Galanin inhibition of enteric cholinergic neurotransmission: guanosine triphosphate-binding protein interactions with adenylate cyclase.

BACKGROUND: The ability of galanin, a unique 29 amino acid peptide, to affect cholinergic neurotransmission was examined in the guinea pig myenteric plexus. METHODS: The effects of galanin on tritiated acetylcholine ([3H]ACh) release were studied with cultured guinea pig myenteric plexus neurons. Functional correlations were made with longitudinal strips of ileal smooth muscle with attached myenteric plexus for examination of isometric contraction. RESULTS: Galanin abolished potassium-stimulated [3H]ACh release (170% +/- 18% of basal vs 109% +/- 16%). Galanin inhibited [3H]ACh release stimulated by forskolin or cholera toxin. [3H]ACh release stimulated by cholecystokin octapeptide, calcitonin gene-related peptide, substance P, or vasoactive intestinal peptide was also suppressed by galanin (10(-8) mol/L). Inhibitory effects were reversed by pertussis toxin preexposure, indicating involvement of guanosine triphosphate-binding proteins. Galanin inhibited contractility of longitudinal smooth muscle strips exposed to cholecystokinin-8 (EC50 7.0 X 10(-9) mol/L for cholecystokinin alone vs 1.3 X 10(-8) mol/L for cholecystokinin-8 plus galanin) and abolished contractile responses to calcitonin gene-related peptide. CONCLUSIONS: Galanin inhibits cholinergic neurotransmission in myenteric plexus neurons. Inhibitory effects involve guanosine triphosphate-binding protein mediation.

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.

C3a57-77, a C-terminal peptide, causes thromboxane-dependent pulmonary vascular constriction in isolated perfused rat lungs.

Pulmonary hypertension occurs after the intravascular activation of complement. However, it is unclear which activated complement fragments are responsible for the pulmonary vascular constriction. We investigated the 21-carboxy-terminal peptide of C3a (C3a57-77) to see if it would cause pulmonary vascular constriction when infused into isolated buffer-perfused rat lungs. Injection of C3a57-77 (225 to 450 micrograms) caused mean pulmonary arterial pressure (Ppa) to rapidly increase. However, the response was transient, with Ppa returning to baseline within 10 min of its administration. C3a57-77 also resulted in an increase in lung effluent thromboxane B2 (TXB2), concomitant with the peak increase in Ppa. C3a57-77 did not affect the amount of 6-keto-PGF1 alpha in the same effluent samples. Indomethacin inhibited the C3a57-77-induced pulmonary artery pressor response and the associated TXB2 production. Indomethacin also decreased lung effluent 6-keto-PGF1 alpha. The thromboxane synthetase inhibitors CGS 13080 and U63,357 inhibited the C3a57-77-induced pulmonary artery pressor response and TXB2 production without affecting 6-keto-PGF1 alpha. These inhibitors did not inhibit pulmonary artery pressor responses to angiotensin II. Tachyphylaxis to C3a57-77 occurred because a second dose of C3a57-77 administered to the same lung failed to cause a pulmonary artery pressor response or TXB2 production. The loss of the pressor response was not due to a C3a57-77-induced decrease in pulmonary vascular responsiveness because pressor responses elicited by angiotensin II were not altered by lung contact with C3a57-77. Thus, C3a57-77 caused thromboxane-dependent pulmonary vascular constriction in isolated buffer perfused rat lungs.

Lung injury caused by cobra venom factor is reduced in rats raised on an essential fatty acid-deficient diet.

