Short-term exposure to carbamazepine causes oxidative stress on common carp (Cyprinus carpio)

The aim of this research was to determine the bioconcentration factor and if subacute exposure to carbamazepine (2 mg L⁻¹) modifies the oxidative state of liver, gills and brain of Cyprinus carpio. This was measured through the following biomarkers: hydroperoxide and protein carbonyl content, lipid peroxidation degree, as well as superoxide dismutase, catalase and glutathione peroxidase activity. Carbamazepine concentration in carp’s tissue was also determined by liquid chromatography with a diode arrangement detector. An increase in lipid peroxidation degree, hydroperoxide and protein carbonyl content, and a decrease in the activity of the antioxidant enzymes (P < 0.05) with respect to control was observed. Also, there is an increase in the concentration of carbamazepina present in the organs with respect to the water in the system, which denotes bioconcentration of the drug. In conclusion, carbamazepine is bioconcentrated and produces oxidative stress on the common carp (C. carpio).     

Neurotoxic,biotransformation, oxidative stress and genotoxic effects in Astyanax altiparanae (Teleostei,Characiformes) males exposed to environmentally relevant concentrations of diclofenac and/or caffeine

The present study evaluated neurotoxic, biotransformation, genotoxic and antioxidant responses to relevant environmental concentrations of diclofenac (0.4 μg L⁻¹) and caffeine (27.5 μg L⁻¹), separate and combined, in adult males of the freshwater fish Astyanax altiparanae after a subchronic exposure (14 days). Fish exposed to diclofenac and caffeine, both separate and combined, revealed a neurotoxic effect through the inhibition of acetylcholinesterase activity in the muscle, while diclofenac alone and in combination caused cyclooxygenase inhibition. Caffeine alone produces genotoxicity on this species but, when combined with diclofenac, it potentiates hepatic lipoperoxidation and the inhibition of oxidative stress enzymes, while diclofenac alone or in combination produces a general inhibition of important enzymes. This study suggests that aquatic contamination produced by these pharmaceuticals has the potential to affect homeostasis and locomotion in A. altiparanae and compromise their immune system and general health.     

Anion transport,chloride cell number and nitrite-induced methaemoglobinaemia in rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio)

The toxic effects of nitrite on ionic transport and haemoglobin function in rainbow trout (Salmo gairdneri Richardson) and carp (Cyprinus carpio L.) were investigated. Under similar environmental conditions the maximal rates of chloride and nitrite uptake in carp (25 and 81 μmol h⁻¹kg⁻¹) were lower than in rainbow trout (368 and 198 μmol h⁻¹kg⁻¹). Carp possess 47% less gill chloride cells than trout and the relationship between anion transport and the chloride cell population is discussed.In vitro, methaemoglobin formation occurs more rapidly in carp blood, 5.58% h⁻¹ compared with trout, 3.39% h⁻¹. The carp haemoglobin molecule is more sensitive to nitrite oxidation than trout haemoglobin and the rate of methaemoglobin reduction is more rapid in carp red cells. The principal reason for carp being more tolerant to nitrite than are trout is because of the lower rate of branchial uptake of nitrite, rather than because of any effects on haemoglobin.     

Biomolecular alterations in the early life stages of four food fish following acute exposure of Triclosan

We investigated the effect of acute concentrations of triclosan (TCS; 96 h exposure and 10d post exposure) on the free amino acid, primary (SDS-PAGE) and secondary (FT-IR) structure of proteins in the embryos/larvae of Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala. A concentration dependent increase in free amino acids, upregulation of polypeptides (100 and 70 kDa in C. carpio, C. idella and L. rohita, 55, 45, 36 kda in C. idella and L. rohita and 22 kDa in all the fish) and a decline in percent area of all the selected peaks of the FT-IR spectra was observed after exposure and recovery period. The decline in percent area was greatest for L. rohita at peak 1080 – 1088 cm⁻¹ (−75.99%) after exposure and at peak 2854 – 2855 cm⁻¹ (−53.59%) after recovery. Curve fitting analysis revealed a decrease in α-helices and increase in β-sheets in all fish after exposure and recovery period. The results suggest that TCS elicits alterations in biomolecules of fish embryos.     

