Subtype-selective Analogs Research Articles

Galanin analogs prevent mortality from seizure-induced respiratory arrest in mice.

ObjectiveSudden Unexpected Death in Epilepsy (SUDEP) accounts for 20% of mortality in those with recurrent seizures. While risk factors, monitoring systems, and standard practices are in place, the pathophysiology of SUDEP is still not well understood. Better knowledge of SUDEP and its potential mechanisms of action is crucial to reducing risk in this patient population and developing potential treatment options. Clinical studies and animal models of SUDEP suggest that diminished post-ictal respiratory control may be the dominant mechanism contributing to mortality. Recently, it was demonstrated that the depletion of the neuropeptide galanin in the amygdala occurs in human SUDEP. The amygdala plays a key role in the central integration of respiratory signaling; the depletion of galanin may represent a critical change that predisposes individuals to SUDEP.Materials and methodsTo evaluate the impact of enhancing galaninergic signaling to potentially protect against SUDEP, we studied seizure-induced respiratory arrest (S-IRA) following central (intracerebroventricular, intra-amygdala) and systemic (intraperitoneal, subcutaneous) administration of galanin analogs. Seizure naïve and seizure experienced (fully kindled) mice were tested.ResultsCentral and systemically administered galanin analogs protect against S-IRA in naïve C57Bl/6J mice. Differential efficacy between receptor subtype-selective analogs varied based on the route of administration. Sub-chronic systemic administration at doses that reduced 6 Hz seizures also protected against S-IRA. Acute treatment benefits also extended to fully kindled mice experiencing tonic extension.SignificanceThese data demonstrate that galanin analogs may be protective against post-ictal respiratory collapse.

Neuropeptide Y Receptors in Primary Human Brain Tumors

Peptide receptors are often overexpressed in tumors, and they may be targeted in vivo. We evaluated neuropeptide Y (NPY) receptor expression in 131 primary human brain tumors, including gliomas, embryonal tumors, meningiomas, and pituitary adenomas, by in vitro receptor autoradiography using the 125I-labeled NPY receptor ligand peptide YY in competition with NPY receptor subtype-selective analogs. Receptor functionality was investigated in selected cases using [35S]GTPgammaS-binding autoradiography. World Health Organization Grade IV glioblastomas showed a remarkably high expression of the NPY receptor subtype Y2 with respect to both incidence (83%) and density (mean, 4,886 dpm/mg tissue); astrocytomas World Health Organization Grades I to III and oligodendrogliomas also exhibited high Y2 incidences but low Y2 densities. In glioblastomas, Y2 agonists specifically stimulated [35S]GTPgammaS binding, suggesting that tumoral Y2 receptors were functional. Furthermore, nonneoplastic nerve fibers containing NPY peptide were identified in glioblastomas by immunohistochemistry. Medulloblastomas, primitive neuroectodermal tumors of the CNS, and meningiomas expressed Y1 and Y2 receptor subtypes in moderate incidence and density. In conclusion, Y2 receptors in glioblastomas that are activated by NPY originating from intratumoral nerve fibers might mediate functional effects on the tumor cells. Moreover, identification of the high expression of NPY receptors in high-grade gliomas and embryonal brain tumors provides the basis for in vivo targeting.

Relevance of the Neuropeptide Y System in the Biology of Cancer Progression

The peptides pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY) share a similar structure, known as PP-fold. Within this family of peptides, NPY, a highly conserved 36-aminoacid residue peptide, is involved in the regulation of a wide range of physiological functions, such as food intake and energy metabolism, as well as in the promotion of some remarkable aspects of tumor progression, including cell proliferation, matrix invasion, metastatization, and angiogenesis. NPY exerts its biological effects through five G-protein coupled receptors, named Y1-, Y2-, Y4-, Y5-, and y6-R, which appear associated with different aspects of oncogenesis. Y1-R seems involved in the modulation of cancer cell proliferation, whereas Y2-R activation appears to promote angiogenesis. The development of NPY receptor subtype selective analogs has helped to elucidate the physiological and pathophysiological role and localization of each receptor and may contribute to a better understanding of the receptor-ligand interaction. The NPY system appears to be variously associated with specific tumors, including neural crest-derived tumors, breast and prostate cancers. In addition to NPY, PYY is also able to affect cancer cell growth in a dose-dependent manner and through Y-Rs. In conclusion, peptides of the NPY family and the related receptors play an important role in the progression of different cancer types, with some molecular specificity according to each step of this process. On this basis, future studies may be directed to the implementation of novel diagnostic and therapeutic approaches targeting this system.

