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Role of Protein Kinase C and Second Messengers in Regulation of the Norepinephrine Transporter
H. Bonisch, R. Hammermann, and M. Bruss Institute of Pharmacology and Toxicology University of Bonn D-53 I I 3 Bonn, Germany
Role of Protein Kinase C and Second Messengers in Regulation of the Norepinephrine Transporter Re-uptake of released norepinephrine (NE)into presynaptic nerve terminals is responsible for the rapid termination of neurotransmission in noradrenergic synapses. Transport of NE by the neuronal NE transporter (NET)is absolutely dependent on extracellular Nat and CI- and is selectively inhibited by, for example, desipramine and nisoxetine, and Na' and Clkdependent binding of these inhibitors to the NET has been used to localize the tissue distribution of NET (1,2). Aside from its physiological role, the NET in the central nervous system is the primary target for tricyclic antidepressants. However, in spite of its physiological and therapeutic importance, little is known about the involvement of second messengers in the regulation of NET expression and function. cDNAs of a series of neurotransmitter transporters (NTTs)have been cloned and the NET has been shown to be a member of the superfamily of structurally related Na+ and CI--dependent NTTs for monoamines (dopamine, serotonin, and NE) and certain amino acids, such as GABA and glycine; transporters of this family are structurallycharacterized by 12 transmembrane domains, intracellular N- and C-termini, and a large second extracellular loop ( 3 ) .Cloning of the human and bovine NET cDNAs (4,5)has revealed one common consensus sequence for phosphorylation by protein kinase C (in the following, termed PKC site) within the second intracellular loop and, for the bovine NET (bNET), two additional PKC sites at the C-terminal end of bNET. In this paper we provide evidence that stimulation of PKC by a phorbol ester causes downregulation of NE transport and that the second-messenger cyclic adenosine monophosphate (CAMP)may be involved in tissue-specific regulation of NETs. To examine whether the PKC sites of NETs are involved in the regulation of NE transport, uptake of [jH]NE was studied in cells expressing the hNET (with one PKC site), bNET (with three PKC sites), and a mutant hNET, hNETSZSYA, in which (by site-directed mutagenesis) serine (at amino acid position 259 of hNET) was exchanged against alanine, resulting in the destruction of the single potential phosphorylation site for PKC of the hNET. For NE transport studies, COS-7 cells were transfected with the corresponding cDNA by means of the calcium phosphate method and used 48 hr posttransfection. The human Advances m Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction 1054-3589198 $ZS.OO
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neuroblastoma cells, SKN-SH-SY5Y (SKN cells), which constitutively express the hNET, were also used in this study. Effects of second messengers on NE transport were studied in SKN cells and in the rNET-expressing rat PC12 pheochromocytoma cells. To determine uptake of NE, cells were washed three times with KrebsRinger-Hepes (KRH) buffer containing 10 p M pargyline (to inhibit monoamine oxidase) and 10 p M U-0521 (to inhibit COMT) and preincubated for 30 min (at 37°C) in this buffer. Therafter, cells were incubated at 37°C for 2-5 min (as indicated) in KRH buffer containing 10 n M [3H]NE (53.4 Cdmmol, NEN) and washed three times with ice-cold KRH buffer to terminate uptake. Specific uptake was defined by subtracting the uptake in the presence of 10 p M nisoxetine from total uptake. When short-term effects of the compounds under study were determined, the compounds were present during incubation and