Toxicological effects of clofibric acid and diclofenac on plasma thyroid hormones of an Indian major carp,Cirrhinus mrigala during short and long-term exposures

In the present investigation, the toxicity of most commonly detected pharmaceuticals in the aquatic environment namely clofibric acid (CA) and diclofenac (DCF) was investigated in an Indian major carp Cirrhinus mrigala. Fingerlings of C. mrigala were exposed to different concentrations (1, 10 and 100 μg L⁻¹) of CA and DCF for a period of 96 h (short term) and 35 days (long term). The toxic effects of CA and DCF on thyroid hormones (THs) such as thyroid stimulating hormone (TSH), thyroxine (T₄) and triiodothyronine (T₃) levels were evaluated. During the short and long-term exposure period TSH level was found to be decreased at all concentrations of CA (except at the end of 14^th day in 1 and 10 μg L^−l and 21^st day in 1 μg L^−l) whereas in DCF exposed fish TSH level was found to be increased when compared to control groups. T₄ level was found to be decreased at 1 and 100 μg L^−l of CA exposure at the end of 96 h. However, T₄ level was decreased at all concentrations of CA and DCF during long-term (35 days) exposure period. Fish exposed to all concentrations of CA and DCF had lower level of T₃ in both the treatments. These results suggest that both CA and DCF drugs induced significant changes (P < 0.01 and P < 0.05) on thyroid hormonal levels of C. mrigala. The alterations of these hormonal levels can be used as potential biomarkers in monitoring of pharmaceutical drugs in aquatic organisms.     

Evaluation of softwood and hardwood sawmill wastes impact on the common carp "Cyprinus carpio" and its aquatic environment: An oxidative stress study

The aquatic pollution due to sawmill wood wastes constitutes a major threat to hydro-chemical and fauna characteristics of the aquatic ecosystems. When this kind of organics wastes enter aquatic environment it can be taken up by aquatic organisms through respiration and/or through their diet. This could concurrently result in oxidative stress and later having adverse effect on physiological and biochemical function. The importance of fish in the society cannot be over emphasized, hence there is the need to know the influence of sawmill wood wastes on the water quality and fish.Therefore, this work aims to study the impact of five species of wood wastes on a type of fish named common carp (Cyprinus carpio) known as the most widely cultured fish species in the world and on its aquatic environment. The monitoring of water parameters showed deterioration in water quality. The activities of superoxide dismutase (SOD), Catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (MDA) were investigated to evaluate the degree of oxidative stress. According to t-student, there was a significant difference compared to control (P < 0.05) in the level of SOD, CAT, GPx and MDA activities in fish exposed to 5 g·l⁻¹ of each sawdust except for GPx, a non-significant difference (p > 0.05) was noted in the case of Beech and Dibetou. When the dispersed amount was about 0.375 g·l⁻¹ we noted a significant difference in the level of SOD and GPx, except for GPx a non-significant difference was detected in the case of Cedar. The level of CAT was significantly difference just in the case of Cedar and Dibetou and that of MDA was significantly difference just in the case of Beech and Mahogany.We conclude therefore that sawmill wood waste not only impact the water quality adversely but also alters the levels of different enzymes activities in Cyprinus Carpio fish by the inhibition of SOD, CAT and GPx activities and by the production of MDA, which reflects response to oxidative stress. This study provides a rational use of these enzymes as suitable biomarkers with different degrees of specificity and as important tool for environmental monitoring.     