Novel chimeric somatostatin analogs: facts and perspectives

Dopamine and somatostatin receptor agonists inhibit hormone secretion by normal pituitary cells and pituitary adenomas. Indeed, initially several dopaminergic drugs, and lately somatostatin analogs, have been developed for the treatment of pituitary adenomas. Recently, it has been demonstrated that subtypes of somatostatin and dopamine receptors may form homo- and hetero-dimers at the membrane level, as part of their normal trafficking and function. Interestingly, a specific ligand for a given receptor may influence the activity of an apparently unrelated receptor, and the association between the two different receptors could be induced by addition of either dopamine or somatostatin. The new properties of these families of G-protein coupled receptors (GPCRs) offer a potential explanation for the apparent conflicting results observed both in vivo and in vitro in human cell systems treated with the presently available analogs. Moreover, this observation not only increases the possibilities of modulating the activities of these receptors, but also raises new questions on the role of associations of specific receptors in the control of cell functions. In fact, results from preclinical studies have shown that receptor activation may not only trigger different intracellular signaling pathways, but also induce a distinct response depending upon the specific cell type. Recently, a number of new interesting compounds (subtype selective analogs and antagonists, as well as bi-specific and hybrid somatostatin/dopamine compounds) have been developed. The effects of these new molecules have been explored in few animal and human cell lines and primary cultures from human tumors, revealing a heterogeneous, but broader, profile of activities. Further studies are certainly needed to fully elucidate the complex interplay between the GPCRs and consequent biological effects, to identify suitable therapies for controlling hormonal secretion of pituitary tumors. However, these recent observations form the basis for the application of new interesting strategies for the treatment of not only pituitary tumors but also other human malignancies.

Neuropeptide Y receptors in renal cell carcinomas and nephroblastomas

Numerous peptide receptors are overexpressed in human cancer, permitting in vivo tumor targeting. Among such receptors, those for the neurotransmitter neuropeptide Y (NPY) are overexpressed in various tumors. Since NPY can play a role in the kidney, NPY receptor expression and/or endogenous production of peptides of the NPY family (NPY, PYY, PP) were evaluated in 40 renal cell carcinomas (RCCs) and 18 nephroblastomas. NPY receptor protein expression was investigated by in vitro autoradiography using (125)I-labeled PYY in competition with NPY receptor subtype-selective analogs. NPY, PYY and PP production was assessed immunohistochemically. Fifty-six percent of RCCs expressed the Y1 receptor subtype in moderate density, and 80% of nephroblastomas expressed Y1 and Y2 subtypes in moderate to high density. Y1 was also highly expressed in intratumoral blood vessels. In selected cases, NPY was observed in nerve fibers in close association with intratumoral blood vessels and in the vicinity of tumor cells, while no PYY or PP was detected immunohistochemically in these sites. NPY receptors on renal tumor cells and tumor blood vessels may therefore be the molecular targets of endogenous NPY released by intratumoral nerve fibers. With regard to clinical applications, NPY receptors may act as in vivo targets for receptor-directed therapy of RCCs and nephroblastomas for which alternative therapeutic approaches are still required.

