Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Iα inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.

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This review focuses on the more recent findings of the structure of sympathetic postganglionic axons and the association of their varicose terminals with vascular smooth muscle. These studies have investigated the innervation of a wide range of vessels from different regions of the vasculature in the rat, guinea pig and rabbit and have predominantly used serial sections and computerised three-dimensional reconstructions of entire varicosities. They have shown, contrary to previous studies conducted in the 1960s and 1970s, that sympathetic axon varicosities commonly form structurally specialised neuromuscular junctions with vascular smooth muscle cells of most resistance arteries and some small veins. In addition, they have shown that most axon varicosities innervating small arterioles and small mesenteric veins form neuromuscular junctions, indicating that neurotransmitter is primarily released at such neuromuscular junctions. This review discusses the structure of sympathetic neuromuscular junctions, their development, structural diversity and distribution on vessels from different regions of the vasculature. These more recent structural findings and their possible significance for our understanding of mechanisms involved in neural transmission in blood vessels is discussed.

The relationship of the varicosities of sympathetic postganglionic nerve terminals to the smooth muscle cells of arterioles in the submucosa of the guinea pig ileum has been investigated quantitatively by electron microscopy. Longitudinal sections were cut through arterioles about 50 micron in diameter after fixation in vitro or in situ under pressure. About 13% of the varicosities in individual ultrathin sections made contact with the outer surface of the smooth muscle cells. The neuromuscular junctions resembled those in skeletal muscle: the basal laminae of the axon bundle and of the smooth muscle were fused, and synaptic vesicles were accumulated close to the region of fusion. When individual varicosities were examined in serial sections, 92% and 83% in two preparations were found to form junctions of this kind. Most of the noncontacting varicosities were bare of Schwann cell toward the arteriolar surface and separated from it by less than 200 nm. Almost all axon profiles contained synaptic vesicles with electron dense cores after exposure to 5-hydroxydopamine. In electrophysiological experiments, ionophoretic application of noradrenaline to the arteriolar surface along the nerve bundles (demonstrated subsequently by fluorescence histochemistry) produced responses resembling those evoked by nerve stimulation. These anatomical and physiological data, taken together with the evidence for quantal release in this preparation (see Hirst et al., '85), suggest that neuromuscular transmission involves the rare release of a quantum of noradrenaline at discrete points on the smooth muscle membrane.