Europium(III) ion probe spectrofluorometric determination of diclofenac sodium

A new method has been devised for the determination of diclofenac sodium in bulk and in pharmaceutical preparations using Eu³⁺ ions as the Fluorescent probe. The technique was built around the hypersensitive property of the transitions of the fluorescent probe ion, Eu³⁺, at 616 nm. This is normally a forbidden transition, but the interaction with diclofenac sodium, which contains a carboxylic group, makes the transition allowed and enhances the intensity of its fluorescence emission. The Eu³⁺ fluorescence emission at 592 nm comes from a non-hypersensitive transition and is not affected by ligation. The intensity ratio, R, defined as I₅₉₂/I₆₁₆, was used as a measure of the percentage of bound probe ions. Diclofenac and Eu(III) forms a (1:1) molar complex.The relative stability constant of the complex was found to be 10⁵. A linear relationship between bound Eu³⁺ and the concentration of diclofenac sodium was found for concentrations from 10 to 200 μg ml⁻¹, with a recovery percentage of 100.22 ± 2.27. The method shows a good agreement with a spectrophotometric method.     

The effects of diclofenac on early life stages of common carp (Cyprinus carpio)

Diclofenac residues have been found in surface water, and thus could present a potential risk to aquatic species. The aim of this study was to assess the impact of diclofenac on the mortality, growth, and development of fish, as well as the impact of the drug on histological changes and selected parameters of oxidative stress in the fish. Subchronic toxic effects of diclofenac at concentrations of 0.015, 0.03, 1, and 3 mg/L on embryos and larvae of common carp (Cyprinus carpio) were investigated during a 30-day toxicity test under experimental conditions. Exposure to diclofenac at 3 mg/L was associated with increased mortality, increased activity of glutathione S-transferase, and decreased activity of glutathione reductase. Decreases in the levels of thiobarbituric-acid-reactive substances were associated with concentrations ≥0.03 mg/L. Based on these results a no observed effect concentration (NOEC) = 0.015 mg/L and lowest observed effect concentration (LOEC) = 0.03 mg/L were generated.     

Toxicity of ammonia,nitrite and nitrate to Litopenaeus vannamei juveniles in low-salinity water in single and ternary exposure experiments and their environmental implications

Information on toxicity of nitrogen compounds for Litopenaeus vannamei in coastal ecosystems and culture under low salinity is scarce. Acute toxicity trials were conducted in L. vannamei to determine the single and combined effects of ammonia, nitrite and nitrate at a salinity of 3 g/L. The 96 h-LC₅₀ was 29.0 mg/L for total ammonia nitrogen (TAN); 10.6 mg/L for nitrogen as nitrite (NO₂⁻-N); and 900 mg/L for nitrogen as nitrate (NO₃⁻-N). The joint effects of ammonia, nitrite and nitrate exposure were antagonistic at 24–72 h; and additive from 72 to 96 h. The proposed safety levels of single exposure to TAN, NO₂⁻-N and NO₃⁻-N for L. vannamei are 1.45, 0.53 and 45.0 mg/L, respectively. When in mixture, the proposed level of TAN/NO₂⁻-N/NO₃⁻-N is 0.05 TU (Toxicity Unit) corresponding to 0.48, 0.08 and 14.6 mg/L of TAN, NO₂⁻-N and NO₃⁻-N, respectively.     

Spectrophotometric study of diclofenac-Fe(III) complex

A multifactor optimisation technique is successfully applied to develop a new spectrophotometric method in which diclofenac sodium is analysed and determined as it's Fe(III) complex. The effect of simultaneously varying the pH, ionic strength and concentration of colour reagents in the reaction mixture were studied. A four-variable two-level factorial design was used to investigate the significance of each variable and interactions between them. A response surface design was used to optimise complex formation and extraction. It was established that diclofenac reacts with Fe(III) chloride, in the presence of ammonium thiocyanate, in the pH range 4.2–6.5, forming a red chloroform extractable (2:1) complex with maximum absorbance at 481 nm. By applying the methods of Sommer and Job involving non-equimolar solutions the conditional stability constant of the complex, at the optimum pH of 6.0 and an ionic strength μ = 0.19M, was found to be 10^6.4. Good agreement with Beer's law was found for diclofenac concentrations up to mmol l⁻¹. The nominal percent recovery of diclofenac was 98.8% (n = 10). The lower limit of sensitivity of the method was found to be 14.7 μg ml⁻¹.     

