Revisiting the usefulness of thromboxane-A2 modulation in the treatment of bronchoconstriction in asthma.

Airway smooth muscle (ASM) is the effector cell in the bronchoconstrictory pathway. It is believed that the bronchoconstriction present in asthma is associated with changes in the airway milieu that affect ASM excitation-contraction coupling and Ca(2+)-handling. Asthmatics also react differently to ventilatory mechanical strain. Deep inspiration (DI), which produces bronchodilation in healthy individuals, is less effective in asthmatics, and even enhances bronchoconstriction in moderate to severely affected patients. Our laboratory has previously studied the mechanotransductory pathway of airway stretch-activated contractions (Rstretch) leading to DI-induced bronchoconstriction. We demonstrated the ability of agonists acting through thromboxane A2 (TxA2) receptors to amplify airway Rstretch responses. Despite the involvement of excitatory prostanoids in bronchoconstriction, clinical trials on treatments targeting TxA2-synthase inhibition and TP-receptor antagonism have produced mixed results. Studies in Western populations produced mostly negative results, whereas studies performed in Asian populations showed mostly positive outcomes. In this review, we discuss the role of TxA2-synthase inhibition and TP-receptor antagonism in the treatment of asthmatics. We present information regarding variations in study designs and the possible role of TP-receptor gene polymorphisms in previous study outcome discrepancies. Perhaps future studies should focus on asthmatic patients with DI-induced bronchoconstriction in particular, planting the seed for the individualized treatments for asthmatics.

L-type Ca(2+) channels, Ca(2+)-induced Ca(2+) release, and BK(Ca) channels in airway stretch-induced contraction.

Resistance arteries constrict in response to mechanical stress. This response is myogenic in nature, and reliant on membrane depolarization, activation of L-type Ca(2+) channels, Ca(2+)-induced Ca(2+)-release and large conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Airway smooth muscle is also affected by mechanical stress: a deep inspiration produces a bronchodilation in healthy individuals, but bronchoconstriction in moderate to severe asthmatics. In this study, our objective was to investigate the regulation of this airway stretch-activated contractile response (R(stretch)), and explore its similarities to the vascular myogenic response. Using a pharmacological approach in intact bovine bronchial segments cannulated horizontally in an organ bath, we showed the ability of carbachol (2-carbamoyloxyethyl-trimethyl-azanium), KCl, neurokinin-A, and U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z, 13E-dien-1-oic acid) to generate R(stretch) in a concentration-dependent manner. R(stretch) was significantly reduced by nifedipine, ryanodine, and iberiotoxin, suggesting that it possesses characteristics similar to those of the vascular smooth muscle myogenic response, such as a role for membrane depolarization, L-type Ca(2+) channel, ryanodine receptors and BK(Ca) channel activation. This study demonstrates a novel role for the L-type Ca(2+) channel in airway smooth muscle and provides new insights into possible mechanisms regulating the deep inspiration-induced bronchoconstriction seen in asthmatics.

Intestinal, airway, and cardiovascular relaxant activities of thymoquinone.

Thymoquinone (TQ) is a bioactive component found in many medicinal herbs. In this study, we report the smooth and cardiac muscle relaxant activities of this compound. TQ concentration dependently suppressed spontaneously contracting rabbit jejunum while also relaxed high K(+)-(80 mM) induced contractions in jejunum and guinea-pig ileum, indicating activity at voltage-operated Ca(++) channels (VOCC). Further, TQ displaced Ca(++) concentration-response curves, obtained in a Ca(++)-free environment, to the right, showing blockade of VOCC. Similar activity was observed with verapamil, a standard VOCC blocker. TQ also exhibited nonadrenergic relaxation of agonist-induced contractions in guinea-pig trachea. When tested in fluo-4-loaded mouse lung slices, TQ inhibited ACh-induced airway narrowing and Ca(++) signalling in airway smooth muscle cells. In endothelium-intact and endothelium-denuded rat aorta, TQ inhibited high K(+)-induced contractions at significantly lower concentrations than phenylephrine-(PE-) (1 microM) induced contractions. Relaxation of PE-induced contractions was resistant to blockade by L-NAME and atropine. In guinea-pig atria, TQ showed noncholinergic relaxation of atrial force and rate of contractions. These data suggest smooth and cardiac muscle relaxant activity of TQ possibly mediated, in part, via blockade of VOCC. The results also justify the use of TQ containing plants in related health disorders like colic, diarrhoea, cough, and asthma.

Thromboxane prostanoid receptor activation amplifies airway stretch-activated contractions assessed in perfused intact bovine bronchial segments.

