Topical mecamylamine for diabetic macular edema.

PURPOSE: Stimulation of nicotinic acetylcholine (nACh) receptors on vascular endothelial cells promotes angiogenesis and vascular permeability in animal models. The safety and bioactivity of topical mecamylamine, an antagonist of nACh receptors, was tested in patients with diabetic macular edema. DESIGN: A multicenter phase I/II clinical trial. METHODS: Twenty-three patients with chronic diabetic macular edema received 1% mecamylamine topically twice daily for 12 weeks, the primary end point. Patients underwent safety assessments, measurement of best-corrected visual acuity (BCVA), and measurement of foveal thickness using optical coherence tomography at baseline, 1, 4, 8, 12, and 16 weeks. RESULTS: Mecamylamine drops were well tolerated and there were no drug-related safety problems. Mean improvement in BCVA at 1, 4, 8, 12, and 16 weeks was 2.8, 1.9, 2.4, 0.8, and 3.1 letters, respectively. There was little change in mean excess foveal thickness. There was substantial heterogeneity in response, because 8 patients showed convincing improvement in BCVA, foveal thickness, or both, 9 patients showed equivocal or no substantial changes, and 4 patients showed worsening. Five patients showed a substantial improvement in BCVA, foveal thickness, or both between their last visit while receiving mecamylamine and 1 month after stopping mecamylamine. CONCLUSIONS: This study suggested that administration of topical mecamylamine, a nonspecific nACh receptor blocker, may have heterogeneous effects in patients with diabetic macular edema. Variable expression of nACh receptor subtypes on endothelial cells that have different effects on permeability would provide an explanation for these results and should be investigated, because more specific nACh receptor blockers may dissociate antipermeability and propermeability effects.

Characteristics of the pulse waveform during altered nitric oxide synthesis in the rabbit.

Nitrovasodilators produce characteristic changes in the shape of the peripheral pulse wave. Similar changes might also be caused by alteration of endogenous NO activity, which would allow such activity to be assessed in vivo. We investigated whether manipulation of the NO pathway influences the pulse waveform, and the mechanisms involved. The pulse wave in the ear of normal rabbits was examined by reflectance photoplethysmography before and during infusion of vasoactive agents. Pulse wave velocity was assessed by using an additional sensor on the rear foot. A diastolic peak was observed in the ear pulse; its timing was consistent with it being a reflection of the systolic peak from the lower body. The height of the dicrotic notch marking the start of this diastolic wave was decreased by acetylcholine or an NO donor, and further decreased by a phosphodiesterase type V inhibitor. The acetylcholine-induced decreases were blocked by inhibiting NO synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME) but were unaffected by the inactive enantiomer D-NAME. These data demonstrate that NO influences the height of the notch in the pulse wave. Heart rate and blood pressure were altered during acetylcholine or L-NAME infusion, but there were no changes in pulse wave amplitude or velocity, or in the timing of the diastolic peak or dicrotic notch. The slope of the pulse wave between the systolic peak and notch changed substantially. These effects are most convincingly explained by changes in wave reflection, not only from the lower body but also from more proximal sites.

Analysis of the signal transduction in the induction of nitric oxide synthase by lipoteichoic acid in macrophages.

1. This study investigates the signal transduction mechanisms leading to the enhanced formation of nitric oxide (NO) due to the induction of NO synthase (iNOS) in murine J774.2 macrophages in culture activated with lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus. 2. LTA (10 microgram ml-1) caused within 24 h an enhanced accumulation of nitrite (an indicator of NO biosynthesis) in the supernatant of J774.2 macrophages which was prevented by the non-selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; IC50: 35 microM) or by the iNOS-selective NOS inhibitor, aminoethyl-isothiourea (AE-ITU; IC50: 6 microM). The inhibition of nitrite formation afforded by these agents was prevented by excess L-arginine (3-30 mM), but not by D-arginine (3-30 mM). Furthermore, the degree of iNOS inhibition was similar when these NOS inhibitors were added to the macrophages 10 h after LTA. 3. Pretreatment of J774.2 macrophages with cyclohexamide or dexamethasone prevented the enhanced formation of nitrite caused by LTA. This inhibition did not occur when dexamethasone or cyclohexamide were added to the cells 10 h after LTA. The increase in nitrite formation stimulated by LTA (10 micrograms ml-1) was not affected by polymyxin B (0.05-0.5 microgram ml-1), an agent which binds and inactivates endotoxin. 4. A specific inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), D609, prevented the increase in nitrite formation (IC50 = 20 micrograms ml-1) caused by LTA. The inhibition afforded by D609 was significantly smaller when this agent was added to the cells 10 h after LTA. 5. The structurally distinct tyrosine kinase inhibitors, erbstatin, genistein, and tyrphostin AG126 prevented the formation of nitrite caused by LTA. The inhibition afforded by these compounds was significantly attenuated when they were added to the cells 10 h after LTA. In contrast, daidzein or tyrphostin A-1, which are inactive analogues of genistein and tyrphostin (up to a concentration of 10 microM) did not affect the nitrite formation caused by LTA. 6. Inhibitors of the activation of the nuclear transcription factor NF-kappa B such as pyrrolidine dithiocarbamate (PDTC; an antioxidant and a metal chelator), butylated hydroxyanisole (BHA; an antioxidant), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), calpain inhibitor I (both I kappa B-protease inhibitors), or rotenone (an antioxidant which inhibits electron transport) prevented the nitrite formation stimulated by LTA. The inhibition afforded by these agents was significantly smaller when they were added to the macrophages 10 h after LTA. 7. Incubation of J774.2 cells with LTA over 24 h resulted in the expression of iNOS protein (130 kDa) as identified by Western blot analysis. The expression of iNOS protein by LTA was significantly attenuated by cyclohexamide, D609, tyrphostin AG126, PDTC or by TPCK. 8. Thus, the signal transduction leading to the expression of iNOS protein and activity caused by LTA in murine J774.2 macrophages involves (i) the activation of PC-PLC, (ii) phosphorylation of tyrosine kinase, and (iii) the activation of the transcription factor NF-kappa B.