Fabrication and Evaluation of Bi-layer Tablet Containing Conventional Paracetamol and Modified Release Diclofenac Sodium

The objectives of present investigation were to achieve immediate release of paracetamol and tailored release of diclofenac sodium from bi-layer tablets. A 2³ full factorial design was adopted using the amount of polyethylene glycol, microcrystalline cellulose and crospovidone as independent variables for fabricating paracetamol tablets. Diclofenac sodium tablets were prepared using hydroxypropyl methylcellulose as a matrixing agent. The results of analysis of variance showed that the friability of paracetamol was distinctly influenced by the formulation variables. The in vitro drug release behaviour of diclofenac tablets was compared with a marketed formulation. The optimized formulations of paracetamol and diclofenac sodium were used for manufacturing of bi-layer tablets. The bi-layer tablets showed immediate release of paracetamol and modified release of diclofenac.     

Individual and combined effects of cadmium and copper on the growth response of Chlorella vulgaris

The individual and combined effects of cadmium and copper on the growth response of the green alga, Chlorella vulgaris, were examined. The effects of pH alone, and in combination with copper were also evaluated. An increase in cadmium and copper concentrations caused a significant reduction in the growth of C. vulgaris cells, and the corresponding EC₅₀ values were 1.02 and 4.01 mg L⁻¹, respectively. For a pH range of 2–7, the inhibitory effect due to increased copper concentrations (coupled with the resulting drop in pH) was significantly higher than the impact due to increased acidity (by addition of HCl) alone. At lower metal concentrations (5 mg L⁻¹ Cu + < 2 mg L⁻¹ Cd or 2.5 mg L⁻¹ Cd + < 4 mg L⁻¹ Cu), a combination of copper and cadmium appeared to have a stronger inhibitory effect on cell growth than that of a single metal. In contrast, at higher metal concentrations (5 mg L⁻¹ Cu + > 2 mg L⁻¹ Cd or 2.5 mg L⁻¹ Cd + > 4 mg L⁻¹ Cu), the effect of a single metal exhibited a significantly stronger effect compared to a combination of the two metals. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 347–353, 1999     

Effect of diclofenac and antidepressants on the inflammatory response in astrocyte cell culture

Central nervous system (CNS) has a completely separate immune system that communicates with the neurons by small molecules called cytokines. Cytokines are involved in many crucial processes in neuron including cell metabolism and neurotransmitter synthesis. It has been reported that cytokine imbalance is involved in the progression of many CNS diseases such as neuropsychiatric disorders (depression, schizophrenia, autism, and bipolar disorder) and neurodegenerative disorders (Parkinson’s and Alzheimer’s disease). Here, the effects of diclofenac, different antidepressants (sertraline, venlafaxine, and fluvoxamine), and vitamin B₆ (pyridoxine) on IL-10 and tumor necrosis factor-α (TNF-α) change with and without immune challenges with lipopolysaccharide (LPS) were investigated in in vitro culture of astrocytes from 2-day-old Swiss-Albino mice. Diclofenac and Sertraline significantly (p < 0.05) improves anti-inflammatory cytokine (IL-10) while suppress (p < 0.05) LPS-induced elevated level of pro-inflammatory mediators (TNF-α) in astrocyte culture. Pyridoxine was not able to reduce (p > 0.05) TNF-α in the astrocyte culture. Antidepressant (sertraline) showed positive effects (increased IL-10 and reduced TNF-α level) possibly through the suppression of Th1 lymphocytes and monocytes and stimulation of Th2 lymphocytes and monocytes/macrophages. NSAID (diclofenac) showed positive immune regulation effect possibly through the inhibition of cyclo-oxygenase enzyme. Based on these findings, it may conclude that, diclofenac and antidepressants (sertraline) may positively contribute in the cytokine production in astrocyte cell culture.     

