[Directed evolution in drug and antibody development : From the Nobel Prize to broad clinical application]. / Gerichtete Evolution in der Wirkstoff- und Antikörperentwicklung : Vom Nobelpreis zur breiten klinischen Anwendung.

Combinatorial procedures have become established in recent years as alternatives to rational design in drug research, particularly when no structural information is available. This article presents the principle that was originally developed by three scientists and was honored with the Nobel Prize for Chemistry in 2018. Furthermore, the application in the field of monclonal antibodies is discussed.

Peptides@mica: from affinity to adhesion mechanism.

Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound.

High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice.

BACKGROUND AND PURPOSE: Human pancreatic polypeptide (hPP) is known to suppress appetite and food intake, thereby representing a potential therapeutic approach against obesity and associated metabolic disorders. The aim of this study was to improve hPP stability by covalent PEGylation with diverse molecular weight polyethylene glycols (PEGs) at two positions using promising lead structures while maintaining target activity. EXPERIMENTAL APPROACH: Modified peptides were synthesized by combined solid-phase and solution-phase peptide synthesis. Their potency was investigated in constitutively expressing human epithelial cells and isolated human colonic mucosa as well as receptor-transfected artificial cell lines. Human blood plasma and porcine liver homogenates were used to examine the in vitro stability of the analogues. The most promising variants were injected s.c. in C57BL/6JRj mice to monitor fasting-induced food intake and bioavailability. KEY RESULTS: In human epithelia and colonic mucosal preparations, activity of the modified hPP peptides depended on the core sequence and latency of the peptides was related to PEG size. Peptides modified with a 22 kDa PEG (PEG22) remained intact in blood plasma and on incubation with liver homogenates for more than 96 h. Finally, hPP2-36 , [K22 (PEG22)]hPP2-36 and [K22 (PEG22),Q34 ]hPP significantly reduced cumulative food intake in mice over 16 h after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Modification with PEG22 at position 22 stabilizes hPP significantly while extending its biological activities and could be used in drug development prospectively.

Pancreatic polypeptide and its central Y4 receptors are essential for cued fear extinction and permanent suppression of fear.

BACKGROUND AND PURPOSE: Avoiding danger and finding food are closely related behaviours that are essential for surviving in a natural environment. Growing evidence supports an important role of gut-brain peptides in modulating energy homeostasis and emotional-affective behaviour. For instance, postprandial release of pancreatic polypeptide (PP) reduced food intake and altered stress-induced motor activity and anxiety by activating central Y4 receptors. EXPERIMENTAL APPROACH: We characterized [K(30) (PEG2)]hPP2-36 as long-acting Y4 receptor agonist and injected it peripherally into wildtype and Y4 receptor knockout (Y4KO) C57Bl/6NCrl mice to investigate the role of Y4 receptors in fear conditioning. Extinction and relapse after extinction was measured by spontaneous recovery and renewal. KEY RESULTS: The Y4KO mice showed impaired cued and context fear extinction without affecting acquisition, consolidation or recall of fear. Correspondingly, peripheral injection of [K(30) (PEG2)]hPP2-36 facilitated extinction learning upon fasting, an effect that was long-lasting and generalized. Furthermore, peripherally applied [K(30) (PEG2)]hPP2-36 before extinction inhibited the activation of orexin-expressing neurons in the lateral hypothalamus in WT, but not in Y4KO mice. CONCLUSIONS AND IMPLICATIONS: Our findings suggests suppression of excessive arousal as a possible mechanism for the extinction-promoting effect of central Y4 receptors and provide strong evidence that fear extinction requires integration of vegetative stimuli with cortical and subcortical information, a process crucially depending on Y4 receptors. Importantly, in the lateral hypothalamus two peptide systems, PP and orexin, interact to generate an emotional response adapted to the current homeostatic state. Detailed investigations of feeding-relevant genes may thus deliver multiple intervention points for treating anxiety-related disorders.

Prenatal notch1 receptor blockade by protein delta homolog 1 (DLK1) modulates adipocyte size in vivo.

INTRODUCTION/OBJECTIVES: The protein delta homolog 1 (DLK1) has been reported to have an important role as inhibitor of adipogenesis. Understanding its mode of action can be a promising approach to cope with the formation of obesity. However, data on DLK1 signaling are not consistent, and especially its role as negative regulator of Notch receptors is discussed controversially. METHODS: DLK1 effects have been investigated in differentiated 3T3-L1 cells by Adipokine Profiler Array, enzyme-linked immunosorbent assay and quantitative real-time PCR (qRT-PCR). In vivo effects of DLK1 on adipogenesis have been studied by the DLK1 treatment of pregnant C57BL/6NTac mice and the phenotypical characterization of the offspring fed on chow or high-fat diet (HFD). Furthermore, gene expression of key adipogenesis genes in adipose tissue (AT) samples was observed by qRT-PCR. RESULTS: In 3T3-L1 cells, DLK1 was found to be an inhibitor of Notch1 signaling. Gene expression of Notch1 and Hes1 was lowered by 53% and 65%, respectively, and the expression of protein target PAI-1 was decreased by 51%. The offspring of DLK1-treated pregnant mice were fed chow or HFD starting from week 4. At week 18, a larger proportion of visceral AT was determined on HFD after DLK1 treatment (P=0.011), whereas adipocyte size was reduced (P=0.007 for maximal size). This was affiliated to an upregulation of adipocyte differentiation. The underlying mechanism was found in an increased expression of the Notch1 receptor gene and protein in AT of the offsprings independently of the diet. However, feeding a chow diet resulted in a decreased expression of Notch1 target genes Hes1 and RBP-Jκ, whereas under HFD these genes were upregulated. CONCLUSIONS: Treatment of mice with recombinant human DLK1 during pregnancy has significant effects on AT of the offspring. This can be associated with counter-regulatory changes in the Notch1 signaling cascade.

Manipulating Y receptor subtype activation of short neuropeptide Y analogs by introducing carbaboranes.

Short selective neuropeptide Y (NPY) analogs are highly attractive because of their facile synthesis. Based on the reduced-size NPY analog [Pro(30), Nle(31), Bpa(32), Leu(34)]NPY 28-36 position 32 was identified as a key position to alter the preferential activation pattern of the human neuropeptide Y receptors (hYRs). By replacing benzoylphenylalanine (Bpa) by a biphenylalanine (Bip) the photostability was first improved while the biological activity was maintained. SAR-studies showed that both aromatic rings have a high influence on the preferential hYR subtype activation. Interestingly, replacement of Bpa(32) by a strongly hydrophobic moiety changed the hYR subtype preference of the analog. Whereas the parent compound is able to activate the human neuropeptide Y1 receptor (hY1R) subtype, the introduction of an N(ε)-ortho-carbaboranyl propionic acid modified lysine resulted in a loss of activity at the hY1R but in an increased activity at both the hY2R and the hY4R. However, subsequent receptor internalization studies with this novel analog revealed that receptor internalization can neither be triggered at the hY2R nor at the hY4R suggesting a biased ligand. Surprisingly, investigations by (1)H NMR spectroscopy revealed structural changes in the side chains of residues Pro(30) and Leu(34) which nicely correlates with the shift from hY1R/hY4R to hY2R/hY4R activation preference. Thus, position 32 has been identified to switch the bioactive conformation and subsequently influences receptor subtype activation behavior.

The selective neuropeptide Y Y5 agonist [cPP(1-7),NPY(19-23),Ala31,Aib32,Gln34]hPP differently modulates emotional processes and body weight in the rat.

The neuropeptide Y (NPY) has been suggested to act as a major regulator of emotional processes and body weight. The full spectrum of biological effects of this peptide is mediated by at least four classes of receptors known as the Y(1), Y(2), Y(4), and Y(5) subtypes. However, the respective contribution of each of these receptor subtypes, especially the Y(5) subtype, in emotional processes is still mostly unknown. In the present study, we investigated the effect of long term administration of a selective Y(5) agonist [cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]hPP on emotional processes and body weight using two rat models of emotional dysfunctions, the corticosterone (CORT)-induced anxiety model as well as the olfactory bulbectomized (OBX) model of depression and anxiety in Wistar and Sprague-Dawley rats, respectively. The sub-chronic administration of the Y(5) agonist reversed the high levels of locomotion, rearing and grooming in the open field test and the impaired social activity induced by OBX, while increased the percentage of entries and time in the open arm of the elevated plus maze in CORT-treated rats. Furthermore, this Y(5) agonist increased body weight in both strains of control rats. These data further demonstrate that Y(5) receptors are not only involved in the control of body weight but also mediate emotional processing under challenged conditions. Thus, the pharmacotherapeutic administration of a Y(5) agonist could be considered as a potentially novel strategy to alleviate some forms of anxiety and depression in humans.

Central vaspin administration acutely reduces food intake and has sustained blood glucose-lowering effects.

AIMS/HYPOTHESIS: Vaspin (visceral adipose tissue-derived serpin) was first identified as an adipokine in a rat model of type 2 diabetes, in which it is predominantly secreted from visceral adipose tissue. Serum concentrations of vaspin show a food intake-related diurnal variation. We therefore tested the hypothesis that vaspin plays a role in the regulation of food intake. METHODS: Vaspin levels in the hypothalamus and human stomach were determined by western blotting. The cerebrospinal fluid concentration of vaspin was measured in five healthy volunteers using an ELISA. Fed 11-week-old female db/db mice were given intraperitoneal injections of 1 mg/kg body weight of vaspin (n = 6) or saline (n = 6) on experimental days 1, 3 and 4. Changes in food intake and fed plasma glucose concentrations were determined after one intracerebroventricular administration of either 1 µg vaspin or artificial cerebrospinal fluid into 11-week-old female db/db (n = 8) and C57BL/6 mice (n = 8) up to 6 days after injection. RESULTS: We detected vaspin in the hypothalamus of db/db and C57BL/6 mice and in the cerebrospinal fluid of healthy individuals. Both peripheral and central vaspin administration decrease food intake in obese db/db and lean C57BL/6 mice. In db/db mice, vaspin treatment is associated with sustained glucose-lowering effects for at least 6 days after injection. In addition, we demonstrated expression of the gene encoding vaspin in the gastric mucosa in humans, and found that this was subject to regional variations. CONCLUSIONS/INTERPRETATION: Our data suggest a previously unrecognised role of vaspin in the regulation of food intake. We postulate that vaspin inhibits a protease that degrades an anti-orexigenic factor.

Functional characterization of the in vitro folded human y(1) receptor in lipid environment.

We describe the recombinant production of the human Y(1) receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y(1) receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Radiometal targeted tumor diagnosis and therapy with peptide hormones.

Radiometal labeled peptide hormones are promising tools for a new generation of radiopharmaceuticals, because their receptors frequently are overexpressed in many human tumors. Furthermore, peptide hormones are characterized by different advantages for clinical application, such as high tumor-to-background ratios as well as rapid blood clearance. Peptidic tumor targeting agents can be sub-divided into the following segments: peptide, spacer, bifunctional chelator and radioisotope. Here the biological and chemical properties of peptide hormones are summarized as well as their prerequisites for their use as tumor targeting agents. Additionally, promising bifunctional chelators and radioisotopes for radiometal labeling are reviewed. Some few special peptide hormones that have been pre-clinically or clinically investigated are furthermore presented, such as somatostatin, bombesin (BBS) / gastrin releasing peptide (GRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). In vitro and in vivo investigations of the binding affinity, selectivity, metabolic stability, bioavailability and biodistribution of radiolabeled peptide hormones could lead to potential peptide-based tumor targeting agents for tumor diagnosis and therapy.

Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation.

In Alzheimer's disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231+pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.

New [99mTc]bombesin analogues with improved biodistribution for targeting gastrin releasing-peptide receptor-positive tumors.

AIM: Bombesin (BBS) receptors are potential targets for diagnosis and therapy of breast and prostate tumors. To overcome the rapid degradation of natural BBS some modifications were introduced at positions 13 and 14. Additionally, a spacer was inserted between the chelator and the binding sequence in order to further improve the in vivo uptake. The analogues were labeled with the [(99m)Tc(CO)(3)]-core and tested. METHODS: Stability was analyzed in vitro in human plasma. Binding affinity and internalization were determined in vitro in prostate carcinoma PC-3 cells. Biodistribution studies and single photon emission computed tomography/X-ray computed tomography (SPECT/CT) imaging were performed in nude mice with PC-3 tumor xenografts. RESULTS: The changes introduced in the BBS(7-14) sequence substantially increased plasma stability. Affinity for gastrin releasing-peptide (GRP) receptors on PC-3 cells was comparable to that of the unmodified analogue with Kd<1 nM. The presence of a spacer in the molecule induced an increment in the in vivo uptake in pancreas and PC-3 xenografts (GRP receptor-positive tissues). The increase in pancreas and tumor uptake was higher when both spacer and stabilization are present in the same molecule. Moreover, in vivo uptake was highly specific. The tumor was clearly visualized by SPECT/CT. CONCLUSIONS: The modifications in the BBS(7-14) sequence led to a higher plasma stability while binding affinity remained unaffected. Stabilization resulted in improved biodistribution with better tumor to non-tumor ratios. However, the insertion of a spacer had a greater influence on the biodistribution. Analogues with both spacer and stabilization are the most promising radiopharmaceuticals for targeting GRP receptor-positive tumors.

