The first luminal domain of vesicular monoamine transporters mediates G-protein-dependent regulation of transmitter uptake

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.     

Regulation of vesicular neurotransmitter transporters

Neurotransmitters are key molecules of neurotransmission. They are concentrated first in the cytosol and then in small synaptic vesicles of presynaptic terminals by the activity of specific neurotransmitter transporters of the plasma and the vesicular membrane, respectively. It has been shown that postsynaptic responses to single neurotransmitter packets vary over a wide range, which may be due to a regulation of vesicular neurotransmitter filling. Vesicular filling depends on the availability of transmitter molecules in the cytoplasm and the active transport into secretory vesicles relying on a proton gradient. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Go2 and Gq, which regulate VMAT activities in brain and platelets, respectively, and may also be involved in the regulation of VGLUTs. It appears that the vesicular content activates the G protein, suggesting a signal transduction form the luminal site which might be mediated by a vesicular G-protein coupled receptor or, as an alternative, possibly by the transporter itself. These novel functions of G proteins in the control of transmitter storage may link regulation of the vesicular content to intracellular signal cascades.     

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands.Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).     

Reduced vesicular storage of dopamine exacerbates methamphetamine-induced neurodegeneration and astrogliosis

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5–10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.     

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1muM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24-48h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI(3)K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.     

Characterization of high-affinity [3H]TBZOH binding to the human platelet vesicular monoamine transporter.

The present study indicates that human platelets can be used as an accessible peripheral model not only for the plasma membrane serotonin transporter, but also for the vesicular monoamine transporter. The vesicular monoamine transporter (VMAT2) is responsible for the accumulation of monoamines in the synaptic vesicles. VMAT2 differs from the plasma membrane transporters in its capability to recognize serotonin, histamine, norepinephrine and dopamine with almost the same affinity. Dihydrotetrabenazine (TBZOH) is a very potent inhibitor of VMAT2 that binds with high affinity to this transporter. [3H]TBZOH has been used as a ligand to label VMAT2 in human, bovine and rodent brain. In this study we characterized the pharmacodynamic and pharmacokinetic parameters of [3H]TBZOH binding in human platelets as compared to rat brain. The density (Bmax) and affinity (Kd) of [3H]TBZOH specific binding was assessed by Scatchard analysis. Association and dissociation rate constants (k(on), K(off)) were assessed by kinetic binding studies. In this study high-affinity and saturable binding sites for [3H]TBZOH were demonstrated in human platelets. Both the affinity of [3H]TBZOH to its binding site in platelets (Kd = 3.2+/-0.5 nM) and the kinetic rate constants (K(on) = 2.8 x 10(7) M(-1) min(-1); K(off) = 0.099 min(-1)) were similar to that in rat brain (Kd(striatum) = 1.5 nM; Kd(cerebral cortex) = 1.35 nM; K(on) = 2 x 10(7) M(-1) min(-1); K(off) = 0.069 min(-1)). Only the VMAT2 blockers tetrabenazine and reserpine inhibited [3H]TBZOH specific binding.     

Tryptophan hydroxylase 1 knockout and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice

Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1(-/-) mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mgxkg(-1)xday(-1)) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele. p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development.     

Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines

The release of monoamine neurotransmitters from cell bodies and dendrites has an important role in behavior, but the mechanism (vesicular or non vesicular) has remained unclear. Because the location of vesicular monoamine transporter 2 (VMAT2) defines the secretory vesicles capable of monoamine release, we have studied its trafficking to assess the potential for monoamine release by exocytosis. In neuroendocrine PC12 cells, VMAT2 localizes exclusively to large dense-core vesicles (LDCVs), and we now show that cytoplasmic signals target VMAT2 directly to LDCVs within the biosynthetic pathway. In neurons, VMAT2 localizes to a population of vesicles that we now find undergo regulated exocytosis in dendrites. Although hippocampal neurons do not express typical LDCV proteins, transfected chromogranins A, B, and brain-derived neurotrophic factor (BDNF) colocalize with VMAT2. VMAT2 thus defines a population of secretory vesicles that mediate the activity-dependent somatodendritic release of multiple retrograde signals involved in synaptic function, growth, and plasticity.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice

The vesicular monoamine transporter 2 (VMAT2) plays a pivotal role in regulating the size of vesicular and cytosolic dopamine (DA) storage pools within the CNS, and can thus influence extracellular DA neurotransmission. Transgenic mice have been generated with a dramatically reduced (by approximately 95%) expression of the VMAT2 gene which, unlike complete knockout lines, survive into adulthood. We compared the pre-synaptic regulation of both impulse-dependent (exocytotic) and carrier-mediated (via reversal of the DA transporter, DAT) DA release in the dorsolateral caudate putamen (CPu) of striatal slices derived from adult homozygous VMAT2 mutant and wild-type mice using fast cyclic voltammetry. Impulse-dependent DA release, evoked by a single electrical pulse, was lower in homozygous (116 nm) than wild-type mice (351 nm) indicating smaller vesicular DA stores, an observation supported by the evanescent effect of amfonelic acid (300 nm) in homozygous mice. Amphetamine (2 microm) increased extracellular DA via DAT reversal in both wild-type (by 459 nm) and VMAT2 mutant (by 168 nm, p < 0.01 vs. wild-type) mice. In both cases, the effect was blocked by the DAT inhibitor GBR12935 (1 microm). Simultaneously, amphetamine decreased impulse-dependent DA release, albeit less in homozygous (by 55%) than in wild-type (by 78%) mice. In wild-types, this decrement was largely reversed by GBR12935 but not by the D2/D3 autoreceptor antagonist (-)sulpiride (1 microm). Conversely, in homozygous VMAT2 mutant mice, it was attenuated by (-)sulpiride but not GBR12935. The D2/D3 receptor agonist quinpirole inhibited impulse-dependent DA release with a lower EC50 value in homozygous mice (12 nm) compared with wild-types (34 nm), indicating the compensatory presence of functionally supersensitive release-regulating autoreceptors. However, analysis of DA reuptake kinetics obtained in the absence and presence of DAT blockade (by cocaine and amfonelic acid) revealed only minor differences in DAT functionality. These results demonstrate that impaired vesicular DA storage constrains extracellular DA levels in the dorsolateral CPu whether induced by either impulse-dependent or carrier-mediated mechanisms and that the relative importance of the DAT and terminal autoreceptors as control mechanisms in the actions of amphetamine are reversed in VMAT2 mutant mice.     

From glutamate co-release to vesicular synergy: vesicular glutamate transporters

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.     

The molecular chaperone Hsc70 interacts with the vesicular monoamine transporter-2

Synaptic transmission depends on the efficient loading of transmitters into synaptic vesicles by vesicular neurotransmitter transporters. The vesicular monoamine transporter-2 (VMAT2) is essential for loading monoamines into vesicles and maintaining normal neurotransmission. In an effort to understand the regulatory mechanisms associated with VMAT2, we have embarked upon a systematic search for interacting proteins. Glutathione-S-transferase pull-down assays combined with mass spectrometry led to the identification of the 70-kDa heat shock cognate protein (Hsc70) as a VMAT2 interacting protein. Co-immunoprecipitation experiments in brain tissue and heterologous cells confirmed this interaction. A direct binding was observed between the amino terminus and the third cytoplasmic loop of VMAT2, as well as, a region containing the substrate binding and the carboxy-terminal domains of Hsc70. Furthermore, VMAT2 and Hsc70 co-fractionated with purified synaptic vesicles obtained from a sucrose gradient, suggesting that this interaction occurs at the synaptic vesicle membrane. The functional significance of this novel VMAT2/Hsc70 interaction was examined by performing vesicular uptake assays in heterologous cells and purified synaptic vesicles from brain tissue. Recombinant Hsc70 produced a dose-dependent inhibition of VMAT2 activity. This effect was mimicked by the closely related Hsp70 protein. In contrast, VMAT2 activity was not altered in the presence of previously denatured Hsc70 or Hsp70, as well as the unrelated Hsp60 protein; confirming the specificity of the Hsc70 effect. Finally, a purified Hsc70 fragment that binds VMAT2 was sufficient to inhibit VMAT2 activity in synaptic vesicles. Our results suggest an important role for Hsc70 in VMAT2 function and regulation.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

Epsilon protein kinase C in pathological myocardial hypertrophy. Analysis by combined transgenic expression of translocation modifiers and Galphaq

The epsilon isoform of protein kinase C (PKC) has a critical cardiotrophic function in normal postnatal developing heart as demonstrated by cardiac-specific transgenic expression of epsilonPKC-selective translocation inhibitor (epsilonV1) and activator (psiepsilonRACK) peptides (Mochly-Rosen, D., Wu, G., Hahn, H., Osinska, H., Liron, T., Lorenz, J. N., Robbins, J., and Dorn, G. W., II (2000) Circ. Res. 86, 1173-1179). To define the role of epsilonPKC signaling in pathological myocardial hypertrophy, epsilonV1 or psiepsilonRACK were co-expressed in mouse hearts with Galpha(q), a PKC-linked hypertrophy signal transducer. Compared with Galpha(q) overexpression alone, co-expression of psiepsilonRACK with Galpha(q) increased epsilonPKC particulate partitioning by 30 +/- 2%, whereas co-expression of epsilonV1 with Galpha(q) reduced particulate-associated epsilonPKC by 22 +/- 1%. Facilitation of epsilonPKC translocation by psiepsilonRACK in Galpha(q) mice improved cardiac contractile function measured as left ventricular fractional shortening (30 +/- 3% Galpha(q) versus 43 +/- 2% psiepsilonRACK/Galpha(q), p < 0.05). Conversely, inhibition of epsilonPKC by epsilonV1 modified the Galpha(q) nonfailing hypertrophy phenotype to that of a lethal dilated cardiomyopathy. These opposing effects of epsilonPKC translocation activation and inhibition in Galpha(q) hypertrophy indicate that epsilonPKC signaling is a compensatory event in myocardial hypertrophy, rather than a pathological event, and support the possible therapeutic efficacy of selective epsilonPKC translocation enhancement in cardiac insufficiency.     