preincubation of the cells. When cells during culture had already been exposed for 24 hr to these compounds, they were not further present during incubation or preincubation. For kinetic analysis, cells were incubated for 2 min in 10 nM [3H]NE with 0.1-2.0 p M unlabeled NE. Binding of [3H]nisoxetine to the NE transporter on intact cell membranes was determined by pretreating the cells are described for NE uptake. Cells were incubated with 0.5-20.0 nM [3H]nisoxetine (83 Ci/mmol, Amersham) at 4°C for 3 hr and then washed three times with KRH buffer. Nonspecific binding was determined in the presence of desipramine (1 pM). At the end of uptake or binding experiments, cells were solubilized in 0.1% Triton X-100 to determine protein content and radioactivity. Short (10 min) exposure of COS-7 cells expressing either the bNET, the hNET, or the mutant hNETszsyAto the PKC-activating phorbol ester PMA (phorbol 12-myristate-13-acetate, 160 nM), the inactive phorbol ester 4aPD (4a-phorbol 12, 13-didecanoate, 160 nM), the PKC inhibitor staurosporine (0.5 pM), or the phosphatase inhibitor okadaic acid (1 p M )did not cause a significant change in [3]NE uptake. When COS-7 cells expressing the various NETS were exposed for 30 min with the aforementioned compounds and with the same concentrations, [3H]NEuptake by hNET, bNET, and hNETS25yA was significantly reduced by PMA to 61.3%, 44.4%, and 71.9%, respectively, of the corresponding control values. On the other hand, uptake of [3H]NEby all three transporters was not affected by 4aPD or staurosporin. Interestingly, uptake of [3]NE by all three transporters was not different from controls if cells were exposed to both PMA and staurosporin; that is, the inhibitory effect of PMA was antagonized by the PKC inhibitor staurosporin. Incubation of COS-7 cells (expressing either hNET or bNET) with okadaic acid affected neither [3H]NE uptake nor the inhibitory effect of PMA. Prolonged treatment with PMA (160 nM) for up to 24 hr of COS-7 cells expressing bNET, hNET, or hNETszsrAresulted in a time-dependent reduction of [3H]NE uptake, which, after about 3 hr, remained at a constant reduced level of 35-70% of corresponding controls. A similar PMA-induced reduction of [3H]NE uptake was also observed in SKN cells. Under the same conditions, 4aPD had no influence on [3H]NE uptake in either of these cells. The PMA-induced reduction of 13H]NE uptake (after 24 hr) was due to a significant reduction in maximum transport velocity (Vmax)with no significant effect on apparent affinity (Km):Vmax was
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reduced to 29.5%, 31.8%, and 28.6% of control values in COS-7 cells expressing bNET, hNET, and hNETsL59A, respectively. In SKN cells, PMA caused a reduction of Vmax from 4.0 pmol/mg/min (control) to 1.9 pmol/mg/min (i.e., to 47.5% of the control value). ['Hlnisoxetine binding was reduced by 24 hr pretreatment of SKN cells with PMA, but it remained unaffected by pretreatment of the cells with 4aPD. Analyses of the binding data revealed no significant differences in Kd values from control cells (controls = 12.1 nM vs 8.8 nM after pretreatment with PMA. However, PMA pretreatment caused a significant reduction in Bmax of ['HH]nisoxetine binding from 529 fmol/mg (controls) to 287 fmol/mg (i.e., to 54.3% of the control value). Thus, the PMA-induced reduction of NE transport was a consequence of the reduction in the number of NET molecules expressed in the plasma membrane of the cells. To study the effects of CAMP on human NET, 2-min uptake of [3H]NE (10 nM) was determined in SKN cells after 15-min or 24-hr exposure of the cells to lipophilic CAMP analogues (8Br-CAMP or db-CAMP) or to adenylate cyclase-stimulating agents (cholera toxin, forskolin). In