Calcitonin-derived carrier peptides.

The recent discovery of carrier peptides offers new opportunities to translocate several bioactive molecules into the cytoplasm. Previous studies have shown that human calcitonin (hCT) and selected C-terminal sequences translocate in nasal epithelium. Moreover, the hCT(9-32) fragment was found to internalize efficiently a number of substances like fluorophores, nucleic acids or the enhanced green fluorescent protein (EGFP). In order to understand the uptake mechanism interactions of hCT(9-32) with membrane models of different lipid compositions have been investigated. From these studies it was possible to shed light on the conformational state of the peptide in the presence of membrane-like conditions. Further insight into the translocation mechanism was provided by fluorescence microscopy of truncated sequences of hCT that were shown to penetrate the plasma membrane and to distribute in a sectoral, punctuated pattern supporting an endocytotic internalization pathway as previously suggested.

PYY3-36 as an anti-obesity drug target.

The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in the physiology of obesity for several decades. More recently ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while at the same time studies finding no anti-obesity effects have surfaced. Out of 41 different rodent studies conducted (in 16 independent labs worldwide), 33 (83%) were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e. adaptation protocols, routes of drug administration and doses, rodent strains, diets, drug vendors, light cycles, room temperatures). Among studies by authors in the original study, procedural caveats are reported under which positive effects may be obtained. Currently, data speak against a sustained decrease in food intake, body fat, or body weight gain following PYY3-36 administration and make the previously suggested role of the hypothalamic melanocortin system unlikely as is the existence of PYY deficiency in human obesity. We review the studies that are in the public domain which support or challenge PYY3-36 as a potential anti-obesity target.

Neuropeptide Y Y5 receptors inhibit kindling acquisition in rats.

Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood-brain barrier, and the selective peptide agonist Ala31Aib34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4-5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala31Aib32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y5 agonist showed stages 4-5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y5 agonist- or antagonist-treated rats had stages 4-5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation.

Biosynthesis of peptide hormones derived from precursor sequences.

The release of hormones is subject to a complex and finely tuned regulation system. The biosynthesis plays a key role by specifically converting the prohormone precursor into its biological active product(s). A family of mammalian proteases could be identified to be responsible for the endoproteolytic processing. These subtilisin/kexin-like prohormone convertases (PC) recognize their substrates at single or pairs of basic residues with a high substrate specificity. The so far known seven members include PC1/3, PC2, furin/PACE, PACE4, PC4, PC5/6 and PC7/SPC7/LPC/PC8. PC1/3 and PC2 are the most important enzymes for the processing of prohormones, whereas furin is the only one that causes lethality in knock-out models. Tissue-specific co-localization of the prohormone and the PC as well as distinct characteristics of both, like the secondary structures, determine the possible conversion processes. Identification of such determinants implies a great potential for the development of novel drug targets. To obtain sufficient amounts for the in vitro characterization of prohormones, chemical and recombinant synthesis methods have been developed. Application of expressed protein ligation lead to the semisynthesis of the first chemically modified analogs of a full-length proneurohormone (pro-neuropeptide Y). Structural analyses mainly on peptides of the pro-oxytocin/neurophysin system and on prosomatostatin highlighted the importance of flexible turn or loop structures adjacent to the cleavage site for the specific substrate-enzyme active site interaction. Prohormones and their processing show multiple functions. Therapeutic application including PC inhibitors is very promising for the treatment of disorders like cancer.

Physiology: does gut hormone PYY3-36 decrease food intake in rodents?

Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.

99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents.