Somatostatin Receptor 1 Selective Analogues: 4. Three-Dimensional Consensus Structure by NMR

The three-dimensional NMR structures of six analogues of somatostatin (SRIF) are described. These analogues with the amino acid 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 exhibit potent and highly selective binding to human SRIF subtype 1 receptors (sst(1)). The conformations reveal that the backbones of these analogues have a hairpin-like structure similar to the sst(2)-subtype-selective analogues. This structure serves as a scaffold for retaining a unique arrangement of the side chains of d-Trp(8), IAmp(9), Phe(7), and Phe(11) or m-I-Tyr(11) (m-I-Tyr = mono-iodo-tyrosine). The conformational preferences and results from biological analyses of these analogues(1,2) allow a detailed study of the structure-activity relationship of SRIF. The proposed consensus pharmacophore of the sst(1)-selective analogues requires a unique set of distances between an indole/2-naphthyl ring, an IAmp side chain, and two aromatic rings. This motif is necessary and sufficient to explain the binding affinities of all of the analogues studied and is distinct from the existing models suggested for sst(4) as well as sst(2)/sst(5) selectivity.

High Expression of Neuropeptide Y Receptors in Tumors of the Human Adrenal Gland and Extra-Adrenal Paraganglia

Recently, a role of neuropeptide Y (NPY) in tumor biology was suggested based on the high density of NPY receptors in breast and ovarian cancers. The high frequency of NPY receptors in steroid hormone-producing ovarian sex cord-stromal tumors, together with the known influence of NPY on steroid hormone and catecholamine secretion in the rodent adrenal gland, led to the investigation of NPY receptor expression in the human adrenal gland and related tumors. Fifteen adrenal cortical tumors, 20 paragangliomas, 23 pheochromocytomas, 20 neuroblastomas, and 8 normal adrenal glands were investigated by in vitro NPY receptor autoradiography using 125I-labeled peptide YY in competition experiments with receptor subtype selective analogs. Ninety three percent of cortical tumors express Y1, 35% of pheochromocytomas and 61% of paragangliomas express Y1 and Y2, and 90% of neuroblastomas express Y2 receptors. The NPY receptors in pheochromocytomas, paragangliomas, and neuroblastomas are often expressed concomitantly with the NPY hormone detected immunohistochemically. The adrenal cortex strongly expresses Y1, whereas no NPY receptors are found in the adrenal medulla. These receptor data suggest a role of NPY in adrenal cortical tumors and, together with the strong NPY innervation of the cortex, a physiologic role in the adrenal gland, mediated by Y1 receptors. These NPY receptors are a potential new molecular target for the therapy of malignant tumors.

Neuropeptide Y receptor expression in human primary ovarian neoplasms

Peptide hormone receptors overexpressed in human malignant neoplasms are potential targets for diagnostic scintigraphy and radiotherapy. One such receptor is the neuropeptide Y (NPY) receptor, mediating primarily feeding behavior in the brain but shown recently to play a role in breast cancer. In this study, the presence of NPY receptors was evaluated in another group of gynecological tumors, namely ovarian tumors, using in vitro receptor autoradiography with 125I-labeled peptide YY and receptor subtype selective analogs. Remarkably, all 10 investigated inhibin-expressing granulosa cell tumors, Leydig cell tumors, and Sertoli–Leydig cell tumors expressed NPY receptors. In contrast, receptors were found in only seven of 22 ovarian adenocarcinomas (32%). Pharmacological characterization of the expressed NPY receptor subtypes in the various tumors revealed the presence of Y1, Y2, or both. In addition, Y1 receptors were observed in intra- and peritumoral blood vessels as well. NPY receptors were not expressed in three ovarian adenomas, three borderline tumors, four fibromas and fibrothecomas, and one dysgerminoma. This is the first time that NPY receptors are described in human ovarian tissue. They may play a role in the pathogenesis and also in the pathophysiology of ovarian malignancies. Moreover, the high incidence and density of NPY receptors in sex cord-stromal tumors suggest that these receptors represent a new potential target for the diagnostic and therapeutic administration of NPY analogs in these tumors.

Novel sst(4)-selective somatostatin (SRIF) agonists. 4. Three-dimensional consensus structure by NMR.