addition, 8Br-cGMP was included in this study. [3H]NE uptake in SKN cells remained unchanged if cells were incubated for 15 min with 8Br-CAMP(1 or 10 mM), choleratoxin (100 mg/ml), forskolin (100 pM), or SBr-cGMP (1mM). These agents, as well as db-CAMP (1 mM), were also without effect on NE transport when present for 24 hr (i.e., during culture of the cells). A more physiological stimulation of adenylate cyclase through activation of the prostaglandin El (PGE, receptor of SKN cells by 10 p M PGE, (present for 24 hr) was also without effect in [3H]NEuptake, and NE transport also remained unchanged if degradation of CAMP was inhibited by IBMX (500 pM). Uptake of [3H]NE under control conditions amounted to 755 fmol/mg protein. In addition, in COS-7 cells expressing the hNET, a 24-hr treatment of the cells with forskolin (100 pM), 8Br-CAMP (1 mM), or 8Br-cGMP (1 mM) also did not affect [3H]NEuptake, which amounted in these cells to 2269 fmollmg protein. Interestingly, 24-hr incubation of PC12 with forskolin (100 pM), cholera toxin (100 ng/ml), or db-CAMP (1 mM) strongly reduced ['HINE uptake to 30.5%, 40.6%, and 36.2%, respectively, of control values (100% = 451 fmol/mg protein). However, NE uptake remained unchanged when PC12 cells were incubated for 24 hr with db-cGMP (1 mM). The results of this study indicate that human NET (and NET in COS-7 cells) is downregulated in a staurosporin-sensitive way by the activation of PKC. Because NE transport in a variant hNET, lacking a potential phosphorylation site for PKC, was also downregulated by the PKC-activating phorbol ester PMA, a direct phosphorylation of the transporter at the PKC site(s) of NETS can be excluded. The PMA-induced reduction in the density of expressed transporters (measured as a decrease in Bmax of nisoxetine binding, with a concomitant decrease in Vmax of NE transport) could be due to PKC-mediated changes in plasma membrane incorporation and/or redistribution of the NET protein. Similar effects of phorbol esters were also observed for other members of the family of Na+/CI- dependent NTTs (3).The effect of CAMP on NE transport seems to be tissue-specific, because CAMPexhibited no effect in SKN cells but decreased NE transport in rat PC12 cells, whereas in rat midbrain neurons, CAMPhad been shown to increase the expression of NET mRNA and protein.
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Louis J. DeFelice and Aurelio Galli
The lack of effect of 8Br-cGMP on NE transport by the human and rat NET suggests a minor role of this second messenger in the regulation of NET. However, further studies with measurement of mRNA and protein expression of NET are necessary to obtain more direct information about the regulation of the NET at the transcriptional or translational level.
Acknowledgment Supported by the Deutsche Forschungsgemeinschaft (SFB 400).
References 1 . Graefe, K-H., and Bonisch, H. (1988).The transport of amines across the axonal membranes of noradrenergic and dopaminergic neurones. In “Handbook of Experimental Pharmacology, 90/I (U. Trendelenburg and N. Weiner, eds.), pp. 193-245. Springer-Verlag, Berlin Heidelberg, New York. 2. Bonisch, H., and Bruss, M. (1994). Catecholamine transporter of the plasma membrane. Ann. N. Y. Acad. Sci. 733, 193-202. 3. Borowsky, B., and Hoffman, B. J. (1995).Neurotransmitter transporters: Molecular biology, function, and regulation. Int. Rev. Neurobiol. 38, 139-1 99. 4. Pacholczyk, T., Blakely, R. D., and Amara, S. G. (1991).Expression cloning of a cocaineand antidepressant-sensitive human noradrenaline transporter. Nature 350, 350-354. 5. Lingen, B., Bruss, M., and Bonisch, (1994).Cloning and expression of the bovine sodiumand chloride-dependent noradrenaline transporter. FEBS Lett. 342, 235-238.
