Secretin depolarizes nucleus tractus solitarius neurons through activation of a nonselective cationic conductance

The recent suggestion that secretin may be useful in treating autism and schizophrenia has begun to focus attention on the mechanisms underlying this gut-brain peptide's actions in the central nervous system (CNS). In vitro autoradiographic localization of (125)I-secretin binding sites in rat brain shows the highest binding density in the nucleus tractus solitarius (NTS). Recent evidence suggests that intravenous infusion of secretin causes fos activation in NTS, a relay station playing important roles in the central regulation of autonomic functions. In this study, whole cell patch-clamp recordings were obtained from 127 NTS neurons in rat medullary slices. The mean resting membrane potential of these neurons was -54.7 +/- 0.3 mV, the mean input resistance was 3.7 +/- 0.2 GOmega, and the action potential amplitude of these neurons was always >70 mV. Current-clamp studies showed that bath application of secretin depolarized the majority (80.8%; 42/52) of NTS neurons tested, whereas the remaining cells were either unaffected (17.3%; 9/52) or hyperpolarized (1.9%; 1/52). These depolarizing effects were maintained in the presence of 5 microM TTX and found to be concentration dependent from 10(-12) to 10(-7) M. Using voltage-clamp techniques, we also identified modulatory actions of secretin on specific ion channels. Our results demonstrate that while secretin is without effect on net whole cell potassium currents, it activates a nonselective cationic conductance (NSCC). These results show that NTS neurons are activated by secretin as a consequence of activation of a NSCC and support the emerging view that secretin can act as a neuropeptide within the CNS.     

Signaling mechanisms of secretin receptor

Secretin, a 27-amino acid gastrointestinal peptide, was initially discovered based on its activities in stimulating pancreatic juice. In the past 20 years, secretin was demonstrated to exhibit pleiotropic functions in many different tissues and more importantly, its role as a neuropeptide was substantiated. To carry out its activities in the central nervous system and in peripheral organs, secretin interacts specifically with one known receptor. Secretin receptor, a member of guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) in the secretin/VIP/glucagon subfamily, possesses the characteristics of GPCR with seven conserved transmembrane domains, a relatively large amino-terminal extracellular domain and an intracellular carboxyl terminus. The structural features and signal transduction pathways of the secretin receptor in various tissues are reviewed in this article.     

Secretin and autism: a basic morphological study about the distribution of secretin in the nervous system

For the first time, the relationship between secretin and autism has been demonstrated by one of us. Intravenous administration of secretin in autistic children caused a fivefold higher pancreaticobiliary fluid secretion than in healthy ones and, at least in some of the patients, better mental functions were reported after the secretin test. Because the precise localization of secretin in the brain is still not completely known, the abovementioned observation led us to map secretin immunoreactivity in the nervous system of several mammalian species. In the present work, the distribution of secretin immunoreactivity in cat and human nervous systems was compared with that of rats using an immunohistochemical approach. Secretin immunoreactivity was observed in the following brain structures of both humans and in colchicine-treated rats: (1) Purkinje cells in the cerebellar cortex; (2) central cerebellar nuclei; (3) pyramidal cells in the motor cortex; and (4) primary sensory neurons. Additionally, secretin immnoreactive cells were observed in the human hippocampus and amygdala and in third-order sensory neurons of the rat auditory system. In cats, secretin was only observed in the spinal ganglia. Our findings support the view that secretin is not only a gastrointestinal peptide but that it is also a neuropeptide. Its presence or the lack of its presence may have a role in the development of behavioral disorders.     

Secretogranin II and its Derivative Peptide,Manserin, are Differentially Localized in Purkinje Cells and Unipolar Brush Cells in the Rat Cerebellum

The cerebellum has long been recognized as the primary center of motor coordination in the central nervous system. Cerebellar neuropeptides have been postulated to be involved in such motor coordination, though this role is not fully understood. We herein investigated the localization of novel neuropeptide, “manserin” in the adult rat cerebellum. Punctate signals of manserin immunoreactivity were observed in the granular layer of the rat cerebellum. Manserin signals were also observed in the fibers and fiber terminals in the granular layer as well as the molecular layer. Manserin did not localize in Purkinje cells. Interestingly, cerebellar manserin was preferentially colocalized with unipolar brush cells, a class of excitatory granular layer interneuron, which are known to be involved in vestibullocerebellar functions. These results indicate that manserin plays pivotal roles in the cerebellar functions.     

Origin of secretin receptor precedes the advent of tetrapoda: evidence on the separated origins of secretin and orexin

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage.     

