Jörg Seebeck

PACAP and VIP stimulate enzyme secretion in rat pancreatic acini via interaction with VIP/PACAP-2 receptors: additive augmentation of CCK/carbachol-induced enzyme release.

The binding and biological effect of pituitary adenylate cyclase activating polypeptide (PACAP), a novel hypothalamic peptide with high sequence homology to vasoactive intestinal polypeptide (VIP), were studied in rat AR 4–2 J pancreatic carcinoma cells and isolated rat pancreatic acini. PACAP(1–27) and analogue PACAP(1–23, VIP-24-28), but not VIP, displaced potently and reversibly 125I-PACAP(1–27) from binding to an abundantly expressed high affinity PACAP-preferring receptor on AR 4–2 J cells, referred to as “PACAP-1 receptor.” High affinity binding was dependent on N-terminal and C-terminal residues of PACAP(1–27): PACAP(1–24,Cys-25) (7.3 ± 1.6 PIM), PACAP(1–23) (8.2 ± 1.5 μM), VIP (>30 μM), PACAP(3–27), PACAP(1–19), PACAP(3–19), PACAP(1–12), and PACAP(18–38) (all >50 μM) showed low or no binding potency. In contrast, high and low affinity binding of 125I-VIP to AR 4–2 J cells was displaced equipotently by PACAP(1–27) and VIP, thus defining on these cells, in addition, two scarcely expressed binding sites, designated “VIPIPACAP-2 receptor,” similar or identical to the previously described high and low affinity acinar VIP receptor. Binding of 125I PACAP(1–27) to a high and low affinity binding site on rat pancreatic acini was inhibited equipotently by PACAP(1–27) and VIP, identifying these sites as VIPPACAP-2 receptors. PACAP(1–23) recognized both type 2 binding sites with only slightly lower affinity. PACAP(1–27), PACAP(1–38), PACAP(1–23,VIP-2428), and PACAP(1–23) equipotently stimulated acinar lipase release and cyclic AMP production in pancreatic acini. Co-incubation of PACAP(1–27) or VIP with cholecystokinin-8 or carbachol revealed additive effects on enzyme secretion. Our results suggest the predominant expression of VIPPACAP-2 receptors on rat pancreatic acini, whereas AR 4–2 J cells express mainly PACAP-1 receptors. PACAP is a potent ligand for both receptor types and has to be regarded as a novel VIP-like pancreatic secretagogue.

Pituitary adenylate cyclase activating polypeptide induces multiple signaling pathways in rat peritoneal mast cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is a high-affinity ligand for at least two types of G-protein coupled receptors, the PACAP type 1 and type 2 receptor. In this study it is demonstrated that the C-terminal PACAP-fragment PACAP(6-27) stimulates serotonin release from rat peritoneal mast cells with higher potency (EC50: 0.2 vs. 2.0 microM) than the PACAP receptor ligand PACAP(1-27). PACAP-induced degranulation of rat peritoneal mast cells was abolished by pertussis toxin and by benzalkonium chloride (IC50: 9.1 microg/ml) indicating the involvement of heterotrimeric G-proteins of the Gi-type. The PACAP effect was also reduced by inhibitors of the phosphatidylinositol specific phospholipase C ((U73122), IC50: 4 microM; (ET-18-O-CH3), IC50: 18 microM), by D609, a specific inhibitor of the phosphatidylcholine specific phospholipase C (IC50: 41 microM), by the protein kinase C-inhibitor staurosporine (IC50: 0.6 microM) and by the lipoxygenase inhibitor nordihydroguaiaretic acid (NGDA) but not by indomethacin. It is concluded that PACAP peptides stimulate secretion in rat peritoneal mast cells in a PACAP receptor-independent manner, probably via direct activation of heterotrimeric G-proteins of the Gi-type; these G-proteins may lead to a sequential activation of different signaling cascades (see above), which may converge at the level of one or more staurosporine-sensitive protein kinase.

Pituitary Adenylate Cyclase Activating Polypeptide Induces Degranulation of Rat Peritoneal Mast Cells via High-Affinity PACAP Receptor-Independent Activation of G Proteins

Abstract: In this study, the secretory effects of PACAP and PACAP analogues on [3H]serotonin‐loaded purified rat peritoneal mast cells (RPMCs) were investigated. PACAP(1‐27) and PACAP(6‐27) stimulated [3H]serotonin release with low potency (ED50: 2 × 10−6 M) but high efficacy. The N‐terminally truncated PACAP form, PACAP(6‐27), stimulated tracer release with an ED50 of 0.2 × 10−6 M, indicating a high‐affinity PACAP receptor‐independent mechanism of action. The secretory response to PACAP(1‐27) could be inhibited by 60‐min preincubation with pertussis toxin (ptx), which inhibits G proteins. U73122, a cell‐permeable phospholipase C inhibitor, dose‐dependently inhibited the secretory effect of 5 μM PACAP(1‐27) with an IC50 value of 4 μM (N= 4; p > 0.006). We conclude that PACAP exerts a secretory effect in RPMCs by high‐affinity PACAP receptor‐independent direct activation of one or more G proteins, which may then activate the PLC‐dependent signal‐transduction pathway.

Stem cell factor influences neuro-immune interactions: The response of mast cells to pituitary adenylate cyclase activating polypeptide is altered by stem cell factor

Mast cells degranulation can be elicited by a number of biologically important neuropeptides, but the mechanisms involved in mast cell-neuropeptide interactions have not been fully elucidated. Stem cell factor (SCF), also known as c-kit or kit ligand, induces multiple effects on mast cells, including proliferation, differentiation, maturation, and prevents apoptosis. We investigated the ability of SCF to affect mast cell responsiveness to the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). PACAP 1-27, PACAP1-38, or VIP failed to induced preformed mediator release from mouse bone-marrow-cultured mast cells (BMCMC) derived in concanavalin A-stimulated spleen conditioned medium (CM). By contrast, BMCMC grown in SCF-containing medium or freshly isolated peritoneal mast cells exhibited significant 3H-hydroxytrypamine (5-HT) release in response to PACAP peptides or VIP. Deoxyglucose and the mitochondrial inhibitor antimycin significantly inhibited PACAP-induced 5-HT release indicating that the central event induced by PACAP peptides was exocytosis. The G(alpha)i inhibitor, pertussis toxin, significantly diminished PACAP-induced 5-HT release from BMCMCs in SCF suggesting the involvement of heterotrimeric G-proteins. Western blot analysis using antibodies directed against the human VIP type I/PACAP type II receptor demonstrated a 70-72 kD immunoreactive protein expressed in greater amounts in BMCMC grown in SCF compared with BMCMC in CM. We conclude that SCF induces a mast cell population that is responsive to PACAPs and VIP involving a heterotrimeric G-protein-dependent mechanism.

The vasorelaxant effect of pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in isolated rat basilar arteries is partially mediated by activation of nitrergic neurons

The structurally related neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are recognised by two G protein-coupled receptors, termed VPAC(1)-R and VPAC(2)-R, with equal affinity. PACAP and VIP have previously been shown to relax cerebral arteries in an endothelium-independent manner. The aim of the present study was to test if intramural neurons are involved in the mediation of PACAP/VIP-induced vasodilatory responses. Therefore, the vascular tone of isolated rat basilar arteries was measured by means of a myograph. The vasorelaxing effect of PACAP was assessed in arteries precontracted by serotonin in the absence or presence of different test compounds known to selectively inhibit certain signaling proteins. The vasorelaxant effect of PACAP could be significantly reduced by the inhibitor of neuronal N-type calcium channels omega-conotoxin GVIA (omega-CgTx), as well as by 3-bromo-7-nitroindazole (3Br-7-Ni), an inhibitor of the neuronal nitric oxide-synthase (nNOS). The localization of N-type calcium channels and VPAC-Rs within the rat basilar artery was investigated by confocal laser scanning microscopy using omega-CgTx- and VIP-analogs labelled with fluorescent dyes. These findings suggest that activation of intramural neurons may represent an important effector mechanism for mediation of the vasorelaxant PACAP-response.

Role of potassium channels in the relaxation induced by the nitric oxide (NO) donor DEA/NO in the isolated rat basilar artery

This study investigates whether potassium ion (K+) channels are involved in the nitric oxide (NO)-induced relaxation in segments of the isolated rat basilar artery, mounted onto a wire myograph. A high extracellular K+ concentration partly inhibited the relaxant effects of the NO donors DEA/NO and SIN-1 (3-morpholino-sydnonimine). Whereas single applications of the K+ channel inhibitors tetraethyl-ammonium (10(-3) M), glibenclamide (10(-6) M), 4-aminopyridine (10(-3) M), or BaCl(2) (5 x 10(-5) M) did not affect the responses to DEA/NO, a combination of these inhibitors reduced the effects of DEA/NO. These data suggest, that the relaxant effects of NO donors are partly mediated via activation of K+channels. Different K+ channel types seem to be involved that function in a redundant manner and compensate for each other.