Louis J. DeFelice and Aurelio Galli Department of Pharmacology and Center for Molecular Neuroscience Vanderbilt University School of Medicine Nashville, Tennessee 37232
Electrophysiological Analysis of Transporter Function Plasma membrane transporters selective for catecholamines efficiently clear these transmitters from the synapse, thereby regulating the spatial and temporal dimensions of synaptic transmission. For the past three decades, cocaine- and antidepressant-sensitive catecholamine transporters have been studied almost Advances m Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98$25.00
Role of Protein Kinase C and Second Messengers in Regulation of the Norepinephrine Transporter Re-uptake of released norepinephrine (NE)into presynaptic nerve terminals is responsible for the rapid termination of neurotransmission in noradrenergic synapses. Transport of NE by the neuronal NE transporter (NET)is absolutely dependent on extracellular Nat and CI- and is selectively inhibited by, for example, desipramine and nisoxetine, and Na' and Clkdependent binding of these inhibitors to the NET has been used to localize the tissue distribution of NET (1,2). Aside from its physiological role, the NET in the central nervous system is the primary target for tricyclic antidepressants. However, in spite of its physiological and therapeutic importance, little is known about the involvement of second messengers in the regulation of NET expression and function. cDNAs of a series of neurotransmitter transporters (NTTs)have been cloned and the NET has been shown to be a member of the superfamily of structurally related Na+ and CI--dependent NTTs for monoamines (dopamine, serotonin, and NE) and certain amino acids, such as GABA and glycine; transporters of this family are structurallycharacterized by 12 transmembrane domains, intracellular N- and C-termini, and a large second extracellular loop ( 3 ) .Cloning of the human and bovine NET cDNAs (4,5)has revealed one common consensus sequence for phosphorylation by protein kinase C (in the following, termed PKC site) within the second intracellular loop and, for the bovine NET (bNET), two additional PKC sites at the C-terminal end of bNET. In this paper we provide evidence that stimulation of PKC by a phorbol ester causes downregulation of NE transport and that the second-messenger cyclic adenosine monophosphate (CAMP)may be involved in tissue-specific regulation of NETs. To examine whether the PKC sites of NETs are involved in the regulation of NE transport, uptake of [jH]NE was studied in cells expressing the hNET (with one PKC site), bNET (with three PKC sites), and a mutant hNET, hNETSZSYA, in which (by site-directed mutagenesis) serine (at amino acid position 259 of hNET) was exchanged against alanine, resulting in the destruction of the single potential phosphorylation site for PKC of the hNET. For NE transport studies, COS-7 cells were transfected with the corresponding cDNA by means of the calcium phosphate method and used 48 hr posttransfection. The human Advances m Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction 1054-3589198 $ZS.OO
111
any form reserved
I83
I84
H. Bonisch et ol.
neuroblastoma cells, SKN-SH-SY5Y (SKN cells), which constitutively express the hNET, were also used in this study. Effects of second messengers on NE transport were studied in SKN cells and in the rNET-expressing rat PC12 pheochromocytoma cells. To determine uptake of NE, cells were washed three times with KrebsRinger-Hepes (KRH) buffer containing 10 p M pargyline (to inhibit monoamine oxidase) and 10 p M U-0521 (to inhibit COMT) and preincubated for 30 min (at 37°C) in this buffer. Therafter, cells were incubated at 37°C for 2-5 min (as indicated) in KRH buffer containing 10 n M [3H]NE (53.4 Cdmmol, NEN) and washed three times with ice-cold KRH buffer to terminate uptake. Specific uptake was defined by subtracting the uptake in the presence of 10 p M nisoxetine from total uptake. When short-term effects of the compounds under study were determined, the compounds were