Tuberoinfundibular peptide of 39 residues- immunoreactive fibers in the zona incerta and the supraoptic decussations terminate in the neuroendocrine hypothalamus.

Tuberoinfundibular peptide of 39 residues (TIP39) is expressed by neurons in the subparafascicular area, the posterior intralaminar complex of the thalamus and the pontine medial paralemniscal nucleus. TIP39-positive fibers from these areas do not form individual bundles or fascicles, they join other pathways to reach their innervated brain areas. Fibers arise from TIP39 perikarya located in the subparafascicular area and the posterior intralaminar complex of the thalamus could be followed to the hypothalamus. After uni- and bilateral posterolateral surgical deafferentations of the hypothalamus, accumulation of TIP39 immunoreactivity was observed in the fibers caudal to the knife cut, while it disappeared completely rostral to the transection. In serial sections of the forebrain, we could follow TIP39-ir fibers coursing within the zona incerta and the supraoptic decussations. TIP39-positive fibers that join the incerto-hypothalamic pathway reach the medio-dorsal part of the hypothalamus and form moderate to high density networks in the dorsomedial and paraventricular nuclei. The other set of TIP39-positive axons from the subthalamic area join the fibers of the supraoptic decussations and run in an antero-medial direction through the most ventral portion of the hypothalamus up to the retrochiasmatic area, where they crossover. A certain portion of these TIP39-positive fibers terminates in the territories of the arcuate and the medial preoptic nuclei, as well as in the retrochiasmatic area.

Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes.

Acetylcholine receptors (AChRs) are packed in the postsynaptic membrane at neuromuscular junctions at a density of approximately 20,000/micron 2, whereas the density a few micrometers away is less than 20/micron 2. To understand how this remarkable distribution comes about during nerve-muscle synapse formation, we have attempted to isolate factors from neural tissue that can promote the accumulation of AChRs and/or alter their distribution. In this paper we report the purification of a polypeptide from chick brains that can increase the rate of insertion of AChR into membranes of cultured chick myotubes at a concentration of less than 0.5 ng/ml. Based on SDS PAGE and the action of neuraminidase, the acetylcholine receptor-inducing activity (ARIA) appears to be a 42,000-D glycoprotein. ARIA was extracted in a trifluoroacetic acid-containing cocktail and purified to homogeneity by reverse-phase, ion exchange, and size exclusion high pressure liquid chromatography. Dose response curves indicate that the activity has been purified 60,000-fold compared with the starting acid extract and approximately 1,500,000-fold compared with a saline extract prepared from the same batch of brains. Although the ARIA was purified on the basis of its ability to increase receptor incorporation, we found that it increased the number and size of receptor clusters as well. It is not yet clear if the two effects are independent. The 42-kD ARIA is extremely stable: it was not destroyed by exposure to intact myotubes, low pH, organic solvents, or SDS. Its action appears to be selective in that the increase in the rate of receptor insertion was not accompanied by an increase in the rate of protein synthesis. Moreover, there was no change in cellular, surface membrane, or secreted acetylcholinesterase. The effect of ARIA is apparently independent of the state of activity of the target myotubes as its effect on receptor incorporation added to that of maximal concentrations of tetrodotoxin.

Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide produced by K cells of the mammalian proximal small intestine and is a potent stimulant of insulin release in the presence of hyperglycemia. However, its relative physiological importance as a postprandial insulinotropic agent is unknown. Using LGIPR2 cells stably transfected with rat GIP receptor cDNA, GIP (1-42) stimulation of cyclic adenosine monophosphate (cAMP) production was inhibited in a concentration-dependent manner by GIP (7-30)-NH2. Competition binding assays using stably transfected L293 cells demonstrated an IC50 for GIP receptor binding of 7 nmol/liter for GIP (1-42) and 200 nmol/liter for GIP (7-30)-NH2, whereas glucagonlike peptide-1 (GLP-1) binding to its receptor on ++betaTC3 cells was minimally displaced by GIP (7-30)-NH2. In fasted anesthetized rats, GIP (1-42) stimulated insulin release in a concentration-dependent manner, an effect abolished by the concomitant intraperitoneal administration of GIP (7-30)-NH2 (100 nmol/ kg). In contrast, glucose-, GLP-1-, and arginine-stimulated insulin release were not affected by GIP (7-30)-NH2. In separate experiments, GIP (7-30)-NH2 (100 nmol/kg) reduced postprandial insulin release in conscious rats by 72%. It is concluded that GIP (7-30)-NH2 is a GIP-specific receptor antagonist and that GIP plays a dominant role in mediating postprandial insulin release.