A deep inspiration (DI) produces bronchodilation in healthy individuals. Conversely, in asthmatics, DIs are less effective in producing bronchodilation and can cause more rapid airway renarrowing and even bronchoconstriction in moderate to severe asthmatics. It is noteworthy that the manner by which a DI is able to cause bronchoconstriction via a stretch-activated contraction (R(stretch)) is thought to correlate positively with airway inflammation. Asthmatic airway inflammation is associated with increased production of thromboxane A(2) (TxA(2)) and subsequent thromboxane prostanoid (TP) receptor activation, causing the heightened contractility of airway smooth muscle. In this study, we sought to investigate the effect of TxA(2) on airway R(stretch) by using bovine bronchial segments. In brief, these intact bronchial segments (2 mm in diameter) were dissected, side branches were ligated, and the tissues were mounted horizontally in an organ bath. R(stretch) was elicited by varying the transmural pressure under isovolumic conditions. Using a pharmacological approach, we showed a reduced R(stretch) response in tissues pretreated with indomethacin, a cyclooxygenase inhibitor, a result mimicked by pretreatment with the TP-selective receptor antagonist 4-(Z)-6-(2-o-chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl)hexenoic acid (ICI 192605) and the selective p42/p44 mitogen-activated protein kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 95089) and by airway epithelial denudation. 9,11-Dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), a TP receptor agonist, elicited enhanced R(stretch) responses in a dose-dependent manner. Pretreatment with 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a prostaglandin E (EP) receptor 1/prostaglandin D2 (DP)-selective receptor antagonist, and 9α,15R-dihydroxy-11.ß-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta-5Z,13E-dien-1-oic acid (AL 8810), a prostaglandin F (FP)-selective receptor antagonist, had no effect, suggesting EP, DP, and FP receptor activation is not involved in amplifying airway smooth muscle R(stretch). These data suggest a role for TP receptor activation and epithelial release of TxA(2) in amplifying airway R(stretch), thus providing novel insights into mechanisms regulating the DI-induced bronchoconstriction seen in asthmatics.

Real-time Imaging of Ca-handling in Intact Renal Glomeruli Using Confocal Microscopy.

Glomeruli are filtering units in the kidneys. Being multicellular and complex in structure, many aspects of glomerular function are yet to be elucidated. Most studies use glomerular cells in culture, which may exhibit altered physiology compared to native cells. Confocal microscopy has opened new avenues in exploring in situ glomerular function and physiology. In this report, we propose experimenting with glomerular cells in renal cortical slices and isolated intact glomeruli for Ca(2+)-handling studies. Cortical slices (100 µm thick) were obtained from mice while intact glomeruli were isolated from rats using the sieving method. These were loaded with fluo-4 and then placed in a confocal microscope. Fluo-4 was excited using a 488 nm photodiode laser and images were collected at 1 frame/sec. Changes in average fluorescence intensity (AFI) were interpreted as changes in [Ca(2+)](i). AFI increased to 37.1 ± 6.7% and 84.3 ± 20.9% with Ang II (0.01 and 0.1 µM respectively). Norepinephrine (10 µM), arginine vasopressin (0.1 µM) and K(+) (30 mM) also elevated AFI by 26.5 ± 6.8%, 22.3 ± 1.0% and 39.8 ± 10.3% respectively in the glomerular cells. Likewise in isolated glomeruli, Ang II (0.1-10 µM), K(+) (30-90 mM) and endothelin-1 (0.01-1 µM), all showed elevation in [Ca(2+)](i). These results give an impetus for future studies examining Ca(2+)-handling by confocal microscopy in glomerular cells using renal cortical slices and isolated intact glomeruli. The results support the utility of this system for study of glomerular physiology and pharmacology.

Involvement of the neurokinin-2 receptor in airway smooth muscle stretch-activated contractions assessed in perfused intact bovine bronchial segments.

The airway response to deep inspirations (DIs) in asthmatics has been shown to be ineffective in producing bronchodilation and can even cause bronchoconstriction. However, the manner by which a DI is able to cause bronchoconstriction remains ambiguous. We sought to investigate the pathway involved in this stretch-activated contraction and whether this contraction is intrinsic to airway smooth muscle (ASM). In brief, intact bovine bronchial segments were dissected, and side branches were ligated and then mounted horizontally in an organ bath. Intraluminal pressure was measured under isovolumic conditions. Instantaneously opening and then closing the tap on a column of fluid 5 to 30 cm high evoked a sudden increase in intraluminal pressure (equivalent to the height of the column of fluid) followed by a stress relaxation response of the ASM. When tissues were stimulated with carbachol (10(-8) M) or serotonin (10(-7) M) for 10 min, and the consequent agonist-evoked pressure response was dissipated manually, the response to the same transmural stretch was accompanied by a slowly developing and prolonged increase in intraluminal pressure. This stretch-activated response was significantly diminished by the stretch-activated cation channel blocker gadolinium (10(-3) M), the L-type Ca2+ channel blockers nifedipine (2 x 10(-6) M), diltiazem (10(-5) M), and verapamil (10(-5) M), the sensory neurotoxin capsaicin (10(-5) M), and the neurokinin (NK)(2) receptor antagonists MEN 10376 ([Tyr(I),d-Trp(6,8,9),Lys(10)]-NKA(4-10)) (10(-5) M) and SR48968 (N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide) (3 x 10(-6) M). These results show the ability of isolated airways to exhibit stretch-activated contractions and suggest a role for stretch-activated cation channels, sensory afferent neurons, the neurotransmitter NKA, and L-type Ca(2+) channels in these isolated airway responses.