present during incubation and preincubation of the cells. When cells during culture had already been exposed for 24 hr to these compounds, they were not further present during incubation or preincubation. For kinetic analysis, cells were incubated for 2 min in 10 nM [3H]NE with 0.1-2.0 p M unlabeled NE. Binding of [3H]nisoxetine to the NE transporter on intact cell membranes was determined by pretreating the cells are described for NE uptake. Cells were incubated with 0.5-20.0 nM [3H]nisoxetine (83 Ci/mmol, Amersham) at 4°C for 3 hr and then washed three times with KRH buffer. Nonspecific binding was determined in the presence of desipramine (1 pM). At the end of uptake or binding experiments, cells were solubilized in 0.1% Triton X-100 to determine protein content and radioactivity. Short (10 min) exposure of COS-7 cells expressing either the bNET, the hNET, or the mutant hNETszsyAto the PKC-activating phorbol ester PMA (phorbol 12-myristate-13-acetate, 160 nM), the inactive phorbol ester 4aPD (4a-phorbol 12, 13-didecanoate, 160 nM), the PKC inhibitor staurosporine (0.5 pM), or the phosphatase inhibitor okadaic acid (1 p M )did not cause a significant change in [3]NE uptake. When COS-7 cells expressing the various NETS were exposed for 30 min with the aforementioned compounds and with the same concentrations, [3H]NEuptake by hNET, bNET, and hNETS25yA was significantly reduced by PMA to 61.3%, 44.4%, and 71.9%, respectively, of the corresponding control values. On the other hand, uptake of [3H]NEby all three transporters was not affected by 4aPD or staurosporin. Interestingly, uptake of [3]NE by all three transporters was not different from controls if cells were exposed to both PMA and staurosporin; that is, the inhibitory effect of PMA was antagonized by the PKC inhibitor staurosporin. Incubation of COS-7 cells (expressing either hNET or bNET) with okadaic acid affected neither [3H]NE uptake nor the inhibitory effect of PMA. Prolonged treatment with PMA (160 nM) for up to 24 hr of COS-7 cells expressing bNET, hNET, or hNETszsrAresulted in a time-dependent reduction of [3H]NE uptake, which, after about 3 hr, remained at a constant reduced level of 35-70% of corresponding controls. A similar PMA-induced reduction of [3H]NE uptake was also observed in SKN cells. Under the same conditions, 4aPD had no influence on [3H]NE uptake in either of these cells. The PMA-induced reduction of 13H]NE uptake (after 24 hr) was due to a significant reduction in maximum transport velocity (Vmax)with no significant effect on apparent affinity (Km):Vmax was
Role of PKC, Second Messengers in Regulation of NET
I85
reduced to 29.5%, 31.8%, and 28.6% of control values in COS-7 cells expressing bNET, hNET, and hNETsL59A, respectively. In SKN cells, PMA caused a reduction of Vmax from 4.0 pmol/mg/min (control) to 1.9 pmol/mg/min (i.e., to 47.5% of the control value). ['Hlnisoxetine binding was reduced by 24 hr pretreatment of SKN cells with PMA, but it remained unaffected by pretreatment of the cells with 4aPD. Analyses of the binding data revealed no significant differences in Kd values from control cells (controls = 12.1 nM vs 8.8 nM after pretreatment with PMA. However, PMA pretreatment caused a significant reduction in Bmax of ['HH]nisoxetine binding from 529 fmol/mg (controls) to 287 fmol/mg (i.e., to 54.3% of the control value). Thus, the PMA-induced reduction of NE transport was a consequence of the reduction in the number of NET molecules expressed in the plasma membrane of the cells. To study the effects of CAMP on human NET, 2-min uptake of [3H]NE (10 nM) was determined in SKN cells after 15-min or 24-hr exposure of the cells to lipophilic CAMP analogues (8Br-CAMP or db-CAMP) or to adenylate cyclase-stimulating agents (cholera toxin, forskolin). In addition, 8Br-cGMP was included in this study. [3H]NE uptake in SKN cells remained unchanged if cells were incubated for 15 min with 8Br-CAMP(1 or 10 mM), choleratoxin (100 mg/ml), forskolin (100 pM), or SBr-cGMP (1mM). These agents, as well as db-CAMP (1 mM), were