Molecular biology of the vesicular ACh transporter.

The cholinergic synapse has long been a model for biochemical studies of neurotransmission. The molecules that are responsible for synaptic transmission are being identified rapidly. The vesicular transporter for ACh, which is responsible for the concentration of ACh within synaptic vesicles, has been characterized recently, both at the molecular and functional level. Definitive identification of the cloned gene involved genetics of Caenorhabditis elegans, the specialized Torpedo electromotor system, and expression in mammalian tissue culture. Comparison of the vesicular transporter for ACh with the vesicular transporters for monoamines demonstrates a new gene family. Gene mapping has demonstrated a unique relationship between the genes for the vesicular ACh transporter and for choline acetyltransferase.

Afferent connections of the subparafascicular area in rat.

The subparafascicular nucleus and the subparafascicular area are the major sites of synthesis of the recently discovered neuropeptide, tuberoinfundibular peptide of 39 residues (TIP39). Better knowledge of the neuronal inputs to the subparafascicular area and nucleus will facilitate investigation of the functions of TIP39. Thus, we have injected the retrograde tracer cholera toxin B subunit into the rostral, middle, and caudal parts of the rat subparafascicular nucleus. We report that the afferent projections to the subparafascicular nucleus and area include the medial prefrontal, insular, and ectorhinal cortex, the subiculum, the lateral septum, the anterior amygdaloid area, the medial amygdaloid nucleus, the caudal paralaminar area of the thalamus, the lateral preoptic area, the anterior, ventromedial, and posterior hypothalamic nuclei, the dorsal premamillary nucleus, the zona incerta and Forel's fields, the periaqueductal gray, the deep layers of the superior colliculus, cortical layers of the inferior colliculus, the cuneiform nucleus, the medial paralemniscal nucleus, and the parabrachial nuclei. Most of these regions project to all parts of the subparafascicular nucleus. However, the magnocellular subparafascicular neurons, which occupy the middle part of the subparafascicular nucleus, may not receive projections from the medial prefrontal and insular cortex, the medial amygdaloid nucleus, the lateral preoptic area, and the parabrachial nuclei. In addition, double labeling of cholera toxin B subunit and TIP39 revealed a remarkable similarity between input regions of the subparafascicular area and the brain TIP39 system. Neurons within regions that contain TIP39 cell bodies as well as regions that contain TIP39 fibers project to the subparafascicular area. Overall, the afferent connections of the subparafascicular nucleus and area suggest its involvement in central reproductive, visceral, nociceptive, and auditory regulation.

Forebrain projections of tuberoinfundibular peptide of 39 residues (TIP39)-containing subparafascicular neurons.

Neurons containing tuberoinfundibular peptide of 39 residues (TIP39) constitute a rostro-caudally elongated group of cells in the posterior thalamus. These neurons are located in the rostral part of the subparafascicular nucleus and in the subparafascicular area, caudally. Projections of the caudally located TIP39 neurons have been previously identified by their disappearance following lesions. We have now mapped the projections of the rat rostral subparafascicular neurons using injections of the anterograde tracer biotinylated dextran amine and the retrograde tracer cholera toxin B subunit, and confirmed the projections from more caudal areas previously inferred from lesion studies. Neurons from both the rostral subparafascicular nucleus and the subparafascicular area project to the medial prefrontal, insular, ecto- and perirhinal cortex, nucleus of the diagonal band, septum, central and basomedial amygdaloid nuclei, fundus striati, basal forebrain, midline and intralaminar thalamic nuclei, hypothalamus, subthalamus and the periaqueductal gray. The subparafascicular area projects more densely to the amygdala and the hypothalamus. In contrast, only the rostral part of the subparafascicular nucleus projects significantly to the superficial layers of prefrontal, insular, ectorhinal and somatosensory cortical areas. Double labeling showed that anterogradely labeled fibers from the rostral part of the subparafascicular nucleus contain TIP39 in many forebrain areas, but do not in hypothalamic areas. Injections of the retrograde tracer cholera toxin B subunit into the lateral septum and the fundus striati confirmed that they were indeed target regions of both the rostral subparafascicular nucleus and the subparafascicular area. In contrast, TIP39 neurons did not project to the anterior hypothalamic nucleus. Our data provide an anatomical basis for the potential involvement of rostral subparafascicular neurons in limbic and autonomic regulation, with TIP39 cells being major subparafascicular output neurons projecting to forebrain regions.

