Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go2, which down-regulates the ...activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5′-(βiγ-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1−/−) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1−/− mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1−/− platelets with serotonin or noradrenaline. Gαq mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Gαq deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A2 receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Gαq.
Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go(2), which down-regulates the ...activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5'-(beta(i)gamma-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1-/-) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1-/- mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1-/- platelets with serotonin or noradrenaline. Galpha(q) mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Galpha(q) deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A(2) receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Galpha(q).
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 Gαo2, Gαq/11, Gβ2 and Gβ5 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 α-, β- and probably γ-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Gαo2 (10 nM) to nearly the same extent as by GMppNp (50 μM) whereas GDPβS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Gαo2 appears to be the main regulator of vesicular momoamine transporter activity.
Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content
may be modulated by vesicle-associated Go 2 , which down-regulates the ...activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and
VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin
O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5â²-(β i γ-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1â/â)
mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did
not influence VMAT2 activity in naive platelets from Tph1â/â mice. GMppNp-mediated inhibition could be reconstituted, however,
when preloading Tph1â/â platelets with serotonin or noradrenaline. Gα q mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Gα q deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A 2 receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle
itself regulates transmitter transporter activity via its content and vesicle-associated Gα q .
Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their α-, β-, and γ-subunits allows numerous combinations thereby confering specificity ...to receptor-G-protein-effector interactions. Using antisera against individual G-protein α-subunits we here present a regional and subcellular distribution of Gβ1, Gβ2, and Gβ5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein β-subunits. Since Gβ-subunits together with a G-protein γ-subunit affect signal cascades we include a distribution of the neuron-specific Gγ2- and Gγ3-subunits in selected brain areas. Gβt1, Gβ2, and Gβ5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gβ2 is highly concentrated in the mossy fibres of dentate gyros neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gβ2 also occur in interneurons innervating spinal cord α-motoneurons. Gβ5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gβ5 in contrast to Gβ2 concentrates around a-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gβ2 and Gβ5 are found in different subcellular compartments. Whereas Gβ5 is restricted to the perikarya, Gβ2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Gγ2 and Gγ3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Gγ2 and Gγ3 in combinations with certain Gαs and/or Gβs.
The widespread but regionally and cellularly rather different distribution of Gpβ- and Gγ2/3-subunits suggests that regionspecific combinations of G-protein subunits mediate signal transduction in the central nervous system. The different subcellular distribution of Gβ-subunits in cultivated neurons reflects that observed in tissue where Gβ5 and Gβ2 associate preferentially with the perikarya and the neuropil, respectively, and suggests an additional association of Gβ2 with secretory vesicles.
Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. ...Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go
2
. We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Gαo
2
, whereas comparable concentrations of Gαo
1
are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go
2
. By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Gαo
2
associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go
2
-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.
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