Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin µO§-GVIIJ.

Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.

Α- and ß-subunit composition of voltage-gated sodium channels investigated with µ-conotoxins and the recently discovered µO§-conotoxin GVIIJ.

We investigated the identities of the isoforms of the α (NaV1)- and ß (NaVß)-subunits of voltage-gated sodium channels, including those responsible for action potentials in rodent sciatic nerves. To examine α-subunits, we used seven µ-conotoxins, which target site 1 of the channel. With the use of exogenously expressed channels, we show that two of the µ-conotoxins, µ-BuIIIB and µ-SxIIIA, are 50-fold more potent in blocking NaV1.6 from mouse than that from rat. Furthermore, we observed that µ-BuIIIB and µ-SxIIIA are potent blockers of large, myelinated A-fiber compound action potentials (A-CAPs) [but not small, unmyelinated C-fiber CAPs (C-CAPs)] in the sciatic nerve of the mouse (unlike A-CAPs of the rat, previously shown to be insensitive to these toxins). To investigate ß-subunits, we used two synthetic derivatives of the recently discovered µO§-conotoxin GVIIJ that define site 8 of the channel, as previously characterized with cloned rat NaV1- and NaVß-subunits expressed in Xenopus laevis oocytes, where it was shown that µO§-GVIIJ is a potent inhibitor of several NaV1-isoforms and that coexpression of NaVß2 or -ß4 (but not NaVß1 or -ß3) totally protects against block by µO§-GVIIJ. We report here the effects of µO§-GVIIJ on 1) sodium currents of mouse NaV1.6 coexpressed with various combinations of NaVß-subunits in oocytes; 2) A- and C-CAPs of mouse and rat sciatic nerves; and 3) sodium currents of small and large neurons dissociated from rat dorsal root ganglia. Our overall results lead us to conclude that action potentials in A-fibers of the rodent sciatic nerve are mediated primarily by NaV1.6 associated with NaVß2 or NaVß4.

Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

Characterization of two neuronal subclasses through constellation pharmacology.

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."

Interactions between corticotropin releasing factor signaling and prophylactic antibiotics on measures of intestinal function in weaned and transported pigs.

The study objective was to evaluate the interaction between corticotrophin releasing factor (CRF) receptor signaling and prophylactic antibiotic administration on intestinal physiology in newly weaned and transported pigs. Pigs (n = 56; 5.70 ± 1.05 kg) were weaned (20.49 ± 0.64 d), a blood sample was taken, and then pigs were given an intraperitoneal injection of saline (SAL; n = 28 pigs) or a CRF receptor antagonist (CRFA; n = 28 pigs; 30 µg/kg body weight; Astressin B), and then were transported in a livestock trailer for 12 h and 49 min. A second and third intraperitoneal injection was given at 4 h 42 min and 11 h 36 min into the transport process, respectively. Following transport, 4 SAL and 4 CRFA pigs were blood sampled and euthanized. The remaining 48 pigs were individually housed and given dietary antibiotics [AB; n = 12 SAL and 12 CRFA pigs; chlortetracycline (441 ppm) + tiamulin (38.6 ppm)] or no dietary antibiotics (NAB; n = 12 SAL and 12 CRFA pigs) for 14 d post-transport. Blood was collected at 12 h and on d 3, 7, and 14, and then pigs were euthanized on d 7 (n = 24) and d 14 (n = 24) post-weaning and transport. Circulating cortisol was reduced (p = 0.05) in CRFA pigs when compared to SAL pigs post-weaning and transport. On d 7, jejunal villus height and crypt depth was greater overall (p < 0.05) in AB-fed pigs versus NAB-fed pigs. On d 14, ileal crypt depth was reduced (p = 0.02) in CRFA pigs when compared to SAL pigs. Jejunal CRF mRNA abundance tended to be reduced (p = 0.09) on d 7 in CRFA pigs versus SAL pigs. On d 14, jejunal tumor necrosis factor-alpha was reduced (p = 0.01) in AB-fed pigs versus NAB-fed pigs. On d 7, change in glucose short-circuit current tended to be increased (p = 0.07) in CRFA pigs fed the AB diet when compared to CRFA pigs fed the NAB diet. In conclusion, CRFA pigs and pigs fed AB had some similar biological intestinal function measures post-weaning and transport.