The possible use of neuropeptide Y (NPY) as a novel radiopeptide has been investigated. NPY is a 36-amino acid peptide of the pancreatic polypeptide family, which is expressed in the peripheral and central nervous system, and is one of the most abundant neuropeptides in the brain. Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines. As structure-activity relationships of NPY are well-known, we could assume where a radionuclide might be introduced without affecting receptor affinity. We applied the novel [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent. The peptides were synthesized by solid-phase peptide synthesis, and PADA was coupled to the side chain of Lys4 of the resin-bound peptide. Upon postlabeling of [K4(PADA)]-NPY, 99mTc(CO)3 did not only bind to the desired PADA, but presumably as well to the His in position 26. Since the replacement of His26 by Ala only slightly decreased binding affinity, [K4(PADA),A26]-NPY was specifically postlabeled, and the 185Re surrogate maintained high binding affinity. Furthermore, the prelabeling approach has been applied for the centrally truncated analogue [Ahx5-24]-NPY, which is highly selective for the Y2 receptor. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, postlabeling was applied for the short analogue as well, again substituting His26 by Ala. Competitive binding assays using (185)Re as a surrogate for 99mTc showed high binding affinity of Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY. Internalization studies with the corresponding 99mTc-labeled analogue revealed receptor-mediated internalization. Furthermore, biodistribution studies were performed in mice, and stability was tested in human plasma. Our centrally truncated analogue revealed a 6-fold increased stability compared to the natural peptide NPY. We conclude that Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY has promising characteristics for future applications in nuclear medicine.

Modulatory effect of two novel CGRP receptor antagonists on nasal vasodilatatory responses to exogenous CGRP, capsaicin, bradykinin and histamine in anaesthetised pigs.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide and potent vasodilatator agent located in sensory C fibres. Several functional studies suggest that CGRP could be involved in the vasodilatation of different vascular beds during neurogenic inflammation. We have studied, in pentobarbital anaesthetised pigs, the antagonistic effect of local intra-arterial (i.a.) pretreatment with the analogues CGRP 8-37, [D31, P34, F35]CGRP 27-37 and [N31, P34, F35]CGRP 27-37 on the vasodilatation of the nasal vascular bed induced by exogenous CGRP, capsaicin, bradykinin (BK) and histamine. The attenuating effect of CGRP 8-37 analogue on exogenous CGRP-induced vasodilatation, previously described in other in vivo animal models, was confirmed in the pig nasal mucosa. It also interfered with BK-and, to a lesser extent, with capsaicin-and histamine-induced decrease in vascular resistance. CGRP 27-37 analogues reduced the duration of CGRP-, capsaicin- and BK-induced vasodilatation by more than 50%. Peak values of vasodilatation were attenuated by more than 25% overall. Attenuation of histamine-induced decrease in vascular resistance was less pronounced. It is concluded that CGRP 27-37 analogues antagonise the action of exogenous CGRP, capsaicin, BK and histamine by attenuating their vasodilatation effect, both in intensity and duration. These results strongly suggest that BK- and histamine-induced vasodilatation is partly mediated by CGRP. CGRP 8-37 and 27-37 appear to be potential contributors to the study of CGRP and its physiological role in neurogenic inflammation. In addition, they may have putative therapeutic applications in the treatment of rhinitic patients suffering from chronic nasal obstruction.

Immunocytochemical localization of angiotensin II receptor subtypes and angiotensin II with monoclonal antibodies in the rat adrenal gland.

Angiotensin II (Ang II), a major regulator of cardiovascular function and body fluid homeostasis, mediates its biological actions via two subtypes of G protein-coupled receptors, termed AT(1) and AT(2). The primary goal of this study was to raise monoclonal anti-peptide antibodies specific to angiotensin AT(1)- and AT(2)-receptor subtypes and to Ang II itself and using these monoclonal antibodies to determine the intraadrenal localization of AT(1) and AT(2) receptors and Ang II in male adult rats. Immunocytochemistry unambiguously demonstrates a regional colocalization of Ang II and angiotensin II receptors in the adrenal gland. The novel antibodies localized Ang II and the AT(1) receptors to the zona glomerulosa of the cortex and to the medulla whereas AT(2) receptors were limited to the medulla. The specificity of immunostaining was documented by pre-adsorption of the antibody with the immunogenic peptide. Our data underscore that AT(1) appears to mediate most of the physiological actions of Ang II in adrenal. Western blot analysis of rat adrenal protein extracts using AT(1) antibody showed a predominant 73-kDa band and a weaker 97-kDa immunoreactive band corresponding to glycosylated forms of the AT(1) receptor. Immunostaining with anti-AT(2) yielded one major immunoreactive band of 73-kDa size and one additional fainter band of 120 kDa. These antibodies may prove of value in unraveling the subcellular localization and intracellular effector pathways of AT(1) and AT(2).