Orlistat reduces gallbladder emptying by inhibition of CCK release in response to a test meal

BACKGROUND AND AIMS Orlistat is a covalent inhibitor of digestive lipase derived from lipstatin, the natural product of Streptomyces toxytricini. By blocking the active site of intestinal lipase, orlistat inhibits hydrolysis of dietary triglycerides and thus reduces the intestinal lipid absorption. It is uncertain whether intestinal inhibition of lipase by orlistat also interferes with nutrient-induced CCK release from intestinal I-cells. The aim of the present study was therefore to assess whether oral administration of orlistat inhibits CCK release in response to a test meal and thus causes impaired gallbladder emptying. METHODS 22 healthy volunteers were given a test meal consisting of 200 ml dairy cream and two teaspoons of chocolate powder (552 kcal=2328 kJ; 56.0 g fat; 5.2 g proteins, 6.6 g carbohydrates), with and without oral application of 120 mg orlistat. Gallbladder volume was determined by ultrasound before and 5, 10, 20, 30 and 40 min after meal ingestion. In parallel, a venous blood sample was collected for the measurement of bioactive CCK. CCK activity was assessed using a bioassay with isolated rat pancreatic acini cells. RESULTS Oral administration of orlistat significantly impairs gallbladder emptying. After ingestion of the test meal the gallbladder contracted by 78.5% in the control group, whereas the test group with orlistat only showed a contraction of 45.7% (p<0.01). Maximal contraction was reached after 35 to 40 min, the maximal gallbladder emptying was delayed up to 10 min by orlistat. Orlistat induced a significant reduction of bioactive CCK levels in response to a test meal (CCK(max) with orlistat=4.1 pmol/l; CCK(max) without orlistat=7.8 pmol/l). CCK levels were reduced by 47% and the onset of maximal CCK secretion was delayed up to 10 min. CONCLUSION The inhibition of intestinal lipolytic activity by orlistat results in reduced gallbladder emptying through inhibition of meal-mediated CCK release. We therefore hypothesize that impaired gallbladder motility may represent a risk factor in chronic treatment of severe obesity using orlistat.

The exocytotic signaling pathway induced by nerve growth factor in the presence of lyso-phosphatidylserine in rat peritoneal mast cells involves a type D phospholipase

Nerve growth factor (NGF) has been previously shown to induce exocytosis in rat peritoneal mast cells (RPMCs) in the presence of lyso-phosphatidylserine (lysoPS) by interacting with high-affinity NGF receptors of the TrkA-type. In RPMCs, type D phosphatidylcholine-selective phospholipases (PLDs) have been postulated to be involved in some exocytotic signaling pathways induced by different agonists. The aim of the present study was to assess a putative functional role of PLD for NGF/lysoPS-induced exocytosis in RPMCs. In 1-[14C]palmitoyl-2-lyso-3-phosphatidylcholine-labelled RPMCs, NGF/lysoPS stimulated the formation of diacylglycerol (DAG) and, in the presence of ethanol (1% [v/v]), phosphatidylethanol (PEtOH). These data indicate PLD-activation by NGF/lysoPS in RPMCs. Preincubation of RPMCs for 2 min with ethanol, an inhibitor of PLD-derived DAG-formation, dose-dependently (IC(50): 0.6% [v/v]) and agonist-selectively inhibited the NGF/lysoPS induced release of [3H]serotonin ([3H]5-HT) in [3H]5-HT-loaded RPMCs, confirming the functional importance of PLD-action. Exocytosis and PEtOH-production was potently inhibited by the broad-spectrum serine/threonine kinase inhibitor staurosporine and activated by the protein kinase C(PKC)-activator PMA (phorbol-12-myristate-13-acetate) suggesting a role for PKC as mediator for NGF/lysoPS-induced activation of PLD.

Inhibition of phospholipase C-independent exocytotic responses in rat peritoneal mast cells by U73122

UNLABELLED The aminosteroid U73122 has been established as potent, selective, and cell-permeable inhibitor C-type phosphatidylinositol-specific phospholipases (PI-PLCs), and has been used to define a contribution of PI-PLCs as part of exocytotic signalling pathways in rat peritoneal mast cells (RPMCs). However, doubts have been raised regarding its PI-PLC selectivity of action. Therefore, in the present study, U73122 was tested in RPMCs under experimental conditions allowing to elicit exocytosis PI-PLC independently (streptolysin O [SLO]-permeabilised cells; stimulated by GTPgammaS; in the presence of low concentrations of free Ca2+). The release of [3H]5-hydroxytryptamine ([3H]5-HT) from [3H]5-HT-loaded RPMCs served as measure of secretion. U73122 potently inhibited the exocytotic response induced by 10 microM GTPgammaS (Ca2+: 10(-6) M) in permeabilised cells (IC50: 0.6 microM, n=5) in an insurmountable manner. In intact RPMCs, with a nearly equal potency (IC50: 4 microM, n=4), U73122 also inhibited the PI-PLC-dependent exocytotic response induced by concomitant application of nerve growth factor and lyso-phosphatidylserine (NGF/lyso-PS). CONCLUSION U73122 exerts potent PI-PLC-independent secretostatic effects, limiting its use to define PI-PLC function within exocytotic processes.