Interactions of human secretin with sterically stabilized phospholipid micelles amplify peptide-induced vasodilation in vivo

Secretin, a 27-amino acid neuropeptide, is a member of the glucagon/secretin/vasoactive intestinal polypeptide (VIP) superfamily of amphipathic peptides that elicits transient vasodilation in vivo. The purpose of this study was to determine whether association of human secretin with sterically stabilized phospholipid micelles (SSM) amplifies the vasorelaxant effects of the peptide in the peripheral microcirculation in vivo. We found that secretin in saline evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch microcirculation (P < 0.05). This response was potentiated and prolonged significantly when secretin was associated with SSM (P < 0.05). Vasodilation evoked by secretin in saline and secretin in SSM was abrogated by VIP(10-28), a VIP receptor antagonist, but not by PACAP(6-38), a PACAP receptor antagonist, or Hoe140, a selective bradykinin B(2) receptor antagonist. Collectively, these data indicate that self-association of human secretin with SSM significantly amplifies peptide vasoreactivity in the intact peripheral microcirculation through activation of VIP receptors. We suggest that the vasoactive effects of human secretin in vivo are, in part, phospholipid-dependent.     

What may be the anatomical basis that secretin can improve the mental functions in autism?

Autism was first described and characterized as a behavioral disorder more than 50 years ago. The major abnormality in the central nervous system is a cerebellar atrophy. The characteristic histological sign is a striking loss or abnormal development in the Purkinje cell count. Abnormalities were also found in the limbic system, in the parietal and frontal cortex, and in the brain stem. The relation between secretin and autism was observed 3 years ago. Clinical observations by Horváth et al. [J. Assoc. Acad. Minor. Physicians 9 (1998) 9] supposed a defect in the role of secretin and its receptors in autism. The aim of the present work was to study the precise localization of secretin immunoreactivity in the nervous system using an immunohistochemical approach. No secretin immunoreactivity was observed in the forebrain structures. In the brain stem, secretin immunoreactivity was observed in the mesencephalic nucleus of the trigeminal nerve, in the superior olivary nucleus, and in scattered cells of the reticular formation. The most intensive secretin immunoreactivity was observed in the Purkinje cells of the whole cerebellum and in some of the neurons of the central cerebellar nuclei. Secretin immunoreactivity was also observed in a subpopulation of neurons in the primary sensory ganglia. This work is the first immunohistochemical demonstration of secretin-immunoreactive elements in the brain stem and in primary sensory ganglia.     

In vitro autoradiographic localization of (125)i-secretin receptor binding sites in rat brain

Although the existence of the receptor for secretin in the brain was suggested, the localization of secretin receptor and the neuronal function of secretin have not been clarified yet. In the present study, the localization of secretin receptor was investigated in the rat brain by using an in vitro autoradiography technique. Frozen section autoradiography with (125)I-secretin showed intense binding in the nucleus of solitary tract, laterodorsal thalamic nucleus, and accumbens nucleus; moderate binding in the hippocampus, caudate/putamen, cerebellum, cingulate and orbital cortices. Scatchard plot analysis gave the Kd value of 125 pM with Bmax of 134 fmol/mg tissue in the hippocampus. The binding specificity was confirmed with secretin and its analogs, VIP, PACAP, and glucagon. These results indicate the secretin receptor system might have some neural functions in the brain, which could give the basis for therapeutic use of secretin in autistic children.     

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.     

Secretin modulation of behavioral and physiological functions in the rat

The effect of secretin on behavioral and physiological functions in the rat was investigated. Secretin injected intracerebroventricularly (ICV) significantly increased defecation and decreased novel-object approaches in rats. The peptide showed no significant effects on stereotypic behavior (gnawing, grooming and rearing), open-field locomotor activity however was significantly decreased, an effect that was probably due to a decreased propensity for the rats to initiate locomotor responses. In addition, secretin showed significant effects on respiration rate in anesthetized rats. When the peptide was injected in the lateral ventricle a decrease in respiration rate occurred, but when the brain was perfused from the lateral ventricle to the cisterna magna increases in respiration rate occurred. These data, combined with the facts that secretin and secretin receptors have been identified in the brain indicate that secretin may play a neurotransmitter or neuroregulator role in the central nervous system.     

Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe(6), Tyr(10), Leu(13), and Met(17) found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water-micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.     

Evidence for the existence of a new receptor for CGRP,which is not CRLR

Receptors for calcitonin gene-related peptide (CGRP), a neuropeptide known to be the most potent vasodilator, are abundantly expressed in cerebellum. A monoclonal antibody to cerebellar CGRP receptors specifically detects a 66 kDa protein from rat cerebellum and other rat and human tissues, but not from SK-N-MC cells which express calcitonin receptor-like receptor (CRLR), a recently described component of CGRP receptors. In contrast, mRNA expression for CRLR was abundant in SK-N-MC cells, but it was undetectable in rat cerebellum. Furthermore, the antibody could not detect any immunoreactive protein in HEK 293 cells transiently transfected with CRLR and receptor activity-modifying protein 1 (RAMP(1)) indicating the possible existence of another CGRP receptor, which does not involve CRLR. Due to the absence of biochemical or structural data on the existence of a CGRP(2) receptor and the new data provided in this paper, we suggest to identify the two CGRP receptors as CGRP-A and CGRP-B.     

Secretin: Hypothalamic Distribution and Hypothesized Neuroregulatory Role in Autism

1. This study aims (1) to determine whether secretin is synthesized centrally, specifically by the HPA axis and (2) to discuss, on the basis of the findings in this and previous studies, secretin's possible neuroregulatory role in autism. 2. An immunocytochemical technique with single-cell resolution was performed in 12 age/weight-matched male rats pretreated with stereotaxic microinjection of colchicine (0.6 microg/kg) or vehicle into the lateral ventricle. Following 2-day survival, rats were anesthetized and perfused for immunocytochemistry. Brain segments were blocked and alternate frozen 30-microm sections incubated in rabbit antibodies against secretin, vasoactive intestinal peptide, glucagon, or pituitary-adenylate-cyclase-activating peptide. Adjacent sections were processed for Nissl stain. Preadsorption studies were performed with members of the secretin peptide family to demonstrate primary antibody specificity. 3. Specificity of secretin immunoreactivity (ir) was verified by clear-cut preadsorption control data and relatively high concentrations and distinct topographic localization of secretin ir to paraventricular/supraoptic and intercalated hypothalamic nuclei. Secretin levels were upregulated by colchicine, an exemplar of homeostatic stressors, as compared with low constitutive expression in untreated rats. 4. This study provides the first direct immunocytochemical demonstration of secretinergic immunoreactivity in the forebrain and offers evidence that the hypothalamus, like the gut, is capable of synthesizing secretin. Secretin's dual expression by gut and brain secretin cells, as well as its overlapping central distribution with other stress-adaptation neurohormones, especially oxytocin, indicates that it is stress-sensitive. A neuroregulatory relationship between the peripheral and central stress response systems is suggested, as is a dual role for secretin in conditioning both of those stress-adaptation systems. Colchicine-induced upregulation of secretin indicates that secretin may be synthesized on demand in response to stress, a possible mechanism of action that may underlie secretin's role in autism.     

Distribution of Neuropeptide Y-Immunoreactive Neurons in the Human Brainstem, Cerebellum, and Cortex During Development

1. Neuropeptide Y is found throughout the central nervous system where it appears to play a wide range of often poorly understood functions. In this study, the distribution of neuropeptide Y immunoreactive (NPY-ir) neurons in the brainstem, cerebellum, and cerebral cortex of human fetuses ranging in age from 11 gestational weeks to term was investigated by immunohistochemistry. 2. The NPY-ir cells were detected in the dorsal and ventral rostral midbrain and the interpeduncular nucleus by 21 weeks and 32 weeks of gestation, respectively. Although no positive cells were found in the pons, the NPY-ir fibers were detected there at 32 gestational weeks. 3. The vagal, hypoglossal, and olivary nuclei of the medulla oblongata contained immunoreactive cells by week 21 and the medullary reticular formation by week 25 of gestation. In most of these locations, both the number and size of neuropeptide Y positive cells were greater at birth and reached maximal values of 100-400 cells per 1 mm2 and 2-5 microm in diameter, respectively. 4. In the cerebellum, numerous NPY-ir horizontal and granule cells, as well as the cells within the dentate nucleus were observed as early as 21 weeks of gestation. 5. The NPY-ir cells were also detected in the developing cerebral cortex, with the earliest activity observed within the temporal cortex at 14 weeks of gestation. By week 21, positive cells appeared in the visual, frontal, sensory, and motor cortices. Most of these cells were bipolar or multipolar in morphology but their numbers at birth were relatively low. 6. Our results show a wide distribution of the NPY-ir cells in the developing human brain and offer supporting evidence for the important modulatory role of NPY in both the fetus and adult.     

Distribution of neuropeptide Y-like immunoreactivity in the brain of Salmo salar and Gambusia affinis.

Through the immunohistochemical PAP technique, the distribution of immune positive neurons and fibres for an antibody anti-NPY in the encephalon of salmon fixed in Bouin have been located and studied. NPY-positive neurons are found forming three important nuclei: in the ventrolateral telencephalon; in the tegmentum mesencephali; and in the locus coeruleus. Neurons in the optic tectum, in the thalamic region and a few in the preoptic recess have also been located. The fibres were found throughout the brain, with the exception of the cerebellum, presenting a greater density in three regions: in the dorsal telencephalon; in the mesencephalon; and in the visceral lobes in the rhombencephalon. With the aim of proving if this distribution is found in other groups of teleosts, we processed, with the same technique, the advanced teleost Gambusia affinis, in order to compare it with the primitive teleost Salmo salar. The results show that in both fish this neuropeptide has the same pattern of distribution. The results also suggest that in fish this neuropeptide can be involved in several functions of the central nervous system, as has been demonstrated for mammals. The innervation of the visceral lobes and also the presence of NPY-fibres in the posterior hypothalamus are anatomical supports of the studies which suggest that NPY is related to the control of the food intake.     

Secretin controls anion secretion in the rat epididymis in an autocrine/paracrine fashion

There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.