NMR structure of the first extracellular domain of corticotropin-releasing factor receptor 1 (ECD1-CRF-R1) complexed with a high affinity agonist.

The corticotropin-releasing factor (CRF) peptide hormone family members coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play important roles within the cardiovascular, gastrointestinal, and central nervous systems, among others. The actions of the peptides are mediated by activation of two G-protein-coupled receptors of the B1 family, CRF receptors 1 and 2 (CRF-R1 and CRF-R2α,ß). The recently reported three-dimensional structures of the first extracellular domain (ECD1) of both CRF-R1 and CRF-R2ß (Pioszak, A. A., Parker, N. R., Suino-Powell, K., and Xu, H. E. (2008) J. Biol. Chem. 283, 32900-32912; Grace, C. R., Perrin, M. H., Gulyas, J., Digruccio, M. R., Cantle, J. P., Rivier, J. E., Vale, W. W., and Riek, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4858-4863) complexed with peptide antagonists provided a starting point in understanding the binding between CRF ligands and receptors at a molecular level. We now report the three-dimensional NMR structure of the ECD1 of human CRF-R1 complexed with a high affinity agonist, α-helical cyclic CRF. In the structure of the complex, the C-terminal residues (23-41) of α-helical cyclic CRF bind to the ECD1 of CRF-R1 in a helical conformation mainly along the hydrophobic face of the peptide in a manner similar to that of the antagonists in their corresponding ECD1 complex structures. Unique to this study is the observation that complex formation between an agonist and the ECD1-CRF-R1 promotes the helical conformation of the N terminus of the former, important for receptor activation (Gulyas, J., Rivier, C., Perrin, M., Koerber, S. C., Sutton, S., Corrigan, A., Lahrichi, S. L., Craig, A. G., Vale, W., and Rivier, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10575-10579).

Amyloid as a depot for the formulation of long-acting drugs.

Amyloids are highly organized protein aggregates that are associated with both neurodegenerative diseases such as Alzheimer disease and benign functions like skin pigmentation. Amyloids self-polymerize in a nucleation-dependent manner by recruiting their soluble protein/peptide counterpart and are stable against harsh physical, chemical, and biochemical conditions. These extraordinary properties make amyloids attractive for applications in nanotechnology. Here, we suggest the use of amyloids in the formulation of long-acting drugs. It is our rationale that amyloids have the properties required of a long-acting drug because they are stable depots that guarantee a controlled release of the active peptide drug from the amyloid termini. This concept is tested with a family of short- and long-acting analogs of gonadotropin-releasing hormone (GnRH), and it is shown that amyloids thereof can act as a source for the sustained release of biologically active peptides.

Early weaning stress impairs development of mucosal barrier function in the porcine intestine.

Early life stress is a predisposing factor for the development of chronic intestinal disorders in adult life. Here, we show that stress associated with early weaning in pigs leads to impaired mucosal barrier function. Early weaning (15- to 21-day weaning age) resulted in sustained impairment in intestinal barrier function, as indicated by reductions in jejunal transepithelial electrical resistance and elevations in mucosal-to-serosal flux of paracellular probes [(3)H]mannitol and [(14)C]inulin measured at 5 and 9 wk of age, compared with that shown in late-weaned pigs (23- to 28-day weaning age). Elevated baseline short-circuit current was observed in jejunum from early-weaned pigs and was shown to be mediated via enhanced Cl(-) secretion. Jejunal barrier dysfunction in early-weaned pigs coincided with increased lamina propria immune cell density particularly mucosal mast cells. The mast cell stabilizer drug sodium cromoglycolate ameliorated barrier dysfunction and hypersecretion in early-weaned pigs, demonstrating an important role of mast cells. Furthermore, activation of mast cells ex vivo with c48/80 and corticotrophin-releasing factor (CRF) in pig jejunum mounted in Ussing chambers induced barrier dysfunction and elevations in short-circuit current that were inhibited with mast cell protease inhibitors. Experiments in which selective CRF receptor antagonists were administered to early-weaned pigs revealed that CRF receptor 1 (CRFr1) activation mediates barrier dysfunction and hypersecretion, whereas CRFr2 activation may be responsible for novel protective properties in the porcine intestine in response to early life stress.