The three-dimensional NMR structures of eight cyclic octapeptide analogues of somatostatin (SRIF) are described. These analogues, with the basic sequence H-c[Cys(3)-Phe(6)-Xxx(7)-Yyy(8)-Lys(9)-Thr(10)-Zzz(11)-Cys(14)]-OH (the numbering refers to the position in native SRIF), with Xxx(7) being Phe/Ala/Tyr, Yyy(8) being Trp/DTrp/D-threo-beta-Me2Nal/L-threo-beta-Me2Nal, and Zzz(11) being Phe/Ala, exhibit potent and highly selective binding to human SRIF type 4 (sst(4)) receptors. The conformations reveal that the backbones of these analogues do not have the usual type-II' beta-turn reported in the literature for sst(2)-subtype-selective analogues. Instead, the structures contain a unique arrangement of side chains of Yyy(8), Lys(9), and Phe(6) or Phe(11). The conformational preferences and results from biological analyses of these analogues (parts 1-3 of this series, Rivier et al., Erchegyi et al., and Erchegyi et al., J. Med. Chem. 2003, preceding papers in this issue) allow a detailed study of the structure-activity relationship of SRIF. The proposed consensus structural motif at the binding pocket for the sst(4)-selective analogues requires a unique set of distances between an indole/2-naphthyl ring, a lysine side chain, and another aromatic ring. This motif is necessary and sufficient to explain the binding affinities of all of the analogues studied and is distinct from the existing model suggested for sst(2)/sst(5) selectivity.

Neuropeptide Y receptor expression in human primary ovarian neoplasms

Peptide hormone receptors overexpressed in human malignant neoplasms are potential targets for diagnostic scintigraphy and radiotherapy. One such receptor is the neuropeptide Y (NPY) receptor, mediating primarily feeding behavior in the brain but shown recently to play a role in breast cancer. In this study, the presence of NPY receptors was evaluated in another group of gynecological tumors, namely ovarian tumors, using in vitro receptor autoradiography with 125I-labeled peptide YY and receptor subtype selective analogs. Remarkably, all 10 investigated inhibin-expressing granulosa cell tumors, Leydig cell tumors, and Sertoli–Leydig cell tumors expressed NPY receptors. In contrast, receptors were found in only seven of 22 ovarian adenocarcinomas (32%). Pharmacological characterization of the expressed NPY receptor subtypes in the various tumors revealed the presence of Y1, Y2, or both. In addition, Y1 receptors were observed in intra- and peritumoral blood vessels as well. NPY receptors were not expressed in three ovarian adenomas, three borderline tumors, four fibromas and fibrothecomas, and one dysgerminoma. This is the first time that NPY receptors are described in human ovarian tissue. They may play a role in the pathogenesis and also in the pathophysiology of ovarian malignancies. Moreover, the high incidence and density of NPY receptors in sex cord-stromal tumors suggest that these receptors represent a new potential target for the diagnostic and therapeutic administration of NPY analogs in these tumors.

Distribution of CCK1 and CCK2 receptors in normal and diseased human pancreatic tissue

Background & Aims: The localization and functional role of cholecystokinin (CCK) receptor proteins in normal and diseased human pancreas, particularly in ductal pancreatic carcinomas, remain unclear. Methods: Tissue samples of normal human pancreas, chronic pancreatitis, and ductal pancreatic carcinomas were investigated under carefully controlled conditions for expression of CCK1 and CCK2 receptor messenger RNA (mRNA) and proteins using in situ hybridization and in vitro CCK receptor autoradiography by means of subtype-selective analogues. Synaptophysin immunohistochemistry was used concomitantly for optimal identification of islets, nerves, and tumor areas with neuroendocrine features. Results: CCK2 receptor mRNA and proteins were found abundantly in human pancreatic islets in normal pancreas and chronic pancreatitis. CCK1 receptor proteins were found occasionally in small-sized pancreatic nerves, whereas acini expressed a low density of CCK2 receptors in a few cases of chronic pancreatitis. Ductal pancreatic carcinomas rarely expressed CCK receptors; a few receptor-positive tumors, often characterized by neuroendocrine differentiation, expressed the CCK2 receptor at the mRNA or protein level. However, the main source of CCK receptors in the pancreatic tumor samples consisted of CCK2-expressing islets and/or CCK1-expressing nerves rather than neoplastic tissue. Conclusions: These data indicate that the presence of CCK receptors in human ductal pancreatic tumor samples is mainly due to CCK2 expression in residual pancreatic islets and CCK1 in pancreatic nerves. Pancreatic acini and ductal pancreatic tumor cells very rarely express CCK2 receptors. These observations suggest that CCK analogues may not be of clinical use to target most of these cancers.

Somatostatin receptors in pituitary and development of somatostatin receptor subtype-selective analogs.

Somatostatin receptor (SSTR) subtypes 1, 2, and 5 are expressed in the normal human pituitary. SSTR2 and SSTR5 are expressed in almost all growth hormone (GH) cell adenomas, and prolactin (PRL)-secreting tumors express SSTR5 more than SSTR2. SSTR4 is not detected in all pituitary adenoma subtypes, and SSTR1 and SSTR3 are expressed in about 50% of tumors. Human GH is regulated through ligand binding to both SSTR2 and SSTR5, but octreotide and lanreotide, the two clinically available somatostatin analogs, bind to human SSTR2 much better than to SSTR5. Novel SSTR2- and SSTR5- selective analogs with improved binding affinity for these receptor subtypes are highly potent in suppressing GH release from cultures of human fetal pituitaries or GH-cell adenomas. Only SSTR5-selective analogs suppress in vitro PRL secretion from cultured prolactinomas. A new SSTR2+5 bispecific analog with high affinity and selectivity for both SSTR2 and SSTR5, and a somatostatin analog with a unique broad receptor (SSTR1, 2, 3, and 5) binding profile, are both able to inhibit in vitro GH release in GH cell adenomas partially sensitive to octreotide. Recently, a somatostatindopamine hybrid molecule was introduced with potentially functional synergy on GH and PRL release. Using the expanding knowledge on SSTRs and their ligand activation, the development of novel pharmacologic concepts may open new opportunities for effective manipulation of this complex intracellular signaling system. These concepts may achieve better control of pituitary hormone hypersecretion, pituitary size, as well as antitumor effects in patients with SSTR-expressing tumors.

Somatostatin receptors in nasopharyngeal carcinoma.

Somatostatin receptors (SS-Rs) are expressed in neuroendocrine tumour tissues where they can be targetted for diagnosis and therapy. This study investigated the presence of SS-Rs in nasopharyngeal carcinoma (NPC), a common cancer in South-East Asia. Nasopharynx biopsy specimens were obtained from 12 NPC patients and 5 patients without tumours. Somatostatin receptor autoradiography was performed using (125)I-labelled [Tyr(3)]-octreotide and (125)I-labelled [Leu(8), DTrp(22), Tyr(25)]-somatostatin-28 as radioligands. Of the 12 NPC samples 9 showed moderate to high expression of SS-Rs. These were of the sst(2) type, based on the rank order of potency of subtype-selective analogues. The 5 non-neoplastic samples, consisting primarily of granulomatous tissue, did not express measurable amounts of SS-Rs. This study demonstrates for the first time the presence of type 2 SS-R in NPC. These receptors may play a role in the management of NPC, as is the case for other somatostatin-expressing tumours.