Multiple regions of ligand discrimination revealed by analysis of chimeric parathyroid hormone 2 (PTH2) and PTH/PTH-related peptide (PTHrP) receptors.

PTH and PTH-related peptide (PTHrP) bind to the PTH/PTHrP receptor and stimulate cAMP accumulation with similar efficacy. Only PTH activates the PTH2 receptor. To examine the structural basis for this selectivity, we analyzed receptor chimeras in which the amino terminus and third extracellular domains of the two receptors were interchanged. All chimeric receptors bound radiolabeled PTH with high affinity. Transfer of the PTH2 receptor amino terminus to the PTH/PTHrP receptor eliminated high-affinity PTHrP binding and significantly decreased activation by PTHrP. A PTH/PTHrP receptor N terminus modified by deletion of the nonhomologous E2 domain transferred weak PTHrP interaction to the PTH2 receptor. Introduction of the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor increased the EC50 for PTH and PTHrP, while preserving high-affinity PTH binding and eliminating high-affinity PTHrP binding. Similarly, transfer of the PTH/PTHrP receptor third extracellular loop preserved high-affinity PTH binding by the PTH2 receptor but decreased its activation. Return of Gln440 and Arg394, corresponding residues in the PTH/PTHrP and PTH2 receptor third extracellular loops, to the parent residue restored function of these receptors. Simultaneous interchange of wild-type amino termini and third extracellular loops eliminated agonist activation but not binding for both receptors. Function was restored by elimination of the E2 domain in the receptor with a PTH/PTHrP receptor N terminus and return of Gln440/Arg394 to the parent sequence in both receptors. These data suggest that the amino terminus and third extracellular loop of the PTH2 and PTH/PTHrP receptors interact similarly with PTH, and that both domains contribute to differential interaction with PTHrP.

Evidence for a parathyroid hormone-2 receptor selective ligand in the hypothalamus.

The PTH2 receptor is expressed in several brain nuclei but we have been unable to detect mRNA encoding PTH, which is the only known ligand for the PTH2 receptor, in the brain. We now have evidence for a PTH2 receptor selective ligand in an acid-acetone extract made from bovine hypothalamus. The partially purified extract activates the PTH2 receptor more effectively than it activates the PTH/PTHrP receptor, while PTH activates these two receptors at similar concentration. The activity appears immunologically distinct from PTH and its effect is potently antagonized by [D-Trp12]bPTH(7-34). These data provide evidence for a biologically active peptide, which may be related to PTH, and which is a potential new neurotransmitter or hormone.

Two receptors for vasoactive intestinal polypeptide with similar specificity and complementary distributions.

Vasoactive intestinal polypeptide (VIP) has a variety of physiological effects. Pharmacological evidence suggesting that VIP acts via multiple receptors has been confirmed by the cloning of two VIP receptors (VIP1 and VIP2) with very different amino acid sequences. At both the VIP1 and the VIP2 receptor VIP, PHI, PACAP38, and PACAP27 have similar potency to each other. Only the VIP1 receptor is activated by secretin. The messenger RNAs (mRNAs) for the two receptors have completely different distributions as mapped by in situ hybridization histochemistry. VIP1 receptor mRNA is predominantly found in the lung, small intestine, thymus, and within the brain in the cerebral cortex and hippocampus. VIP2 receptor mRNA is present in a number of areas where VIP acts but VIP1 receptor mRNA is not present, including the stomach and testes. In the CNS VIP2 receptor mRNA is exclusively present in areas associated with neuroendocrine function, including several hypothalamic nuclei. In the periphery, it is also present in the pituitary and in pancreatic islets.

Comparison of rat and human parathyroid hormone 2 (PTH2) receptor activation: PTH is a low potency partial agonist at the rat PTH2 receptor.

The human PTH2 receptor, expressed in tissue culture cells, is selectively activated by PTH. Detailed investigation of its anatomical and cellular distribution has been performed in the rat. It is expressed by neurons in a number of brain nuclei; by endocrine cells that include pancreatic islet somatostatin cells, thyroid parafollicular cells, and peptide secreting cells in the gastrointestinal tract; and by cells in the vasculature and heart. The physiological role of the PTH2 receptor expressed by these cells remains to be determined. All pharmacological studies performed to date have used the human receptor. We have now isolated a complementary DNA including the entire coding sequence of the rat PTH2 receptor and compared its pharmacological profile with that of the human PTH2 receptor when each is expressed in COS-7 cells. PTH-based peptides, including rat PTH(1-84), rat PTH(1-34), and human PTH(1-34), have low potency at the rat PTH2 receptor for stimulation of adenylyl cyclase (EC50 = 19-140 nM). When compared with the effect of a bovine hypothalamic extract, PTH-based peptides are partial agonists at the rat PTH2 receptor. This suggests that PTH is unlikely to be a physiologically important endogenous ligand for the PTH2 receptor. A peptide homologous to an activity detected in a bovine hypothalamic extract is a good candidate for the endogenous PTH2 receptor ligand.