Switch from antagonist to agonist after addition of a DOTA chelator to a somatostatin analog.

PURPOSE: Peptide receptor targeting has become an increasingly attractive method to target tumors diagnostically and radiotherapeutically. Peptides linked to a variety of chelators have been developed for this purpose. They have, however, rarely been tested for their agonistic or antagonistic properties. We report here on a somatostatin antagonist that switched to an agonist upon coupling to a DOTA chelator. METHODS: Two novel somatostatin analogs, 406-040-15 and its DOTA-coupled counterpart 406-051-20, with and without cold Indium labeling, were tested for their somatostatin receptor subtypes 1-5 (sst(1)-sst(5)) binding affinity using receptor autoradiography. Moreover, they were tested functionally for their ability to affect sst(2) and sst(3) internalization in vitro in HEK293 cells stably expressing the human sst(2) or sst(3) receptor, using an immunofluorescence microscopy-based internalization assay. RESULTS: All three compounds were characterized as pan-somatostatin analogs having a high affinity for all five sst. In the sst(2) internalization assay, all three compounds showed an identical behavior, namely, a weak agonistic effect complemented by a weak antagonistic effect, compatible with the behavior of a partial agonist. Conversely, in the sst(3) internalization assay, 406-040-15 was a full antagonist whereas its DOTA-coupled counterpart, 406-051-20, with and without Indium labeling, switched to a full agonist. CONCLUSION: Adding the DOTA chelator to the somatostatin analog 406-040-15 triggers a switch at sst(3) receptor from an antagonist to an agonist. This indicates that potential radioligands for tumor targeting should always be tested functionally before further development, in particular if a chelator is added.

Ring size in octreotide amide modulates differently agonist versus antagonist binding affinity and selectivity.

H-DPhe (2)-c[Cys (3)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Thr (15)-NH2 (1) (a somatostatin agonist, SRIF numbering) and H-Cpa (2)-c[DCys (3)-Tyr (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Nal (15)-NH2 (4) (a somatostatin antagonist) are based on the structure of octreotide that binds to three somatostatin receptor subtypes (sst 2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the backbone flexibility, resulting in loss of binding affinity at all sst s. The introduction of Hcy at positions 3 and 14 improved selectivity for sst 2 as a result of significant loss of binding affinity at the other sst s. Substitution by DHcy at position 3 in the antagonist scaffold (5), on the other hand, resulted in a significant loss of binding affinity at sst 2 and sst 3 as compared to the different affinities of the parent compound (4). The 3D NMR structures of the analogues in dimethylsulfoxide are consistent with the observed binding affinities.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity.

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).

Safety, Biodistribution, and Radiation Dosimetry of 68Ga-OPS202 in Patients with Gastroenteropancreatic Neuroendocrine Tumors: A Prospective Phase I Imaging Study.