Immunohistochemical Detection of Somatostatin Receptors in Human Ovarian Tumors

Objective. It is well established that many human tumors can express somatostatin receptors. This is the molecular basis for the application of long-acting somatostatin analogues for therapeutic and diagnostic purposes. However, there are discrepancies in earlier studies regarding the prevalence of somatostatin receptor expression in human ovarian tumors detected using autoradiographic binding studies or reverse transcription-polymerase chain reaction.Methods. We have previously developed a panel of somatostatin receptor subtype-specific antibodies that effectively stain formalin-fixed, paraffin-embedded human tumor tissue. In the present study, we have used these antibodies to determine the somatostatin receptor status of 47 randomly selected human ovarian tumors.Results. Somatostatin receptor-like immunoreactivity was in most cases located at the plasma membrane as well as in the cytoplasm of the tumor cells and was completely blocked with antigenic peptide. The pattern of expression of somatostatin receptor subtypes varied greatly between individual tumors. Of the 47 ovarian tumor specimens analyzed, 9 tumors (∼19%) were positive for sst1, 13 (∼28%) revealed immunoreactive sst2A receptors, 20 (∼42%) showed sst3-like immunoreactivity, 8 (∼17%) revealed immunoreactive sst4 receptors, and 10 (∼21%) were positive for sst5. Whereas 18 tumors (∼38%) revealed no somatostatin receptor immunoreactive staining, 14 (∼30%) expressed more than one somatostatin receptor subtype. The expression of somatostatin receptor subtypes was independent of patient age, diagnosis, and histological grade.Conclusion. We provide direct evidence of somatostatin receptor protein expression in human ovarian tumors. Our findings suggest that a subgroup of receptor-positive ovarian tumors may be a potential target for treatment with somatostatin receptor subtype-selective analogs. However, due to the wide variations in the somatostatin receptor status of similar cases, each tumor specimen must be tested individually for the presence of particular somatostatin receptor subtypes.

Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands.

Somatostatin receptors are known to be expressed in a large number of human tumours and represent the basis for in vivo tumour targeting. Stable somatostatin derivatives such as octreotide or lanreotide are the most frequently used radiopharmaceuticals acting through specific binding to somatostatin receptors; however, they do not bind with high affinity to all five receptor subtypes. Whereas the mRNAs for most receptor subtypes have been detected in tumours, it is in most cases unclear which of the receptor subtype proteins are expressed. Since in vitro receptor binding methods are close correlates and predictors of in vivo peptide receptor targeting, we took advantage of the recently developed subtype-selective analogues and evaluated approximately 200 tumours for their receptor subtype protein expression in specific binding assays using autoradiography with 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 and displacement by subtype-selective analogues. The majority of the tested neuroblastomas, meningiomas, medulloblastomas, breast carcinomas, lymphomas, renal cell carcinomas, paragangliomas, small cell lung carcinomas and hepatocellular carcinomas predominantly expressed sst2. The prostate carcinomas and sarcomas preferentially expressed sstl, while a majority of inactive pituitary adenomas displayed sst3 and, to a lesser extent, sst2. Growth hormone-secreting pituitary adenomas preferentially expressed sst2 and sst5; gastroenteropancreatic tumours and phaeochromocytomas frequently displayed sst2 and/or sstl. Non-neoplastic human tissues such as vessels, nerve plexus, pancreatic islets, prostatic stroma, adrenal medulla, spleen and germinal centres of the lymphoid tissues preferentially expressed sst2. However, the human gastric mucosa predominantly expressed sst1 while colonic mucosa displayed sst2. Interestingly, a minority of tumours showed a strong 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 binding, of which less than 50% could be displaced by the sum of the five subtype-selective analogues. This observation suggests the existence of an as yet unknown subtype in selected tumours. This study is the first report to analyse the somatostatin receptor subtype expression in tumours with binding methods. We conclude that sst2, with high affinity for current radiopharmaceuticals such as Octreoscan, is predominantly expressed in a majority of tumours. Fewer tumour types (sarcomas, prostate cancers, inactive pituitary adenomas) preferentially express another subtype. This information is of importance with regard to the clinical applications and development of somatostatin analogues with distinct receptor subtype selectivities.

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism.

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.

Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms.