Distribution of parathyroid hormone-2 receptor messenger ribonucleic acid in rat.

The PTH2 receptor is a recently identified G protein-coupled receptor activated by PTH. Its amino acid sequence is most similar to the PTH/PTHrP receptor, but unlike the PTH/PTHrP receptor, it is activated by PTH and not by PTH-related peptide. We previously demonstrated using Northern blots that expression of PTH2 receptor messenger RNA was greatest within the brain and occurred at lower levels in pancreas, testis, and placenta. We have now obtained a complementary DNA encoding the rat PTH2 receptor and used it to study the distribution of the PTH2 receptor using in situ hybridization histochemistry. PTH2 receptor messenger RNA is abundantly expressed in arterial and cardiac endothelium and at lower levels in vascular smooth muscle. It is also abundant in the lung, both within bronchi and in the parenchyma, and is present within the exocrine pancreas. It is expressed by sperm in the head of the epididymis. A small number of cells associated with the vascular pole of renal glomeruli express the receptor. These data suggest that the PTH2 receptor may be responsible for PTH effects in a number of physiological systems.

Evaluating the ligand specificity of zebrafish parathyroid hormone (PTH) receptors: comparison of PTH, PTH-related protein, and tuberoinfundibular peptide of 39 residues.

Homologs of mammalian PTH1 and PTH2 receptors, and a novel PTH3 receptor have been identified in zebrafish (zPTH1, zPTH2, and zPTH3). zPTH1 receptor ligand specificity is similar to that of mammalian PTH1 receptors. The zPTH2 receptor is selective for PTH over PTH-related protein (PTHrP); however, PTH produces only modest cAMP accumulation. A PTH2 receptor-selective peptide, tuberoinfundibular peptide of 39 residues (TIP39), has recently been purified from bovine hypothalamus. The effect of TIP39 has not previously been examined on zebrafish receptors. The zPTH3 receptor was initially described as PTHrP selective based on comparison with the effects of human PTH. We have now examined the ligand specificity of the zebrafish PTH-recognizing receptors expressed in COS-7 cells using a wide range of ligands. TIP39 is a potent agonist for stimulation of cAMP accumulation at two putative splice variants of the zPTH2 receptor (EC50, 2.6 and 5.2 nM); in comparison, PTH is a partial agonist [maximal effect (Emax) of PTH peptides ranges from 28-49% of the TIP39 Emax]. As TIP39 is much more efficacious than any known PTH-like peptide, a homolog of TIP39 may be the zPTH2 receptor's endogenous ligand. At the zPTH3 receptor, rat PTH-(1-34) and rat PTH-(1-84) (EC50, 0.22 and 0.45 nM) are more potent than PTHrP (EC50, 1.5 nM), and rPTH-(1-34) binds with high affinity (3.2 nM). PTH has not been isolated from fish. PTHrP-like peptides, which have been identified in fish, may be the natural ligands for zPTH1 and zPTH3 receptors.

Distribution of the parathyroid hormone 2 receptor in rat: immunolocalization reveals expression by several endocrine cells.

The PTH2 receptor is a G protein-coupled receptor selectively activated by PTH. We are studying the receptors distribution to guide the investigation of its physiological function. We have now generated an antibody from a C-terminal peptide sequence of the PTH2 receptor and used this to study its cellular distribution. Labeling with the antibody identified a number of endocrine cells expressing the PTH2 receptor, including thyroid parafollicular cells, pancreatic islet D cells, and some gastrointestinal peptide synthesizing cells. There was complete overlap of PTH2 receptor labeling with somatostatin in pancreatic islets, and partial overlap with somatostatin in thyroid parafollicular cells and in the gastrointestinal tract. Furthermore, observations made previously by in situ hybridization histochemistry, including expression throughout the cardiovascular system, as well as by discrete populations of cells within the gastrointestinal tract and reproductive system were confirmed. These data suggest a broad role for the PTH2 receptor, especially within the endocrine system, and provide a basis for experimental exploration of its physiology.

Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain.

Gastric inhibitory polypeptide (GIP), or glucose-dependent insulinotropic peptide, is released from endocrine cells in the small intestine after meals. It is involved in several facets of the anabolic response and is thought to be particularly important in stimulating insulin secretion. We have cloned, functionally expressed, and mapped the distribution of the receptor for GIP. It is a member of the secretin-vasoactive intestinal polypeptide family of G-protein-coupled receptors. When expressed in tissue culture cells, it stimulates cAMP production (EC50 0.3 nM) and also increases intracellular calcium accumulation. GIP receptor mRNA is present in the pancreas as well as the gut, adipose tissue, heart, pituitary, and inner layers of the adrenal cortex, whereas it is not found in kidney, spleen, or liver. It is also expressed in several brain regions, including the cerebral cortex, hippocampus, and olfactory bulb. These results suggest that GIP may have previously undescribed actions. GIP receptor localization in the adrenal cortex suggests that it may have effects on glucocorticoid metabolism. Neither GIP nor its effects have been described in the central nervous system, and mRNA for the known peptide ligand for the receptor cannot be detected in the brain by in situ hybridization or polymerase chain reaction. This suggests that a novel peptide may be present in the brain.

The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.

We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.

Pseudohypoparathyroidism 1b: exclusion of parathyroid hormone and its receptors as candidate disease genes.

Pseudohypoparathyroidism 1b (PHP 1b) is characterized by specific resistance of target tissues to PTH, but no mutations in the PTH/PTH-related peptide (PTHrP) receptor gene have been identified. To investigate the basis for defective PTH signaling, we used polymorphic markers in or near the genes encoding PTH and its receptors to perform linkage analysis between these loci and PHP 1b. Two multiplex PHP 1b families (families M and K) were informative for an intragenic polymorphism in exon 13 of the PTH/PTHrP receptor gene detected by PCR amplification and resolved by denaturing gradient gel electrophoresis. Linkage analysis revealed discordance of the PTH/PTHrP receptor with PHP1b. One PHP 1b kindred (family M) was informative for a intragenic polymorphism in exon 3 of the PTH gene detected by PCR amplification and resolved by denaturing gradient gel electrophoresis. The PTH gene polymorphism segregation was discordant with PHP 1b. Probands from each family had normal PTH genes by direct sequence analysis. In three PHP 1b kindreds, we analyzed simple sequence polymorphisms in three microsatellite markers flanking the PTH type 2 receptor locus located at 2q33. Linkage analysis demonstrated no linkage. In conclusion, neither the PTH gene nor the PTH receptor genes (type 1 and 2) are linked to PHP 1b.

Food-dependent Cushing's syndrome: characterization and functional role of gastric inhibitory polypeptide receptor in the adrenals of three patients.

In the present work, the presence of gastric inhibitory polypeptide (GIP) receptors and their functional role in the adrenal cells of three patients with food-dependent Cushing's syndrome were studied. RT-PCR and in situ hybridization studies demonstrated the presence of GIP receptor in the adrenals of the three patients. The presence of this receptor was also demonstrated in two human fetal adrenals, but not in two normal adult human adrenals or in the adrenals of one patient with nonfood-dependent Cushing's syndrome. Freshly isolated cells from patient adrenals responded in a dose-dependent manner to the steroidogenic action of both ACTH and GIP, whereas cells from normal adrenals responded only to ACTH. Treatment of cultured normal adrenal cells with ACTH, but not with GIP, increased the messenger ribonucleic acid (mRNA) levels of cholesterol side-chain cleavage cytochrome P-450, P450c17, and 3beta-hydroxysteroid dehydrogenase, whereas both hormones enhanced these mRNAs in patients' adrenal cells, although the effects of ACTH were greater than those of GIP. Moreover, pretreatment with ACTH enhanced the steroidogenic responsiveness of both normal and patient adrenal cells, whereas GIP caused homologous desensitization, and this was associated with a marked reduction of GIP receptor mRNA levels, as demonstrated by RT-PCR and in situ hybridization. Finally, both ACTH and GIP inhibited DNA synthesis in one patient's adrenal cells, whereas in normal adrenal cells only ACTH had this effect. In conclusion, the present data demonstrate that ectopic expression of functional GIP receptors is the main cause of food-dependent Cushing's syndrome.