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms.

Endothelin-1 mediates the increase in ex vivo coronary vascular resistance induced by hemorrhagic shock in the anesthetized rat.

Cytokines such as tumor necrosis factor-alpha (TNF-alpha), given exogenously or liberated endogenously, cause the release of endothelin (ET) into the circulation. In the rat, increase in ET causes marked ex vivo coronary vasoconstriction. Hemorrhage also increases the circulating levels of cytokines such as TNF-alpha. Here we show that in rats subjected to hemorrhagic shock there is a marked increase in ex vivo coronary vascular resistance, which is mediated by ET. Hemorrhagic shock was induced in anesthetized rats by withdrawing sufficient blood to reduce mean arterial blood pressure to 40 mm Hg for a period of 30 min, after which all the withdrawn blood was retransfused over a period of 15 min. Hearts were perfused ex vivo at a constant flow of 10 ml/min according to the Langendorff technique. After 90 min in vitro, the coronary perfusion pressure in hearts removed from control rats was 76 +/- 1 mm Hg (n = 5), whereas in hearts taken from rats after hemorrhage it was 114 +/- 6 mm Hg (n = 5; p < 0.05 from control). After the same time in vitro, the coronary perfusion pressure of hearts from rats treated with TNF-alpha (4 micrograms/kg, i.v.) was 122 +/- 4 mm Hg (n = 4; p < 0.05 from control). The increases in coronary perfusion pressure caused by hemorrhagic shock or TNF-alpha were abolished by pretreating rats with the nonselective ET receptor (ETA/ETB) antagonist SB209670 (3 mg/kg, i.v.) (coronary perfusion pressure at 90 min 80 +/- 1 mm Hg after hemorrhage; 73 +/- 4 mm Hg after TNF-alpha, p < 0.05 compared to hemorrhage or TNF-alpha controls, respectively; n = 3-5). Interestingly, pretreatment with polyclonal antibodies to TNF-alpha (3 mg/kg, i.v.) did not significantly attenuate the rise in coronary perfusion pressure caused by hemorrhage. Therefore, hemorrhage followed by retransfusion causes marked coronary vasoconstriction assessed ex vivo owing to the release of ET by factors including TNF-alpha.

pKI values of prazosin and idazoxan for receptors stimulated by neuronally released transmitter in the epididymal portion of rat isolated vas deferens.

1. A new method has been used to measure pKI values of prazosin and idazoxan against neuronally-released transmitter in the epididymal portion of the rat isolated vas deferens. The most reproducible results were obtained with a prolonged antagonist equilibration time (1 h). 2. Under these conditions the pKI of prazosin was practically unaffected by addition of alpha, beta-methylene-adenosine-5'-triphosphate (10 microM) to desensitize purinoceptors. Addition of desmethylimipramine (DMI) (0.3 microM) produced a small, but statistically non-significant, reduction. 3. The same method has been used to measure the pKI of prazosin against exogenous noradrenaline. In the latter case addition of DMI (0.3 microM) and corticosterone (30 microM) together produced a statistically significant reduction in the apparent pKI of prazosin. 4. The new method for estimating pKI values shows that DMI itself acts either pseudo-irreversibly or non-competitively and may be reducing the apparent pKI of prazosin. 5. The pKI values obtained for prazosin and idazoxan against neuronally-released transmitter are in good agreement with those obtained by other workers for the actions of these drugs on alpha-adrenoceptors.