Peptides (v.25, #3)

IFC (IFC).

Twenty-five years of peptides by Abba J Kastin (315-317).

Corticotropin releasing hormone receptors: two decades later by Greti Aguilera; Maria Nikodemova; Peter C Wynn; Kevin J Catt (319-329).
Hypothalamic corticotropin releasing hormone (CRH) regulates pituitary ACTH secretion and mediates behavioral and autonomic responses to stress, through interaction with type 1 plasma membrane receptors (CRHR1) located in pituitary corticotrophs and the brain. Although CRHR1 are essential for ACTH responses to stress, their number in the pituitary gland does not correlate with corticotroph responsiveness, suggesting that activation of a small number of receptors is sufficient for maximum ACTH production. CRH binding and hybridization studies in adrenalectomized, glucocorticoid-treated or stressed rats revealed divergent changes in CRH receptors and CRHR1 mRNA in the pituitary, with a reduction in receptor binding but normal or elevated expression of CRHR1 mRNA levels. Western blot analysis of CRHR1 protein in pituitary membranes from adrenalectomized rats showed unchanged receptor mRNA levels and increased CRHR1 protein, despite the binding down-regulation, suggesting that decreased binding is due to homologous desensitization, rather than reduced receptor synthesis. In contrast, decreased CRH binding following glucocorticoid administration is associated with a reduction in CRHR1 protein, suggesting inhibition of CRHR1 mRNA translation. The regulation of CRHR1 translation may involve binding of cytosolic proteins, and a minicistron in the 5′-UTR of the CRHR1 mRNA. It is likely that post-transcriptional regulatory mechanisms that permit rapid changes in CRH receptor activity are important for adaptation of corticotroph responsiveness to continuous changes in physiological demands.
Keywords: Pituitary corticotroph; Corticotropin releasing hormone (CRH); Type 1 CRH receptor (CRHR1); CRHR1 mRNA; CRHR1 regulation; Translation;

The many lives of leptin by William A Banks (331-338).
Leptin is a 16,000-Da protein which is secreted by fat but acts within the brain to regulate adiposity. Our Peptides Classic addressed the mystery of how such a large molecule could negotiate the blood–brain barrier (BBB), a structure which normally excludes proteins from the brain. We found that leptin was transported across the BBB by a saturable transport system. This finding was important to understanding how satiety-related peptides and proteins worked, but it was also important to the concept that the BBB is a regulatory interface important in brain–body communication. Obesity in humans and many animals is associated with a leptin resistant state rather than a leptin deficiency. Subsequent work has shown that a defect in the BBB transport of leptin is key in producing and reinforcing this state of resistance. Leptin is pluripotent and the concept of it being primarily an adipostat is being discarded for more encompassing views. Consideration of the BBB data would favor the view that ancestral levels of leptin were much lower than those currently considered normal and are consistent with leptin acting as a metabolic switch, informing the brain when fat reserves are adequate to direct energy expenditures towards activities other than seeking calories.
Keywords: Leptin; Protein; Adipostat; Blood-brain barrier; Obesity; Anorexia;

A 25 year adventure in the field of tachykinins by Jean-Claude Beaujouan; Yvette Torrens; Monique Saffroy; Marie-Louise Kemel; Jacques Glowinski (339-357).
Several aspects of our 25 year adventure in the field of tachykinins will be successively described. They concern: substance P (SP) synthesis and release in the basal ganglia, the identification and pharmacological characterization of central tachykinin NK1, NK2 and NK3 binding sites and their topographical distribution, the description of some new biological tests for corresponding receptors, the identification of tachykinin NK1 receptor subtypes or conformers sensitive to all endogenous tachykinins (substance P, neurokinin A (NKA), neurokinin B (NKB), neuropeptide γ (NPγ) and neuropeptide K (NPK)) and finally, the functional involvement of these receptors and their subtypes in tachykinin-induced regulations of dopamine and acetylcholine release in the striatum.
Keywords: Tachykinins; Substance P; Tachykinin receptors; NK1, NK2, NK3 receptors; NK1 receptor subtypes; Binding sites; Septide-sensitive binding sites; Autoradiography; Biosynthesis; Release; Brain; Biological activities;

Neuropeptide Y, ubiquitous and elusive by Bibie M Chronwall; Zofia Zukowska (359-363).
This paper reviews aspects of NPY research that were emerging in 1985, shortly after the isolation and characterization of the peptide. NPY had become known for its widespread distribution especially in the central and peripheral nervous systems, but also in the gastro-intestinal and respiratory tracts and in fibers innervating smooth muscle around blood vessels. Consistent with its distribution, it was determined that NPY is a potent vasoconstrictor, affects neuroendocrine systems and is involved in appetite regulation—areas of research still relevant today. Through advances in technology knowledge about NPY’s role in these and newly discovered physiological functions has deepened considerably. Successful cloning of a series of NPY receptors has opened up new and complex research vistas. Lately, the creation of mice genetically modified for NPY as well as for several receptor subtypes has brought many puzzling observations—followed by questions yet to be answered.
Keywords: Neuropeptide Y; NPY receptor; Cardiovascular; Neuroendocrine distribution;

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y1, Y2, Y4, Y5 and y6. All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.
Keywords: Receptor autoradiography; Agonists; Antagonists;

Neurotransmitters co-existing with VIP or PACAP by Jan Fahrenkrug; Jens Hannibal (393-401).
It is now recognized that a neuron can produce, store and release more than one transmitter substance, and a number of examples of co-existing transmitters, particularly a neuropeptide together with a classical transmitter, have been reported. The present paper deals with transmitter substances, peptides or classical transmitters, co-existing with the two structurally related peptides VIP and PACAP and the possible functional implications of this co-existence.
Keywords: Co-existence; Co-release; Neuropeptides; Neurotransmitters; PACAP; VIP;

After commenting on some perceived reasons why our review may have been relatively frequently cited, a brief overview is presented that first summarizes what we knew 25 years ago about the dynamic neuronal–astroglial interactions that occur in response to changes in the physiological state of the animal. The brain system in which these dynamic interactions were studied was the magnocellular hypothalamo-neurohypophysial system (mHNS) of the rat. The mHNS developed as and continues to be the model system yielding the most coherent picture of dynamic morphological changes and insights into their functional consequences. Many other brain areas, however, have more recently come under scrutiny in the search for glial–neuronal dynamisms. Outlined next are some of the questions concerning this phenomenon that led to the research efforts immediately following the initial discoveries, along with the answers, both complete and incomplete, obtained to those research questions. The basis for this first wave of follow-up research can be characterized by the phrase “what we knew we didn’t know at that time.” The final section is an update and brief overview of highlights of both “what we know now” and “what we now know that we don’t know” about dynamic neuronal–astroglial interactions in the mHNS.
Keywords: Neuronal–glial interactions; Physiological state; mHNS;

Substance P in the baroreceptor reflex: 25 years by Cinda J Helke; Jeanne L Seagard (413-423).
Twenty-five years ago, very little was known about chemical communication in the afferent limb of the baroreceptor reflex arc. Subsequently, considerable anatomic and functional data exist to support a role for the tachykinin, substance P (SP), as a neuromodulator or neurotransmitter in baroreceptor afferent neurons. Substance P is synthesized and released from baroreceptor afferent neurons, and excitatory SP (NK1) receptors are activated by baroreceptive input to second-order neurons. SP appears to play a role in modulating the gain of the baroreceptor reflex. However, questions remain about the specific role and significance of SP in mediating baroreceptor information to the central nervous system (CNS), the nature of its interaction with glutaminergic transmission, the relevance of colocalized agents, and complex effects that may result from mediation of non-baroreceptive signals to the CNS.
Keywords: Substance P; Baroreceptor; Reflex arc; Baroreflex; Nucleus of the solitary tract; Nodose ganglion; Petrosal ganglion;

Just cool it! by Gloria E Hoffman; Wei Wei Le (425-431).
Immunohistochemical techniques offer specificity as well as flexibility for visualizing antigens. Their use with freely floating sections provides a high signal-to-noise ratio and has become a gold standard for brain and a number of other tissues. Yet this approach initially suffered from inability to keep the antigenicity in tissue sections and required immediate processing of all cut sections. Use of sucrose solutions enabled storage at refrigerator temperatures for a few days but longer-term storage was risky and either bacterial/fungal growth or evaporation of the storage solution compromised the integrity of the tissue. Our discovery 25 years ago that tissue sections can be stored for many years at −20 °C in an anti-freeze cryoprotectant solution with no loss of antigenicity solved this problem and has become widely used. More recently the utility of tissue stored for many years in anti-freeze cryoprotectant was pushed to new levels by testing new non-radioactive in situ hybridization (ISH) techniques that are based on modern immunocytochemistry. This review touches upon these advances in immunocytochemical technology using examples from neuroscience applications.
Keywords: Cryoprotectant anti-freeze; Immunocytochemistry; In situ hybridization;

Galanin in the brain: chemoarchitectonics and brain cartography—a historical review by David M Jacobowitz; Adelheid Kresse; Gerhard Skofitsch (433-464).
We present a review of galanin in the brain from a historical perspective of the development of “chemoarchitectonics” and “brain cartography” accomplished in the Histopharmacology Section at the National Institutes of Health. It was the mapping of potential brain neuroregulators that served as a springboard of ideas from which behavioral studies emanate. The integration of the known localization of neurotransmitter/neuromodulatory nerves (“chemoarchitectonic maps”) and receptor binding sites with biochemical data derived from brain micropunches coupled with behavioral analysis at the level of discrete brain allows one to define the anatomical circuits which support behavioral changes and which ultimately will improve our understanding of mental disorders.
Keywords: Galanin; Brain; Histochemistry; Brain cartography; Chemoarchitectonics; Brain maps; Neuropeptides; In situ hybridization histochemistry; Brain atlas; Colocalization; Gene expression;

Although a dynamic link between the two innate drives, appetite for food and the urge to reproduce, in vertebrate evolution has been known for a long time, a distinct neurochemical pathway mediating this integration has only recently been appreciated. Study of the precise anatomy of the neural track began in the early to mid 20th century after the sites of genesis of the two instincts were localized to the hypothalamus. This report narrates the birth and fruition to maturity of insights into the commonality of hypothalamic neuropeptide Y (NPY) signaling for the two instinctual drives along two distinct pathways.
Keywords: NPY; Hypothalamus; Appetite;

Hypothalamic control of energy balance: different peptides, different functions by Sarah F Leibowitz; Katherine E Wortley (473-504).
Energy balance is maintained via a homeostatic system involving both the brain and the periphery. A key component of this system is the hypothalamus. Over the past two decades, major advances have been made in identifying an increasing number of peptides within the hypothalamus that contribute to the process of energy homeostasis. Under stable conditions, equilibrium exists between anabolic peptides that stimulate feeding behavior, as well as decrease energy expenditure and lipid utilization in favor of fat storage, and catabolic peptides that attenuate food intake, while stimulating sympathetic nervous system (SNS) activity and restricting fat deposition by increasing lipid metabolism. The equilibrium between these neuropeptides is dynamic in nature. It shifts across the day–night cycle and from day to day and also in response to dietary challenges as well as peripheral energy stores. These shifts occur in close relation to circulating levels of the hormones, leptin, insulin, ghrelin and corticosterone, and also the nutrients, glucose and lipids. These circulating factors together with neural processes are primary signals relaying information regarding the availability of fuels needed for current cellular demand, in addition to the level of stored fuels needed for long-term use. Together, these signals have profound impact on the expression and production of neuropeptides that, in turn, initiate the appropriate anabolic or catabolic responses for restoring equilibrium. In this review, we summarize the evidence obtained on nine peptides in the hypothalamus that have emerged as key players in this process. Data from behavioral, physiological, pharmacological and genetic studies are described and consolidated in an attempt to formulate a clear statement on the underlying function of each of these peptides and also on how they work together to create and maintain energy homeostasis.
Keywords: Neuropeptide Y; Agouti-related protein; Melanin-concentrating hormone; Galanin; Orexins; Melanocortins; Galanin-like peptide; Cocaine- and amphetamine-regulated transcript; Corticotropin-releasing factor; Obesity; Diet;

Our journey with neuropeptide Y: effects on ingestive behaviors and energy expenditure by Allen S Levine; David C Jewett; James P Cleary; Catherine M Kotz; Charles J Billington (505-510).
Clark and colleagues first described the robust orexigenic effects of neuropeptide Y (NPY) in 1984. Our group as well as Stanley et al. confirmed these effects in the same year. During the next 20 years, we investigated the effects of NPY on diet preferences, opioid-related feeding, distributed neural feeding networks, energy metabolism, motivation and discriminative stimulus effects. These data together with data from other laboratories indicate that NPY increases feeding, even when rats work for food; that NPY decreases energy expenditure, particularly by altering thermogenesis; and that NPY’s effects on energy metabolism are mediated by a widely distributed neural network involving other neuroregulators known to be involved in energy regulation.
Keywords: NPY; Neuroregulators; Thermogenesis; Neuropeptides;

As we commemorate the 25th anniversary of the journal Peptides, it is timely to review the functional significance of the bombesin (BB)-like peptides and receptors in the CNS. Over two decades ago we published an article in the journal Peptides demonstrating that BB-like peptides are present in high densities in certain rat brain regions (such as the paraventricular nucleus of the hypothalamus). Subsequently, one of the mammalian forms of BB, gastrin-releasing peptide (GRP) containing cell bodies were found in the suprachiasmatic nucleus of the hypothalamus and nucleus of the solitary tract of the hindbrain. Another related peptide, namely neuromedin (NM)B, was detected in the olfactory bulb and dentate gyrus. BB and GRP bind with high affinity to BB2 receptors, whereas NMB binds with high affinity to BB1 receptors. The actions of BB or GRP are blocked by BB2 receptor antagonists such as (Psi13,14-Leu14)BB whereas PD168368 is a BB1 receptor antagonist. Exogenous administration of BB into the rat brain causes hypothermia, hyperglycemia, grooming and satiety. BB-like peptides activate the sympathetic nervous system and appear to modulate stress, fear and anxiety responses. GRP and NMB modulate distinct biological processes through discrete brain regions or circuits, and globally these peptidergic systems may serve in an integrative or homeostatic function.
Keywords: Bombesin; Gastrin-releasing peptide; Neuromedin B; Rat CNS; Behavior;

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met11-NH2. The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7, Phe7-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio=4:1). The hydrolysis was Cl dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu5-enkephalin. The K i values in μM were as follows: for ACE, bradykinin=0.4, angiotensin I=4, SP=25, SP free acid=2, NT=14, and Met5-enkephalin=450, and for NEP, bradykinin=162, angiotensin I=36, SP=190, NT=39, and Met5−enkephalin=22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.
Keywords: Angiotensin converting enzyme; Neprilysin; Neuropeptides hydrolysis; Kininase II; Bradykinin; Substance P; Enkephalins; Carboxypeptidase; Deamidase;

Practical bradykinin antagonists were discovered in 1984 by Vavrek and Stewart and reported in “Peptides.” At that time there was already much evidence for involvement of bradykinin in inflammation and pain, so the specific, competitive antagonists were widely accepted and applied. The key to conversion of bradykinin into an antagonist was replacement of the proline residue at position 7 with a d-aromatic amino acid. Other modifications converted the initial weak antagonists into modern peptides which are totally resistant to all degrading enzymes, are orally available, and have been used in clinical trials. Non-peptide bradykinin antagonists have also been developed.
Keywords: Bradykinin antagonists; Cancer; Inflammation;