1 The purpose of this study was to investigate the antitussive activity and sites of action of the NK 1 and NK 2 tachykinin receptor antagonists, 994, SR 48968, and the racemate of SR 48968, SR 48212A in the cat and guinea-pig. 2 Guinea-pigs were dosed subcutaneously (s.c.) with CP-99,994, SR 48212A or SR 48968 one hour before exposure to aerosols of capsaicin (0.3 mM) to elicit coughing. Coughs were detected with a microphone and counted. 3 Intracerebroventricular (i.c.v.) cannulae were placed in the lateral cerebral ventricles of anaesthetized guinea-pigs. Approximately one week later, the animals were dosed with 994 or SR 48212A (i.c.v.) and exposed to aerosols of capsaicin (0.3 mM) to elicit coughing. 4 Cough was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was monitored from electromyograms of respiratory muscle activity. Cannulae were placed for intravenous (i.v.) or, in separate groups of animals, intravertebral arterial (i.a.) administration of CP-99,994, SR 48212A or SR 48968. Dose-response relationships for i.v. and i.a. administration of each drug were generated to determine a ratio of i.v. ED 50 to i.a. ED 50 , known as the e ective dose ratio (EDR). The EDR will be 20 or greater for a centrally active drug and less than 20 for a peripherally active drug. 5 In the guinea-pig, CP-99,994 (0.1 ± 30 mg kg 71 , s.c.), SR 48212A (1.0 ± 30 mg kg 71 , s.c.), and SR 48968 (0.3 ± 3.0 mg kg 71 , s.c.) inhibited capsaicin-induced cough in a dose-dependent manner. Capsaicininduced cough was also inhibited by i.c.v. administration of CP-99,994 (10 and 100 mg) or SR 48212A (100 mg). 6 In the cat, both CP-99,994 (0.0001 ± 0.3 mg kg 71 , i.a., n=5; 0.003 ± 3.0 mg kg 71 , i.v., n=5) and SR 48212A (0.003 ± 1.0 mg kg 71 , i.a., n=5; 0.01 ± 3.0 mg kg 71 , i.v., n=5) inhibited mechanically induced cough by either the i.v. or i.a. routes in a dose-dependent manner. SR 48968 (0.001 ± 0.3 mg kg 71 , i.a., n=5; 0.03 ± 1.0 mg kg 71 , i.v., n=5) inhibited cough when administered by the i.a. route in a dosedependent manner, but had no e ect by the i.v. route up to a dose of 1.0 mg kg 71 . Intravenous antitussive potencies (ED 50 , 95% con®dence interval (CI)) of these compounds were: CP-99,994 (0.082 mg kg 71 , 95% CI 0.047 ± 0.126), SR 48212A (2.3 mg kg 71 , 95% CI 0.5 ± 20), and SR 48968 (41.0 mg kg 71 , 95% CI not determined). The intra-arterial potencies of these compounds were: CP-99,994 (1.0 mg kg 71 , 95% CI 0.4 ± 1.8), SR 48212A (25 mg kg 71 , 95% CI 13 ± 52), and SR 48968 (8.0 mg kg 71 , 95% CI 1 ± 32). The derived EDRs for each compound were: 994, 82; SR 48212A, 92; and SR 48968, 4125. 7 We concluded that CP-99,994 and SR 48968 inhibit cough in the guinea-pig and cat by a central site of action. In the cat, the antitussive action of these compounds appears to be solely by a central site.

1 The GABA-B receptor agonists baclofen and 3-aminopropylphosphinic acid (3-APPi) have antitussive activity in the cat and guinea pig. The purpose of this study was to investigate the sites of action of these GABA-B receptor agonists to inhibit the cough reflex. 2 Single intracerebroventricular (i.c.v.) cannulas were placed in the lateral ventricles of anaesthetized guinea pigs. Approximately 1 week later, the animals were exposed to aerosols of capsaicin (0.3 mM) to elicit coughing. Coughs were detected with a microphone and counted. 3 Cough was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was recorded from electromyograms of respiratory muscle activity. Cannulas were placed for intravenous (i.v.) or, in separate groups of animals, intravertebral arterial (i.a.) administration of baclofen, 3-APPi, the centrally active antitussive drug codeine or the peripherally active antitussive drug BW443c. Dose-response relationships for i.v. and i.a. administration of each drug were generated to determine a ratio of i.v. ED" to i.a. ED50, known as the effective dose ratio (EDR). The EDR will be 20 or greater for a centrally acting drug. 4 In the guinea pig, baclofen (3 mg kg-', s.c.) and 3-APPi (10 mg kg-', s.c.) inhibited capsaicininduced cough by 50% and 35% respectively. The antitussive activity of baclofen was completely blocked by i.c.v. administration of the GABA-B receptor antagonist CGP 35348 (10 jig). Conversely, the antitussive effect of 3-APPi was unaffected by i.c.v. CGP 35348. However, systemic administration of CGP 35348 (30 mg kg-', s.c.) completely blocked the antitussive activity of 3-APPi (10 mg kg-', s.c.). In separate experiments baclofen alone (1 gpg, i.c.v.) inhibited capsaicin-induced cough by 78%. 3-APPi (10 and 100 gg, i.c.v.) had no effect on capsaicin-induced cough in the guinea pig. 5In the cat, potencies (ED50) of the standards and GABA-B agonists by the i.v. route were: codeine (0.34 mg kg-'), BW443C (0.17 mg kg-'), baclofen (0.63 mg kg-') and 3-APPi (2.3 mg kg-'). Potencies of these drugs by the i.a. route were: codeine, 0.013 mg kg-'; BW443C, 0.06mg kg-'; baclofen, 0.016mg kg-'; and 3-APPi, 0.87 mg kg'. The EDRs for each drug were: codeine, 26; BW443C, 3; baclofen, 39; and 3-APPi, 3. 6 We conclude that in both the cat and guinea pig baclofen inhibits cough by a central site of action, while 3-APPi inhibits cough by a peripheral site of action.

Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1β-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.