also without effect on NE transport when present for 24 hr (i.e., during culture of the cells). A more physiological stimulation of adenylate cyclase through activation of the prostaglandin El (PGE, receptor of SKN cells by 10 p M PGE, (present for 24 hr) was also without effect in [3H]NEuptake, and NE transport also remained unchanged if degradation of CAMP was inhibited by IBMX (500 pM). Uptake of [3H]NE under control conditions amounted to 755 fmol/mg protein. In addition, in COS-7 cells expressing the hNET, a 24-hr treatment of the cells with forskolin (100 pM), 8Br-CAMP (1 mM), or 8Br-cGMP (1 mM) also did not affect [3H]NEuptake, which amounted in these cells to 2269 fmollmg protein. Interestingly, 24-hr incubation of PC12 with forskolin (100 pM), cholera toxin (100 ng/ml), or db-CAMP (1 mM) strongly reduced ['HINE uptake to 30.5%, 40.6%, and 36.2%, respectively, of control values (100% = 451 fmol/mg protein). However, NE uptake remained unchanged when PC12 cells were incubated for 24 hr with db-cGMP (1 mM). The results of this study indicate that human NET (and NET in COS-7 cells) is downregulated in a staurosporin-sensitive way by the activation of PKC. Because NE transport in a variant hNET, lacking a potential phosphorylation site for PKC, was also downregulated by the PKC-activating phorbol ester PMA, a direct phosphorylation of the transporter at the PKC site(s) of NETS can be excluded. The PMA-induced reduction in the density of expressed transporters (measured as a decrease in Bmax of nisoxetine binding, with a concomitant decrease in Vmax of NE transport) could be due to PKC-mediated changes in plasma membrane incorporation and/or redistribution of the NET protein. Similar effects of phorbol esters were also observed for other members of the family of Na+/CI- dependent NTTs (3).The effect of CAMP on NE transport seems to be tissue-specific, because CAMPexhibited no effect in SKN cells but decreased NE transport in rat PC12 cells, whereas in rat midbrain neurons, CAMPhad been shown to increase the expression of NET mRNA and protein.
I86
Louis J. DeFelice and Aurelio Galli
The lack of effect of 8Br-cGMP on NE transport by the human and rat NET suggests a minor role of this second messenger in the regulation of NET. However, further studies with measurement of mRNA and protein expression of NET are necessary to obtain more direct information about the regulation of the NET at the transcriptional or translational level.
Acknowledgment Supported by the Deutsche Forschungsgemeinschaft (SFB 400).
References 1 . Graefe, K-H., and Bonisch, H. (1988).The transport of amines across the axonal membranes of noradrenergic and dopaminergic neurones. In “Handbook of Experimental Pharmacology, 90/I (U. Trendelenburg and N. Weiner, eds.), pp. 193-245. Springer-Verlag, Berlin Heidelberg, New York. 2. Bonisch, H., and Bruss, M. (1994). Catecholamine transporter of the plasma membrane. Ann. N. Y. Acad. Sci. 733, 193-202. 3. Borowsky, B., and Hoffman, B. J. (1995).Neurotransmitter transporters: Molecular biology, function, and regulation. Int. Rev. Neurobiol. 38, 139-1 99. 4. Pacholczyk, T., Blakely, R. D., and Amara, S. G. (1991).Expression cloning of a cocaineand antidepressant-sensitive human noradrenaline transporter. Nature 350, 350-354. 5. Lingen, B., Bruss, M., and Bonisch, (1994).Cloning and expression of the bovine sodiumand chloride-dependent noradrenaline transporter. FEBS Lett. 342, 235-238.
Louis J. DeFelice and Aurelio Galli Department of Pharmacology and Center for Molecular Neuroscience Vanderbilt University School of Medicine Nashville, Tennessee 37232
Electrophysiological Analysis of Transporter Function Plasma membrane transporters selective for catecholamines efficiently clear these transmitters from the synapse, thereby regulating the spatial and temporal dimensions of synaptic transmission. For the past three decades, cocaine- and antidepressant-sensitive catecholamine transporters have been studied almost Advances m Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98$25.00