Food-dependent Cushing's syndrome resulting from abundant expression of gastric inhibitory polypeptide receptors in adrenal adenoma cells.

We studied a 45-yr-old woman with food-dependent Cushing's syndrome. Plasma cortisol levels were subnormal (4-47 nmol/L) after an overnight fast and increased after a mixed meal to values between 500-1000 nmol/L. There was a close correlation between circulating gastric inhibitory polypeptide (GIP) and cortisol levels during normal food intake (r = 0.92; P < 0.0002). Plasma corticotropin (ACTH) levels were undetectable. Nonfasting plasma cortisol levels were not suppressed by low or high doses of dexamethasone. Plasma ACTH and cortisol levels did not increase after human CRH administration, but fasting plasma cortisol levels increased after ACTH treatment. The infusion of GIP increased plasma cortisol levels to 7.8 times above baseline. Radiological and cholesterol uptake studies pointed to a unilateral adrenal adenoma. Treatment with octreotide initially prevented the meal-induced increases in cortisol and GIP levels and decreased urinary cortisol excretion. Unilateral adrenalectomy was performed. Cortisol production by cultured adrenal adenoma cells from the patient was stimulated by GIP and ACTH. In situ hybridization studies using a GIP receptor probe showed an abundant expression of GIP receptor messenger ribonucleic acid in the adrenocortical adenoma. We conclude that food-dependent Cushing's syndrome results from the expression of GIP receptors on adrenocortical adenoma cells.

Cloning and expression of the vesamicol binding protein from the marine ray Torpedo. Homology with the putative vesicular acetylcholine transporter UNC-17 from Caenorhabditis elegans.

Complementary DNA clones corresponding to a messenger RNA encoding a 56 kDa polypeptide have been obtained from Torpedo marmorata and Torpedo ocellata electric lobe libraries, by homology screening with a probe obtained from the putative acetylcholine transporter from the nematode Caenorhabditis elegans. The Torpedo proteins display approximately 50% overall identity to the C. elegans unc-17 protein and 43% identity to the two vesicle monoamine transporters (VMAT1 and VMAT2). This family of proteins is highly conserved within 12 domains which potentially span the vesicle membrane, with little similarity within the putative intraluminal glycosylated loop and at the N- and C-termini. The approximately 3.0 kb mRNA species is specifically expressed in the brain and highly enriched in the electric lobe of Torpedo. The Torpedo protein, expressed in CV-1 fibroblast cells, possesses a high-affinity binding site for vesamicol (Kd = 6 nM), a drug which blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles.

Molecular mechanisms of ligand recognition by parathyroid hormone 1 (PTH1) and PTH2 receptors.

The mammalian parathyroid hormone (PTH) / PTH receptor family includes PTH1 and PTH2 receptors and three related ligands (PTH, PTH-related protein (PTHrP) an d tuberoinfundibular peptide of 39 residues (TIP39)). Here we comparatively and systematically review the pharmacological properties of PTH receptors and ligands, structure of the ligands, and molecular mechanisms of receptor-ligand interaction. The PTH1 receptor is activated by PTH and PTHrP but not by TIP39. The PTH2 receptor is activated by TIP39 but not by PTHrP. PTH strongly activates the human PTH2 receptor but is a weak partial agonist for rat and zebrafish PTH2 receptors. Receptor-G-protein interaction increases the receptor binding selectivity of PTHrP and TIP39. Despite different primary structures, the secondary structures of PTH, PTHrP and TIP39 are quite similar. Each ligand contains an N-terminal and a C-terminal alpha-helix in secondary structure-inducing conditions. Receptor-bound ligand structure is less well-characterized. The orientation of receptor-ligand interaction is highly similar for PTH and PTHrP binding to the PTH1 receptor and TIP39 interaction with the PTH2 receptor. Ligands bind according to a 'two-site' mechanism, in which the C-terminal portion of the ligand binds the extracellular N-terminal domain of the receptor (N-interaction), and the N-terminal ligand portion binds to the juxtamembrane receptor domain (J-interaction). The N-interaction provides most of the PTH1-receptor binding energy for PTH and PTHrP but provides less energy for PTH2 receptor-TIP39 interaction. The J-interaction stimulates G-protein activation. For the PTH-PTH1 receptor interaction, the efficacy-generating component of the J-interaction is independent of the N-domain of the receptor and C-terminal portion of the ligand. This finding suggests that it might be possible to design low molecular-weight PTH1 receptor agonists, which could be bone anabolic agents and used for the treatment of osteoporosis.