported in vitro toxicity of dopamine oxidized and metabolic product dopamine, no evidence of in vitro toxicity was observed at up to 72 hrs in cell cultures at the EC50 of IPX-750 for increasing intracellular cAMP. IPX-750 was evaluated in the Parkinsons disease animal models, including MPTP mouse model, the 6-hydroxydopamine (6-OHDA) rat model and the Nurr1(+/−) knockout mouse model. In MPTP-lesioned and Nurr1+/ − knockout mice, IPX-750 significantly increased Rota-rod time. In 6-OHDA-lesioned rats, IPX-750 significantly decreased apomorphine (APO)-induced rotation. Worthy of note, after cessation of IPX-750 treatments the anti-parkinsonian activity in MPTP-lesioned and Nurr1+/ − mice required about 2 weeks to washout, suggesting a possible biologic reservoir of drug. In addition, after eight weeks of twice daily administration of 20 mg/kg IPX-750, mice did not show statistical difference in the total number of TH-positive neurons in substantia nigra (SN). These combined results suggest (i) that stereo-specific glycoconjugation may be an effective method to improve penetrability of drugs through the blood brain barrier; (ii) treatment with bioavailable IPX-750 in vitro did not show evidence for neurotoxicity; and, (iii) IPX-750 possesses dopaminergic properties and exerts anti-parkinsonian effects in three different PD rodent models, suggesting therapeutic potential for this new class of drugs in treating dopamine deficiency diseases.

A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line1

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney‐derived COS‐7, COS‐1 and HEK‐293 but not in other mammalian cell lines (CHO, Y1, Ltk−), we have characterized a putative functional dopamine transporter displaying a high affinity (K m∼250 nM) and a low capacity (∼0.1 pmol/105 cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated K i values of numerous substrates and inhibitors for the COS‐dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium‐sensitive and reduced by phorbol ester pre‐treatment. Two mRNA species of 3.8 and 4.0 kb in COS‐7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse‐transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of ∼1.3 kb, isolated from a COS‐1 cDNA library and encoding transmembrane domains 1–6, displayed a deduced amino acid sequence homology of ∼96% to the human dopamine transporter. Taken together, the data suggest the existence of a non‐neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.