This study demonstrates the involvement of the MT2 (Mel1b) melatonin receptor in mediating phase advances of circadian activity rhythms by melatonin. In situ hybridization histochemistry with digoxigenin-labeled oligonucleotide probes revealed for the first time the expression of mt1 and MT2 melatonin receptor mRNA within the suprachiasmatic nucleus of the C3H/HeN mouse. Melatonin (0.9 to 30 microg/mouse, s.c.) administration during 3 days at the end of the subjective day (CT 10) to C3H/HeN mice kept in constant dark phase advanced circadian rhythms of wheel running activity in a dose-dependent manner [EC50=0.72 microg/mouse; 0.98+/-0.08 h (n=15) maximal advance at 9 microg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P-ADOT and 4P-PDOT (90 microg/mouse, s.c.) nor luzindole (300 microg/mouse, s.c.), which shows 25-fold higher affinity for the MT2 than the mt1 subtype, affected the phase of circadian activity rhythms when given alone at CT 10. All three antagonists, however, shifted to the right the dose-response curve to melatonin, as they significantly reduced the phase shifting effects of 0.9 and 3 microg melatonin. This is the first study to demonstrate that melatonin phase advances circadian rhythms by activation of a membrane-bound melatonin receptor and strongly suggests that this effect is mediated through the MT2 melatonin receptor subtype within the circadian timing system. We conclude that the MT2 melatonin receptor subtype is a novel therapeutic target for the development of subtype-selective analogs for the treatment of circadian sleep and mood-related disorders.

Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors.

Previously, we have shown somatostatin receptor (SSTR) subtype-specific regulation of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL) secretion in human fetal pituitary cultures, where GH and thyroid-stimulating hormone are mediated by both SSTR2 and SSTR5, whereas SSTR2 preferentially mediates PRL secretion. We now tested SSTR subtype-selective analogues in primary human GH- and PRL-secreting pituitary adenoma cultures. Analogue affinities determined by membrane radioligand binding in cells stably expressing human SSTR forms were either SSTR2 or SSTR5-selective. Analogues preferential either for SSTR2, including octreotide, lanreotide, and novel compounds with improved affinity for SSTR2, or new SSTR5-selective compounds suppressed GH in tumor cell cultures (up to 44% of control; P < 0.0005). However, novel analogues from both groups were 30-40% more potent than octreotide and lanreotide in suppressing GH (P < 0.05). Heterologous analogue combinations containing both SSTR2- and SSTR5-selective compounds were more potent in decreasing GH than analogues used alone (P < 0.05), or than combinations of compounds specific for the same receptor subtype (P < 0.005). In contrast, SSTR2-selective analogues did not suppress PRL release from six cultured prolactinomas studied. However, new SSTR5-selective analogues suppressed in vitro PRL secretion (30-40%; P < 0.05) in four of six prolactinomas. These results suggest that both SSTR2 and SSTR5 are involved in GH regulation in somatotroph adenoma cells, whereas SSTR5 exclusively regulates PRL secretion from prolactinoma cells. Thus, somatostatin analogues with improved selective binding affinity for these receptor subtypes may be effective in the treatment of either GH- or PRL-secreting adenomas.

Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor.

We have identified subtype selective agonists, partial agonists and antagonists, which distinguish the human recombinant Mel1a and Mel1b melatonin receptors expressed in COS-7 cells. Melatonin receptor agonists showed higher affinity for competition of 2-[125I]-iodomelatonin binding for the Mel1b than the Mel1a melatonin receptor. The dissociation constants (Ki) of 16 agonists determined on the recombinant human Mel1a and Mel1b melatonin receptor subtypes showed a significant correlation (r2 = 0.85, slope = 0.97, P < 0.0001, n = 16). However, six agonists showed 10 to 60 fold higher affinity for the Mel1b melatonin receptor as indicated by the affinity selectivity ratios (Mel1a/Mel1b) [8-methoxy-2-acetamidotetraline (11); S20098 (14); 8-methoxy-2-propionamidotetraline (20); 6, 7 di-chloro-2-methylmelatonin (21); 6-chloromelatonin (57); 6-methoxymelatonin (59)]. Dissociation constants for competition of 11 partial agonists and antagonist for 2-[125I]-iodomelatonin binding were between 15.5 (luzindole, pKi: 7.7) to 362 (4-phenyl-2-chloroacetamidotetraline, pKi: 9.1) fold higher for the Mel1b than for the Mel1a melatonin receptor. The lack of correlation between the pKi values (r2 = 0.23, P > 0.1, n = 11) strongly suggest that the two human melatonin receptor subtypes can be distinguished pharmacologically. The partial agonist: 5-methoxyluzindole (pKi: 9.6) and the competitive melatonin receptor antagonists: GR128107 (pKi: 9.6), 4-phenyl-2-chloroacetamidotetraline (pKi: 9.1), 4-phenyl-2-acetamidotetraline (pKi: 8.9) and 4-phenyl-2-propionamidotetraline (pKi: 8.8) are selective Mel1b melatonin receptor analogues as their affinity selectivity ratios (Mel1a/Mel1b) are bigger than 100. We conclude that the 40% overall amino acid difference in the sequence of the human recombinant Mel1a and Mel1b melatonin receptors is reflected in distinct pharmacological profiles for the subtypes. We compared the pharmacological profile of the presynaptic ML1 melatonin heteroreceptor of rabbit retina mediating inhibition of the calcium-dependent release of dopamine to that of the recombinant Mel1a and Mel1b melatonin receptors. Melatonin inhibited [3H]dopamine release by 50% (1C50) at 20 pM with a maximal inhibitory effect (80%) at 1 nM. The partial agonists, i.e., N-acetyltryptamine (1C50 5.6, maximal inhibition 55%) and 5-methoxyluzindole (1C50: 1.3, maximal inhibition 40%) showed various degrees of efficacy while none of the competitive melatonin receptor antagonists did inhibit [3H]dopamine release on their own. The potency (1C50) of full melatonin receptor agonists significantly correlated with their affinity to compete for 2-[125I]-iodomelatonin binding to either the Mel1a (r2 = 0.76, slope = 0.77, P < 0.0001, n = 17) or Mel1b (r2 = 0.63, slope = 0.75, P < 0.001, n = 17) human melatonin receptors. By contrast, the apparent dissociation constants (KB) for partial agonists and antagonists to antagonize the inhibition of [3H]dopamine release mediated by activation of the ML1 heteroreceptor by melatonin, significantly correlated with the affinity constants (Ki) for 2-[125I]-iodomelatonin binding determined of the Mel1b (r2 = 0.77, slope = 0.55, P < 0.001; n = 11) but not the Mel1a (r2 = 0.27, P < 0.1, n = 11) subtype. Together these results demonstrate that the pharmacological profile of the human recombinant Mel1b melatonin receptor is similar to that of the functional presynaptic melatonin heteroreceptor of rabbit retina, which we referred as an ML1B subtype. We conclude that the selective Mel1b melatonin partial agonists and antagonists described here can be used to identify melatonin receptor subtypes in native tissues and to search for subtype selective analogues with therapeutic potential.

Fluorescent Labelled Analogues of Neuropeptide Y for the Characterization of Cells Expressing NPY Receptor Subtypes

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, and subtype selective analogues have been synthesized by solid phase peptide synthesis. The peptides were labelled with Cy3TM, a commercially available fluorescent marker based on a cyanine dye, by solid phase strategy. During the cleavage α partial fragmentation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled analogues of NPY showed high affinity to the NPY receptor subtypes Y1 and Y2. Thus, Cy3-NPY. Y1-selective Cy3-[Pro34] NPY and Y2 selective Cy3-[Ahx5–24] NPY were used to label SK-N-MC- and SMS-KAN-cells, which are stably expressing the Y1-(SK-N-MC) and the Y2-receptor subtype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe27] NPY, which showed high affinity to both receptor subtypes was labelled with Cy3 in solution. Whereas the fluorescent labelling of the cells with analogues without photoactivatable amino acid was reversible, successful photocrosslinking could be investigated by the irreversible staining of the cells using Cy3-[(Tmd)Phe27] NPY. These subtype selective analogues are exciting tools to trace receptors in tissues and to identify the pharmacologically characterized subtypes without radioactivity.