Arachidonate metabolites appear to be involved in lung injury caused by cobra venom factor (CVF)-induced complement and polymorphonuclear leukocyte (PMN) activation. These studies were designed to assess the effects of a dietary-induced deficiency of arachidonic acid on CVF-induced lung injury. Rats raised on an essential fatty acid-deficient (EFAD) diet exhibited the expected changes in fatty acid composition including decreased plasma levels of arachidonic acid and increased levels of 5,8,11-eicosatrienoic acid. In intact rats raised on the EFAD diet, CVF-induced lung injury was attenuated. When blood and excised lungs from rats raised on the normal diet were used, CVF caused pulmonary vascular constriction and acute lung injury, as evidenced by increased 125I-labeled bovine serum albumin accumulation in lung parenchyma and alveolar lavage fluid. The CVF-induced pulmonary artery pressor response and lung injury were reduced when blood perfusate or blood perfusate and excised lungs were obtained from rats raised on the EFAD diet. The pulmonary vascular constriction and lung injury were not attenuated when the blood perfusate was obtained from rats raised on the normal diet, irrespective of whether the excised lungs were obtained from rats raised on the normal or EFAD diet. PMNs obtained from rats raised on the EFAD diet demonstrated decreased superoxide production as well as impaired random migration and chemotaxis in vitro. In contrast, beta-glucuronidase release was quantitatively similar to PMNs from control rats. These data indicate that the EFAD diet-induced attenuation of CVF-induced pulmonary hypertension and acute lung injury is due to defective effector cells in blood rather than modified pulmonary target tissue.

Cytoplasts and Mo1-deficient neutrophils pretreated with plasma and LPS induce lung injury.

We hypothesized that neutrophil adhesion and lung injury could occur independent of the surface receptor glycoprotein, Mo1 (C3bi receptor). We investigated whether preincubation of human neutrophil-derived cytoplasts (cell fragments that lack nuclei and granules and have a fixed number of surface Mo1 receptors) with plasma and lipopolysaccharide (LPS) would augment the cytoplasts' ability to cause lung injury when activated. We also investigated whether preincubating normal human neutrophils treated with anti-Mo1 antibody with plasma and LPS would increase the neutrophils' ability to adhere and cause lung injury. Human neutrophils infused into isolated salt-perfused rat lungs subsequently stimulated with phorbol 12-myristate 13-acetate (PMA) resulted in lung injury as assessed by the accumulation of 125I-bovine serum albumin in the lung parenchyma. The infusion of cytoplasts resulted in significantly less injury. Cytoplasts preincubated in 20% human plasma and LPS caused an increase in lung injury. Similarly, neutrophils treated with plasma, LPS, and anti-Mo1 antibody or neutrophils congenitally deficient in the Mo1 surface receptor and treated with plasma and LPS augmented lung injury. Plasma and LPS preincubation also increased anti-Mo1 antibody-treated neutrophil adhesion to endothelial cell monolayers after activation by PMA. Thus, plasma and LPS increase adhesion and lung injury caused by neutrophils or neutrophil fragments that share defects in Mo1 receptor expression.

Eicosanoids are involved in the permeability changes but not the pulmonary hypertension after systemic activation of complement.

Intravenous injection of the complement activator, cobra venom factor (CVF), produces acute lung injury that is neutrophil-dependent and oxygen radical mediated. Using the ex vivo model of lung perfusion, the current studies were designed to measure the appearance of eicosanoids in relation to the development of pulmonary arterial hypertension and vascular permeability. Inhibitors of the cyclooxygenase and lipoxygenase pathways were also employed to assess the possible role of eicosanoids in these two functional responses. Ten minutes after infusion of CVF, when the pulmonary hypertensive changes were maximal, no increases in eicosanoids could be detected in whole lung lavage fluid (TXB2, 6-keto-PGF1 alpha, LTB4, LTC4) or plasma (TXB2, 6-keto-PGF1 alpha) and the inhibitors failed to affect the pressor response. In contrast, lung injury as defined by increased vascular permeability was temporally associated with the appearance in whole lung lavage fluid of TXB2, LTB4 and LTC4, the presence of which was blocked by the relevant inhibitors. Lung injury was attenuated by both cyclooxygenase and lipoxygenase inhibitors. This effect was not peculiar to the isolated lung model since cyclooxygenase (ibuprofen, indomethacin) and lipoxygenase (nafazatrom, U66,855) inhibitors also attenuated the CVF-induced increased vascular permeability in intact rats. These data suggest that in the model system employed eicosanoid production is linked to increases in lung vascular permeability but not to pulmonary artery hypertension.