Single-Molecule Microscopy Reveals Dynamic FLNA Interactions Governing SSTR2 Clustering and Internalization.

The cytoskeletal protein filamin A (FLNA) has been suggested to play an important role in the responsiveness of GH-secreting pituitary tumors to somatostatin receptor subtype 2 (SSTR2) agonists by regulating SSTR2 expression and signaling. However, the underlying mechanisms are unknown. In this study, we use fast multicolor single-molecule microscopy to image individual SSTR2 and FLNA molecules at the surface of living cells with unprecedented spatiotemporal resolution. We find that SSTR2 and FLNA undergo transient interactions, which occur preferentially along actin fibers and contribute to restraining SSTR2 diffusion. Agonist stimulation increases the localization of SSTR2 along actin fibers and, subsequently, SSTR2 clustering and recruitment to clathrin-coated pits (CCPs). Interfering with FLNA-SSTR2 binding with a dominant-negative FLNA fragment increases SSTR2 mobility, hampers the formation and alignment of SSTR2 clusters along actin fibers, and impairs both SSTR2 recruitment to CCPs and SSTR2 internalization. These findings indicate that dynamic SSTR2-FLNA interactions critically control the nanoscale localization of SSTR2 at the plasma membrane and are required for coupling SSTR2 clustering to internalization. These mechanisms explain the critical role of FLNA in the control of SSTR2 expression and signaling and suggest the possibility of targeting SSTR2-FLNA interactions for the therapy of pharmacologically resistant GH-secreting pituitary tumors.

Somatostatin immunoreactivity in axon terminals in rat nucleus tractus solitarii arising from central nucleus of amygdala: coexistence with GABA and postsynaptic expression of sst2A receptor.

Axon terminals synapsing on neurones in the nucleus tractus solitarii (NTS) that originate from the central nucleus of the amygdala (CeA) have been shown to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here we investigated whether such terminals also contain somatostatin (SOM), a neuropeptide found in axons distributed throughout the NTS and in somata in the CeA, and known to modulate cardiovascular reflexes when microinjected into the NTS. With fluorescence microscopy, SOM immunoreactivity was seen in the varicosities of some axons throughout the NTS that were anterogradely labelled with biotin dextran amine injected into the CeA. Such varicosities were frequently observed in close proximity to dendrites of NTS neurones that were immunoreactive for the SOM receptor sst(2A) subtype, and in many cases also for catecholamine synthesising enzymes. In the caudal, cardioregulatory zone of NTS, SOM immunoreactivity was localised by electron microscopic pre-embedding gold labelling to boutons containing dense-cored and clear pleomorphic vesicles and forming symmetrical synapses, mostly onto dendrites. Additional post-embedding gold labelling for GABA suggested that a subpopulation (29%) of GABAergic terminals sampled in this area of NTS contained SOM. Almost all boutons anterogradely labelled from the amygdala were GABA-immunoreactive (-IR) and 21% of these were SOM-IR. A similar proportion of these boutons (22%) formed synapses onto dendrites containing immunoreactivity for the SOM receptor sst(2A) subtype. These observations provide evidence that some of the GABAergic projection neurones in the CeA that inhibit baroreceptor reflex responses in the NTS in response to fear or emotional stimuli could release SOM, which might modulate the activity of NTS neurones via an action on sst(2A) receptors.

Somatostatin receptor localization of pancreatic endocrine tumors.

Gastroenteropancreatic endocrine tumors are difficult to localize. At the same time the tumor is localized, though, there is an opportunity for cure or to remove tumor tissue. In this study we have prospectively examined the ability of 111In-octreotide scintigraphy, magnetic resonance imaging (MRI), and computed tomography (CT) to localize tumor lesions in 24 patients with a biochemical or histologic diagnosis of neuroendocrine tumor. In eight patients a surgical assessment of the imaging results was prospectively performed. Planar and abdominal single-photon emission tomography (SPET) images acquired 4 and 24 hours after 180 to 220 MBq of 111In-octreotide injection were evaluated and compared with conventional imaging techniques. SPET scintigraphy visualized more presumed tumor lesions (n = 39) than conventional imaging studies (MRI,n = 25; CT,n = 13); 23 of 24 patients had positive octreotide scintigraphy, 17 of 24 had positive MRI-scans, and 12 of 24 patients had positive CT scans. It was concluded that 111In-octreotide scintigraphy combined with conventional imaging improves the preoperative localization of presumably tumorous lesions in patients with gastroenterohepatic endocrine tumors.