Stimulation of natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in murine leukocytes by bombesin-related peptides requires the presence of adherent cells

Bombesin and the two mammalian bombesin-related peptides, gastrin-releasing peptide (GRP) and neuromedin C, at physiological concentrations have been previously shown to stimulate significantly in vitro the antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activities in BALB/c mouse leukocytes from axillary nodes, spleen and thymus. In the present work we have shown that adherent cells are required in leukocyte samples for stimulation of cytotoxicity by the neuropeptides, which suggests that this effect may be mediated by those cells. Here we demonstrate the specificity of the effects by reversing them in the presence of the bombesin-antagonist (Leu¹³-ΨCH₂NH-Leu¹⁴)-BN, and by detecting specific receptors for GRP on macrophages of high and low affinity. Using the same binding technics, no receptors for this neuropeptide were found in non-adherent leukocytes.     

Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes

Calcitonin gene-related peptide (CGRP) exerts a positive contractile response directly in rat ventricular cardiomyocytes. This response is mediated by receptors of the CGRP₁-subtype. Amylin is 46% homologous with CGRP and binds to receptors selective for CGRP in a range of tissues. The ability of amylin to influence ventricular contractility has been assessed using cardiomyocytes isolated from the ventricles of adult rats. Cardiomyocytes were subjected to biphasic electrical stimulation at 0.5 Hz. CGRP produced a concentration-dependent positive contractile response which became maximal 4 min after initial stimulation. CGRP increased the contractile amplitude maximally at 1 nM and to a value which was 23.3% greater than in the absence of peptide (EC₅₀ VALUE = 21 pM). Amylin increased the contractile amplitude maximally at 20 nM and to a value which was 17.3% greater than in the absence of peptide (EC₅₀ VALUE = 216 pM). In the presence of amylin (20 nM), the concentration-dependence of the contractile response to CGRP was shifted to the left, so that the response became maximal when CGRP was present at 50 pM. In the presence of CGRP_8–37 (100 nM), a selective antagonist at CGRP₁-preferring receptors, the concentration-dependence of the contractile response to CGRP was shifted to the right (dose RATIO = 54). Similarly, in the presence of CGRP_8–37 (100 nM), the contractile response to amylin was inhibited significantly (P ≤ 0.01). Amylin_8–37 (100 nM) did not inhibit the concentration-dependence of the contractile responses to CGRP and amylin significantly (dose RATIOS = 4.2 and 2.4, respectively). In conclusion, these data indicate that amylin exerts a contractile response directly in rat ventricular cardiomyocytes via CGRP₁-preferring receptors. This effect could assume greater significance in non-insulin-dependent diabetes mellitus and in hypertensive states, in which the concentration of amylin is elevated in plasma.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), the 38- and 27-amino-acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100- to 1,000-fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP-selective type I receptor. PACAP38 elicited a sustained seven- to ninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro-NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro-NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Differential Calcitonin Gene-Related Peptide (CGRP) and Amylin Binding Sites in Nucleus Accumbens and Lung: Potential Models for Studying CGRP/Amylin Receptor Subtypes

Abstract: Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[¹²⁵I]iodohistidyl¹⁰)humanCGRPα ([¹²⁵I]hCGRPα) binding (IC₅₀ = 0.4–7.7 nM), which was displaced by hCGRP_8–37α with equally high affinity (IC₅₀ = 0.4–7.3 nM). High-affinity binding for [¹²⁵I]Bolton-Hunter human amylin ([¹²⁵I]BH-h-amylin) was also observed in these tissues (IC₅₀ = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC₅₀ = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin_8–37 competed for [¹²⁵I]hCGRPα binding with higher affinity (IC₅₀ = 5.4 nM) compared with [¹²⁵I]BH-h-amylin binding (IC₅₀ = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC₅₀ values for rat amylin_8–37 were 117 and 12 nM against [¹²⁵I]hCGRPα and [¹²⁵I]BH-h-amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC₅₀ = 0.3 nM), h-amylin (EC₅₀ = 150 nM), and salmon calcitonin (EC₅₀ = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP_8–37α. The affinity of hCGRP_8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1-type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes.     

A VIP hybrid antagonist: From developmental neurobiology to clinical applications

Summary 1. The 28 amino acid vasoactive intestinal peptide, VIP, was originally isolated from the intestine, following a bioassay measuring vasodilating properties. Immunocytochemistry, receptor binding assays and in situ hybridizations have demonstrated VIP abundance in the nervous system, suggesting multiple bioactivities.2. A pharmacological approach was chosen to dissect VIP activities and a prototype VIP antagonist (Met-Hybrid) consisting of a carboxyl fragment of VIP_7–28 and a six amino acid fragment of neurotensin, neurotensin_6–11-VIP_7–28 was synthesized.3. This hybrid peptide was designed to maintain the binding capacity of one parent molecule (VIP), while loosing the agonistic properties, representing a classical competitive receptor antagonist. Furthermore, the new molecule exhibited increased specificity to central nervous system VIP receptors.4. The Met-Hybrid was originally discovered as a potent inhibitor of VIP functionin vivo. In the adult rodent, acute administration of the antagonist resulted in blockade of VIP-mediated potentiation of sexual behavior and chronic intracerebroventricular application impaired VIP-associated learning abilities. During ontogeny, chronic injections of the molecule resulted in neuronal damage, disruption of the diurnal rhythmicity of motor behavior, and retardation in the acquisition of neonatal reflexes in the rat.5. During gestation, severe microcephaly was induced by acute administration of the Met-Hybrid to pregnant mice. The hybrid antagonist inhibited VIP-stimulated mitosis in whole embryo cultures and in a variety of cancer cell linesin vitro andin vivo, suggesting therapeutical potential.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids