Vasoactive Intestinal Peptide (VIP) Inhibits Substrate Adherence Capacity of Rat Peritoneal Macrophages by a Mechanism That Involves cAMP

In this study, vasoactive intestinal peptide (VIP) is shown to inhibit substrate adherence capacity of rat peritoneal macrophages. The inhibitory response occurred in the 0.1-1, 000 nM range of VIP concentrations and it was a time-dependent process. At 15 min, half maximal inhibition (ICw) was obtained at 0.37 ± 0.26 nM and maximal inhibition (53.8%) at 10^-6 M VIP. The inhibitory effect of VIP was correlated with the stimulation by this peptide of cyclic AMP (cAMP) production in rat peritoneal macrophages. Moreover, agents that inhibited VIP-stimulated cAMP production, such as the VIP-antagonist [4-Cl-D-Phe₆ Leu₁₇]-VIP and somatostatin, also decreased the inhibitory effect of VIP on substrate adherence capacity of macrophages. On the contrary, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the lipid-soluble derivative of cAMP N⁶ 2'-O-dibutyryl cAMP (Bu-cAMP) inhibited the adherence of macrophages to substrate and potentiated the inhibitory action of VIP. These results demonstrate that VIP inhibits substrate adherence capacity of rat peritoneal macrophages by a mechanism that involves cAMP, and show, for the first time, an action of VIP on the function of peritoneal macrophages.     

Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages.

Functional vasoactive intestinal peptide (VIP) receptors have been characterized in rat peritoneal macrophages. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of two classes of binding sites: a class with high affinity (kd = 1.1 +/- 0.1 nM) and low capacity (11.1 +/- 1.5 fmol/10(6) cells), and a class with low affinity (kd = 71.6 +/- 10.2 nM) and high capacity (419.0 +/- 80.0 fmol/10(6) cells). Structural requirements of these receptors were studied with peptides structurally or not structurally related to VIP. Several peptides inhibited 125I-VIP binding to rat peritoneal macrophages with the following order of potency: VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, somatostatin, pancreastatin and octapeptide of cholecystokinin (CCK 26-33) were ineffective. VIP induced an increase of cyclic AMP production. Half-maximal stimulation (ED50) was observed at 1.2 +/- 0.5 nM VIP, and maximal stimulation (3-fold above basal levels) was obtained between 0.1-1 microM. Properties of these binding sites strongly support the concept that VIP could behave as regulatory peptide on the macrophage function.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15°C, the response occurred in the 1·10⁻¹⁰−10⁻⁷ M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1·10⁻⁶ M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

The synthetic peptide octraphin TPLVTLFK is a selective agonist of nonopioid β-endorphin receptor

The peptide TPLVTLFK (coined by the authors “octarphin”), corresponding to the amino acid sequence of β-endorphin fragment 12–19, and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL) were synthesized. The peptide octarphin was labeled with tritium (specific activity, 28 Ci/mol) and its binding to the rat brain cortex membranes and mouse peritoneal macrophages was studied. [³H]Octarphin was found to bind to brain membranes and macrophages with high affinity (K _d = 2.6 ±0.2 and 2.3 +0.2 nM, respectively) and specificity. The specific binding of [³H]octarphin with rat brain membranes and mouse macrophages was inhibited by unlabeled β-endorphin (K _i = 2.4 +0.2 and 2.7 +0.2 nM, respectively) and selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.9 +0.2 and 2.4 +0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met⁵]enkephalin (K _i > 10 mM). Inhibiting activity of unlabeled analogs of octarphin was more than 100 times lower than that of the unlabeled octarphin. Octarphin was shown to stimulate activity of mouse immunocompetent cells in vitro: at the concentration of 1 nM it enhanced the capacity of peritoneal macrophages to digest bacteria Salmonella typhimurium virulent strain 415 in vitro. Thus, octarphin is a selective agonist of nonopioid (insensitive to the opioid antagonist naloxone) β-endorphin receptor of rat brain cortex membranes and mouse peritoneal macrophages.     

Duck Pancreatic Acinar Cell as a Unique Model for Independent Cholinergic Stimulation–Secretion Coupling

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 μM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose–response curve. ACh dose dependently induced increases in cytosolic Ca²⁺ concentration ([Ca²⁺] _c ), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 μM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca²⁺] _c increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.     

Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate

Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of ¹²⁵I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15°C. At apparent equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native VIP in the 1·10⁻¹⁰−10⁻⁷ M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a K_d = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a K_d = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium.     

Properties and mechanism of the action of the synthetic peptide octarphin

The peptide TPLVTLFK, whose amino acid sequence corresponds to the 12–19 fragment of β-endorphin (the author’s name for the peptide octarphin), and its analogues (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, and TPLVTLFL) have been synthesized. Tritium-labeled octarphin (specific activity of 28 Ci/mol) has been obtained, and its binding to murine peritoneal macrophages has been studied. It was found that [³H]octarphin binds to macrophages with a high affinity (K _d 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin to macrophages was inhibited by the unlabeled β-endorphin and the selective agonist of the nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met5]enkephalin (K _i > 10 μM). The inhibitory activity of the octarphin analogues was more than 100 times lower than that of octarphin. It was shown that octarphin stimulates the activity of mouse immunocompetent cells in vitro and in vivo; at a concentration of 1–10 nM, it increased the adhesion and spreading of peritoneal macrophages and their ability to digest the bacteria of the Salmonella typhimurium virulent strain 415 in vitro. The intraperitoneal injection of the peptide at a dose of 20 μg/animal on day 7, 3, and 1 prior to the isolation of cells led to an increase in the activity of the peritoneal macrophages and the Tand B lymphocytes of the spleen.     

Binding of synthetic fragments of β‐endorphin to nonopioid β‐endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [³H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (K_d = 2.0 ± 0.1 nM ). The [³H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (K_i = 2.9 ± 0.2 nM ) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met⁵]enkephalin (K_i > 10 µM ). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [³H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the author's name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (K_i = 3.1 ± 0.3 nM ). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [³H]Octarphin was found to bind to macrophages with high affinity (K_d = 2.3 ± 0.2 nM ). The specific binding of [³H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (K_i = 2.4 ± 0.2 and 2.7 ± 0.2 nM , respectively). Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

Binding of β-endorphin and its fragments to the nonopiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid β-endorphin receptor the decapeptide immunorphin ([³H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [³H]immunorphin binds with a high affinity to the non-opioid β-endorphin receptor of mouse peritoneal macrophages (K _d 2.4 ± 0.1 nM). The specific binding of [³H]immunorphin to macrophages was inhibited by unlabeled β-endorphin (K _i of the [³H]immunorphin-receptor complex 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met⁵]enkephalin (K _i > 10 μM). Thirty fragments of β-endorphin were synthesized, and their ability to inhibit the specific binding of [³H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as β-endorphin is its fragment 12–19 (K _i 3.1 ± 0.3 nM).     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15 degrees C, the response occurred in the 1 X 10(-10)-10(-7)M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1 X 10(-6)M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

Interaction of vasoactive intestinal peptide (VIP) with a mouse adrenal cell line (Y-1): specific binding and biological effects.

Y-1 cells specifically bind radiolabelled vasoactive intestinal peptide (VIP) with a dissociation constant of about 10^?9 M. [¹²⁵I]-VIP bound was not displaced by ACTH. VIP stimulates both steroid and cAMP production, with half-maximal stimulation at 10^?9 and 10^?8 M, respectively. At maximal concentration VIP produces the same stimulation of steroidogenesis as ACTH, but induced three times lower production of cAMP than ACTH. Y-1 DNA synthesis is inhibited by VIP in a dose-dependent manner with half-maximal inhibition at 10^?8 M. At submaximal concentrations the effects of VIP and ACTH on cAMP and steroid production and on inhibition of DNA synthesis are additive. Similar additive effects on cAMP production and on inhibition of DNA synthesis were observed at submaximal ACTH and maximal VIP concentration, but the phenomenon was no longer seen at maximal concentrations of both peptides. These data suggest that in Y-1 cells VIP stimulates, through its own distinct receptors, only a part of the pool of adenylate cyclase sensitive to ACTH.     

Morphological and functional correlates of VIP neurons in cerebral cortex

Vasoactive Intestinal Polypeptide (VIP) promotes the hydrolysis of ³H-glycogen newly synthesized from ³H-glucose by mouse cortical slices. This effect occurs rapidly, approximately 50% of the maximal effect being reached within one minute. The maximal effect is achieved after 5 minutes and maintained for at least 25 minutes. Furthermore the glycogenolytic effect of VIP is reversible, and pharmacologically specific. Thus several neuropeptides present in cerebral cortex such as cholecystokinin-8, somatostatin-28, somatostatin-14, met-enkephalin, leu-enkephalin, do not affect ³H-glycogen levels. VIP fragments 6–28, 16–28 and 21–28 are similarly inactive. Furthermore, among the peptides which share structural homologies with VIP, such as glucagon, secretin, PHI-27 and Gastric Inhibitory Peptide, only secretin and PHI-27 promote ³H-glycogen hydrolysis, with EC₅₀ of 500 and 300 nM respectively, compared to an EC₅₀ of 25 nM for VIP. Immunohistochemical observations indicate that each VIP-containing bipolar cell is identified with a unique radial cortical volume, which is generally between 15–60 μm in diameter and overlaps with the contiguous domains of neighbouring VIP-containing bipolar cells. Thus this set of biochemical and morphological observations support the notion that VIP neurons have the capacity to regulate the availability of energy substrates in cerebral cortex locally, within circumscribed, contiguous, radial domains.     

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [³H]Octarphin was found to bind to macrophages with high affinity (K _d = 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met⁵]enkephalin (K _i > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 M GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0±7.0 nM VIP, whereas the maximal activity (at 1 M VIP)corresponded to an increase of about 140% with respect to basal values (7.5±0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED₅₀=1.8±1.4 nM)>VIP(ED₅₀=25.0±7.0 nM)>PHI (ED₅₀=725.0±127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of _s and _i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of¹²⁵I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd=0.43 M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization,immunocytochemistry and radioimmunoassay

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt₂cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNA_s (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt₂cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt₂cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt²cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9-39) amide, a specific exendin receptor antagonist.

Exendin-3 increased cellular cAMP levels and amylase release from dispersed acini from guinea pig pancreas. Low concentrations (0.1-3 nM) caused a 12-fold increase in cAMP, whereas higher concentrations (0.3-3 microM) caused an additional 24-fold increase in cAMP. Maximal cAMP with the highest concentration tested was the same as the maximal response with secretin, vasoactive intestinal peptide (VIP), peptide histidine isoleucine, helodermin, or helospectin-I. In terms of amylase release, exendin-3 had the same efficacy but was the least potent of these peptides. Exendin-3-induced increases in amylase release were inhibited by VIP receptor antagonists and the new peptide (greater than 0.1 microM) competed with radiolabeled VIP for binding sites on dispersed acini. Increasing concentrations of an exendin-3 fragment, exendin-3(9-39) amide, did not increase cAMP or amylase release but inhibited the increase in cAMP observed with 0.1-3 nM exendin-3. The fragment did not alter the effects of other peptides that are known to increase acinar cAMP. We conclude that exendin-3 interacts with at least two receptors on guinea pig pancreatic acini; at high concentrations (greater than 100 nM) the peptide interacts with VIP receptors, thereby causing a large increase in cAMP and stimulating amylase release; at lower concentrations (0.1-3 nM) the peptide interacts with a putative exendin receptor, thereby causing a smaller increase in cAMP of undetermined function. Exendin-3(9-39) amide is a specific exendin receptor antagonist.     

Acute stimulation of ganglionic tyrosine hydroxylase activity by secretin,VIP and PHI

We have examined the ability of a number of neuropeptides to increase tyrosine hydroxylase (TH) activity in the superior cervical ganglion in vitro. Secretin and vasoactive intestinal peptide (VIP) both increased TH activity, whereas angiotensin II, bombesin, bradykinin, cholecystokinin octapeptide, insulin, luteinizing hormone-releasing hormone, [D-Ala², Met³]enkephalinamide, motilin, neurotensin, somatostatin, and substance P produced no effects. Secretin and VIP increased TH activity with an EC₅₀ of 5 nM and 0.5 μM, respectively. The effects of these peptides were not altered by prior decentralization of the ganglia, by addition of hexamethonium (3 mM) and atropine (6 μM), or by lowering the concentration of calcium in the medium to 0.1 mM. Addition of carbachol (3 μM) potentiated the effects of both secretin and VIP on TH activity. Several gastrointestinal peptides with structural similarities to secretin and VIP were examined for their ability to increase TH activity. Glucagon, gastric inhibitory peptide and human pancreatic tumor growth hormone-releasing factor produced no effect at a concentration of 10 μM, while PHI increased enzyme activity.