Preclinical and preliminary clinical evidence indicates that radiolabeled somatostatin (sst) receptor antagonists perform better than agonists in detecting neuroendocrine tumors (NETs). We performed a prospective phase I/II study to evaluate the sst receptor antagonist 68Ga-OPS202 (68Ga-NODAGA-JR11; NODAGA = 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid and JR11 = Cpa-c(dCys-Aph(Hor)-dAph(Cbm)-Lys-Thr-Cys)-dTyr-NH2)) for PET imaging. Here, we report the results of phase I of the study. Methods: Patients received 2 single 150-MBq intravenous injections of 68Ga-OPS202 3-4 wk apart (15 µg of peptide at visit 1 and 50 µg at visit 2). At visit 1, a dynamic PET/CT scan over the kidney was obtained during the first 30 min after injection, and static whole-body scans were obtained at 0.5, 1, 2, and 4 h after injection; at visit 2, a static whole-body scan was obtained at 1 h. Blood samples and urine were collected at regular intervals to determine 68Ga-OPS202 pharmacokinetics. Safety, biodistribution, radiation dosimetry, and the most appropriate imaging time point for 68Ga-OPS202 were assessed. Results: Twelve patients with well-differentiated gastroenteropancreatic (GEP) NETs took part in the study. 68Ga-OPS202 cleared rapidly from the blood, with a mean residence time of 2.4 ± 1.1 min/L. The organs with the highest mean dose coefficients were the urinary bladder wall, kidneys, and spleen. The calculated effective dose was 2.4E-02 ± 0.2E-02 mSv/MBq, corresponding to 3.6 mSv, for a reference activity of 150 MBq. Based on total numbers of detected malignant lesions, the optimal time window for the scan was between 1 and 2 h. For malignant liver lesions, the time point at which most patients had the highest mean tumor contrast was 1 h. 68Ga-OPS202 was well tolerated; adverse events were grade 1 or 2, and there were no signals of concern from laboratory blood or urinalysis tests. Conclusion:68Ga-OPS202 showed favorable biodistribution and imaging properties, with optimal tumor contrast between 1 and 2 h after injection. Dosimetry analysis revealed that the dose delivered by 68Ga-OPS202 to organs is similar to that delivered by other 68Ga-labeled sst analogs. Further evaluation of 68Ga-OPS202 for PET/CT imaging of NETs is therefore warranted.

Common and divergent structural features of a series of corticotropin releasing factor-related peptides.

Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.

Delayed satiety-like actions and altered feeding microstructure by a selective type 2 corticotropin-releasing factor agonist in rats: intra-hypothalamic urocortin 3 administration reduces food intake by prolonging the post-meal interval.

Brain corticotropin-releasing factor/urocortin (CRF/Ucn) systems are hypothesized to control feeding, with central administration of 'type 2' urocortins producing delayed anorexia. The present study sought to identify the receptor subtype, brain site, and behavioral mode of action through which Ucn 3 reduces nocturnal food intake in rats. Non-food-deprived male Wistar rats (n=176) were administered Ucn 3 into the lateral (LV) or fourth ventricle, or into the ventromedial or paraventricular nuclei of the hypothalamus (VMN, PVN) or the medial amygdala (MeA), regions in which Ucn 3 is expressed in proximity to CRF(2) receptors. LV Ucn 3 suppressed ingestion during the third-fourth post-injection hours. LV Ucn 3 anorexia was reversed by cotreatment with astressin(2)-B, a selective CRF(2) antagonist and not observed following equimole subcutaneous or fourth ventricle administration. Bilateral intra-VMN and intra-PVN infusion, more potently than LV infusion, reduced the quantity (57-73%) and duration of ingestion (32-68%) during the third-fourth post-infusion hours. LV, intra-PVN and intra-VMN infusion of Ucn 3 slowed the eating rate and reduced intake by prolonging the post-meal interval. Intra-VMN Ucn 3 reduced feeding bout size, and intra-PVN Ucn 3 reduced the regularity of eating from pellet to pellet. Ucn 3 effects were behaviorally specific, because minimal effective anorectic Ucn 3 doses did not alter drinking rate or promote a conditioned taste aversion, and site-specific, because intra-MeA Ucn 3 produced a nibbling pattern of more, but smaller meals without altering total intake. The results implicate the VMN and PVN of the hypothalamus as sites for Ucn 3-CRF(2) control of food intake.

Synthesis of protected norcysteines for SPPS compatible with Fmoc-strategy.

We report the synthesis of racemic Alloc-Ncy(Tmob)-OH, the resolution of its methyl ester, and demonstrate its application to form a norcystine bridge in octreotide-amide using the Fmoc-strategy on solid phase. N-Alloc and S-Tmob protections of norcysteine (Ncy) were found to be a preferred choice for Fmoc-strategy over three other protected norcysteines synthesized i.e. Fmoc-Ncy(tBu)-OH, Alloc-Ncy(tBu)-OH and Alloc-Ncy(Trt)-OH.

Prospective Clinical Applications of CRF Peptide Antagonists.

It is noteworthy that thirty three years of efforts in corticotropin releasing factor (CRF) research by academia and the pharmaceutical industry resulted in several thousand papers and patents, yet little progress has been made to identify and market diagnostic or therapeutic CRF peptides and small molecule ligands. We document the potential relevance of CRF peptide antagonists to reinvigorate stress/anxiety affected "anatomy systems" in order to boost their efficacy.

Distribution of corticotropin-releasing factor (CRF) receptor binding in the mouse brain using a new, high-affinity radioligand, [125 I]-PD-Sauvagine.

The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.

Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists.

UNLABELLED: The uptake of radiolabeled somatostatin analogs by tumor cells through receptor-mediated internalization is a critical process for the in vivo targeting of tumoral somatostatin receptors. In the present study, the somatostatin receptor internalization induced by a variety of somatostatin analogs was measured with new immunocytochemical methods that allow characterization of trafficking of the somatostatin receptor subtype 2 (sst2), somatostatin receptor subtype 3 (sst3), and somatostatin receptor subtype 5 (sst5) in vitro at the protein level. METHODS: Human embryonic kidney 293 (HEK293) cells expressing the sst2, sst3, or the sst5 were used in a morphologic immunocytochemical internalization assay using specific sst2, sst3 and sst5 antibodies to qualitatively and quantitatively determine the capability of somatostatin agonists or antagonists to induce somatostatin receptor internalization. In addition, the internalization properties of a selection of these agonists have been compared and quantified in sst2-expressing CHO-K1 cells using an ELISA. RESULTS: Agonists with a high sst2-binding affinity were able to induce sst2 internalization in the HEK293 and CHO-K1 cell lines. New sst2 agonists, such as Y-DOTA-TATE, Y-DOTA-NOC, Lu-DOTA-BOC-ATE (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TATE is [Tyr3, Thr8]-octreotide; NOC is [1-NaI3]-octreotide; and BOC-ATE is [BzThi3, Thr8]-octreotide), iodinated sugar-containing octreotide analogs, or BIM-23244 were considerably more potent in internalizing sst2 than was DTPA-octreotide (where DTPA is diethylenetriaminepentaacetic acid). Similarly, compounds with high sst3 affinity such as KE108 were able to induce sst3 internalization. In sst2- or sst3-expressing cell lines, agonist-induced receptor internalization was efficiently abolished by sst2- or sst3-selective antagonists, respectively. Antagonists alone had no effect on sst2 or sst3 internalization. We also showed that somatostatin-28 and somatostatin-14 can induce sst5 internalization. Unexpectedly, however, potent sst5 agonists such as KE108, BIM-23244, and L-817,818 were not able to induce sst5 internalization under the same conditions. CONCLUSION: Using sensitive and reproducible immunocytochemical methods, the ability of various somatostatin analogs to induce sst2, sst3, and sst5 internalization has been qualitatively and quantitatively determined. Whereas all agonists triggered sst2 and sst3 internalization, sst5 internalization was induced by natural somatostatin peptides but not by synthetic high-affinity sst5 agonists. Such assays will be of considerable help for the future characterization of ligands foreseen for nuclear medicine applications.