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose

ObjectiveTo investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition.MethodsThe hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmol L⁻¹), the PPD low dose group (1 μmol L⁻¹, PPD-L), the PPD middle dose group (5 μmol L⁻¹, PPD −M) and the PPD high dose group (10 μmol L⁻¹, UCN-H). The GMC were incubated for 48 h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca²⁺]_і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method.ResultsThe viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P < 0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P < 0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca²⁺]_і transient was reduced (P < 0.05 or P < 0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups.ConclusionThe protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca²⁺]_і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.     

Assessment of biochemical,hematological and behavioral biomarkers of Cyprinus carpio on exposure to a type-II pyrethroid insecticide Alpha-cypermethrin

This study assessed some important physiological biomarkers of freshwater edible fish Cyprinus carpio following exposure to 10 % (T1) and 20 % (T2) sublethal concentrations of Alpha-cypermethrin (A-cyp) over a total period of 45 days. Behavioral responses were noticed and Kaplan-Meier survival curves were prepared during acute toxicity study. Total serum protein concentration, total erythrocyte count, hemoglobin, packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and total leukocytes count were decreased significantly (p < 0.05), while the blood glucose, total serum lipid concentration, and clotting time were increased significantly (p < 0.05) over control. The most affected fish group and most significantly altered biomarker under toxic stress of A-cyp were identified using integrated biomarker response (IBR). The biomarker response index (BRI) values measured the overall health status of the treated fish and indicated that moderate adverse effects were exerted on the fish group exposed to T2 for 45 days.     

Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels

Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages. While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in macrophages this K⁺ current is generated by the association of Kv1.3 and Kv1.5. Patients with autoimmune diseases show a high number of effector memory T cells that are characterized by a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca²⁺-activated K⁺ channels (K_Ca3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.     

A study of genotoxicity and oxidative stress induced by mercuric chloride in the marine polychaete Perinereis aibuhitensis

The marine polychaete worm Perinereis aibuhitensis was used to study the genotoxic effects of mercuric chloride by means of the comet assay and micronucleus (MN) test. P. aibuhitensis was subjected in vivo to two different concentrations of mercuric chloride (0.05 mg L⁻¹ and 0.5 mg L⁻¹) for 96 h. The comet assay of coelomocytes demonstrated that TailDNA% values increased with extended exposure to or increased concentrations of HgCl₂ (p < 0.01). The frequency of MNs was the highest in the treatment with 96 h of exposure at all concentrations (p < 0.01). The genotoxic effect of HgCl₂ was both dose- and time-dependent in exposed P. aibuhitensis. The activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidases (GPx) were also estimated. Significant variations in antioxidant enzyme activities depended on the sampling time and the concentrations of mercuric chloride. Compared with the control, the activities of the antioxidant enzymes (SOD and GPx) were elevated at the lower concentration of mercuric chloride (0.05 mg L⁻¹) (p < 0.05) for shorter exposure periods (24 h and 72 h). At the higher concentration of mercury (0.5 mg L⁻¹), the activities of GPx and SOD were inhibited; no variation was observed. These results proved that the use of the comet assay and MN test in coelomocytes of P. aibuhitensis is appropriate for determining the levels of DNA damage and that P. aibuhitensis is a species that is sensitive to mercury pollutants. This species may be considered a suitable candidate for monitoring marine heavy metal pollution.     