A rapid oxidation and persistent decrease in the vesicular monoamine transporter 2 after methamphetamine

Methamphetamine (METH) produces long-term decreases in markers of dopamine (DA) terminals in animals and humans. A decrease in the function of the vesicular monoamine transporter 2 (VMAT2) has been associated with damage to striatal DA terminals caused by METH; however, a possible mechanism for this decrease in VMAT2 function has not been defined. The current study showed that METH caused a rapid decrease to 68% of controls in VMAT2 protein immunoreactivity of the vesicular fraction from striatal synaptosomes within 1 h after a repeated high-dose administration regimen of METH. This decrease was associated with a 75% increase in nitrosylation of VMAT2 protein in the synaptosomal fraction as measured by nitrosocysteine immunoreactivity of VMAT2 protein. The rapid decreases in VMAT2 persisted when evaluated 7 days later and were illustrated by decreases in VMAT2 immunoreactivity and DA content of the vesicular fraction to 34% and 51% of control values, respectively. The decreases were blocked or attenuated by prior injections of the neuronal nitric oxide synthase inhibitor, S-methyl-l-thiocitrulline. These studies demonstrate that METH causes a rapid neuronal nitric oxide synthase-dependent oxidation of VMAT2 and long-term decreases in VMAT2 protein and function. The results also suggest that surviving DA terminals after METH exposure may have a compromised capacity to buffer cytosolic DA concentrations and DA-derived oxidative stress.     

In vivo imaging of the vesicular acetylcholine transporter and the vesicular monoamine transporter.

Validation of the vesicular acetylcholine transporter (VAChT) and the neuronal vesicular monoamine transporter (VMAT2) as important molecular targets in the cholinergic and dopamine neurons, respectively, has sparked interest in the development of radiotracers for studying these markers in vitro and in vivo. Currently, a number of selective high-affinity radiotracers are available for studying these targets in vivo with positron emission tomography (PET) or single photon emission computed tomography (SPECT). PET studies of VMAT2 in neuropathology reveal changes in the density of this marker that can be verified independently. Similarly, in vivo studies with VAChT ligands suggest that the latter are potentially useful in detecting cholinergic lesions in vivo; however, additional development is required to fully realize the potential of these radioligands.     

Heterotrimeric G proteins of the Gq/11 family are crucial for the induction of maternal behavior in mice

Heterotrimeric G proteins of the G(q/11) family transduce signals from a variety of neurotransmitter receptors and have therefore been implicated in several functions of the central nervous system. To investigate the potential role of G(q/11) signaling in behavior, we generated mice which lack the alpha-subunits of the two main members of the G(q/11) family, Galpha(q) and Galpha(11), selectively in the forebrain. We show here that forebrain Galpha(q/11)-deficient females do not display any maternal behavior such as nest building, pup retrieving, crouching, or nursing. However, olfaction, motor behavior and mammary gland function are normal in forebrain Galpha(q/11)-deficient females. We used c-fos immunohistochemistry to investigate pup-induced neuronal activation in different forebrain regions and found a significant reduction in the medial preoptic area, the bed nucleus of stria terminalis, and the lateral septum both in postpartum females and in virgin females after foster pup exposure. Pituitary function, especially prolactin release, was normal in forebrain Galpha(q/11)-deficient females, and activation of oxytocin receptor-positive neurons in the hypothalamus did not differ between genotypes. Our findings show that G(q/11) signaling is indispensable to the neuronal circuit that connects the perception of pup-related stimuli to the initiation of maternal behavior and that this defect cannot be attributed to either reduced systemic prolactin levels or impaired activation of oxytocin receptor-positive neurons of the hypothalamus.     

Norepinephrine transporter expression in cholinergic sympathetic neurons: differential regulation of membrane and vesicular transporters

Sympathetic neurons that undergo a noradrenergic to cholinergic change in phenotype provide a useful model system to examine the developmental regulation of proteins required to synthesize, store, or remove a particular neurotransmitter. This type of change occurs in the sympathetic sweat gland innervation during development and can be induced in cultured sympathetic neurons by extracts of sweat gland-containing footpads or by leukemia inhibitory factor. Sympathetic neurons initially produce norepinephrine (NE) and contain the vesicular monoamine transporter 2 (VMAT2), which packages NE into vesicles, and the norepinephrine transporter (NET), which removes NE from the synaptic cleft to terminate signaling. We have used a variety of biochemical and molecular techniques to test whether VMAT2 and NET levels decrease in sympathetic neurons which stop producing NE and make acetylcholine. In cultured sympathetic neurons, NET protein and mRNA decreased during the switch to a cholinergic phenotype but VMAT2 mRNA and protein did not decline. NET immunoreactivity disappeared from the developing sweat gland innervation in vivo as it acquired cholinergic properties. Surprisingly, NET simultaneously appeared in sweat gland myoepithelial cells. The presence of NET in myoepithelial cells did not require sympathetic innervation. VMAT2 levels did not decrease as the sweat gland innervation became cholinergic, indicating that NE synthesis and vesicular packaging are not coupled in this system. Thus, production of NE and the transporters required for noradrenergic transmission are not coordinately regulated during cholinergic development.