Potential protective effect of sunitinib after administration of diclofenac: biochemical and histopathological drug–drug interaction assessment in a mouse model

Sunitinib is a tyrosine kinase inhibitor for GIST and advanced renal cell carcinoma. Diclofenac is used in cancer pain management. Coadministration may mediate P450 toxicity. We evaluate their interaction, assessing biomarkers ALT, AST, BUN, creatinine, and histopathological changes in the liver, kidney, heart, brain, and spleen. ICR mice (male, n?=?6 per group/dose) were administered saline (group A) or 30 mg/kg diclofenac ip (group B), or sunitinib po at 25, 50, 80, 100, 140 mg/kg (group C) or combination of diclofenac (30 mg/kg, ip) and sunitinib (25, 50, 80, 100, 140 mg/kg po). Diclofenac was administered 15 min before sunitinib, mice were euthanized 4 h post-sunitinib dose, and biomarkers and tissue histopathology were assessed. AST was 92.2?±?8.0 U/L in group A and 159.7?±?14.6 U/L in group B (p?<?0.05); in group C, it the range was 105.1–152.6 U/L, and in group D, it was 156.0–209.5 U/L (p?<?0.05). ALT was 48.9?±?1.6 U/L (group A), 95.1?±?4.5 U/L (p?<?0.05) in group B, and 50.5–77.5 U/L in group C and 82.3–115.6 U/L after coadministration (p?<?0.05). Renal function biomarker BUN was 16.3?±?0.6 mg/dl (group A) and increased to 29.9?±?2.6 mg/dl in group B (p?<?0.05) and it the range was 19.1–33.3 mg/dl (p?<?0.05) and 26.9–40.8 mg/dl in groups C and D, respectively. Creatinine was 5.9 pmol/ml in group A; 6.2 pmol/ml in group B (p?<?0.01), and the range was 6.0–6.2 and 6.2–6.4 pmol/ml in groups C and D, respectively (p?<?0.05 for D). Histopathological assessment (vascular and inflammation damages) showed toxicity in group B (p?<?0.05) and mild toxicity in group C. Damage was significantly lesser in group D than group B (p?<?0.05). Spleen only showed toxicity after coadministration. These results suggest vascular and inflammation protective effects of sunitinib, not shown after biomarker analysis.     

The effects of salmeterol and salbutamol on ciliary beat frequency of cultured human bronchial epithelial cells,in vitro

Studies investigating mechanisms of mucociliary clearance have suggested that β₂-adrenergic agents may significantly influence ciliary activity of epithelial cells and therefore play a vital role in the maintenance of functional integrity of the airways. We have cultured human bronchial epithelial cells, from surgical explants and investigated the effects of salbutamol and salmeterol, in a time- and dose-dependent manner, on the ciliary beat frequency (CBF) of these cells. Prior to and at several times after exposure to either salbutamol (10⁻⁸ to 10⁻³ m) or salmeterol (10⁻⁸ to 10⁻⁴ m), the epithelial cells were monitored for CBF and on the basis of data obtained from these studies, the effect of 10⁻⁶ m propranolol was investigated in the presence of optimal concentrations of salbutamol and salmeterol. Salbutamol was optimally active at a concentration of 10⁻⁴m and caused a transient but significant increase in the CBF from baseline level of 8.6 ± 0.4 to 9.6 ± 0.5 Hz (P < 0.05), after 2 h incubation. In contrast, salmeterol was maximally active at a concentration of 10⁻⁶m and caused a significantly rapid and prolonged increase in CBF from a baseline value of 9.2 ± 0.4 to 10.9 ± 0.6 Hz (P < 0.02) and 10.6 ± 0.8 Hz (P < 0.05) after 15 min and 24 h incubation, respectively. Propranolol (10⁻⁶ m) abrogated the salbutamol- but not the salmeterol-induced increases in CBF. Analysis of cultures for cAMP demonstrated that both salbutamol and salmeterol led to a significant release of cAMP into the culture medium and increased the amounts of CAMP from a baseline value of 4.5 ± 0.7 fmol/μg protein to 137.4 ± 38.7 and 151.8 ± 49.1 fmol/μg protein, respectively, after 24 h incubation (P < 0.001). Propranolol significantly attenuated both the salbutamol- and salmeterol-induced release of cAMP (P < 0.001 and P < 0.01, respectively). These results suggest that salmeterol may be of particular significance in the management of airway disease due to its role in mucociliary clearance, in addition to bronchodilation.