This is the first study to show lipid-soluble smoking particles "DSP" to cause a bronchial hyperresponsiveness to endothelin-1, a strong airway constrictor, and to S6c, a specific ETB receptor agent. Since no change in mRNA was found, except in protein expression, the effect can be considered to be mediated by an increase of the number endothelin receptors via translation. Smoking has been shown in the long run to result in airway distortion and an increase in airflow resistance, and to lead to the airways becoming muscularized and fibrotic. Similar changes can be observed after several decades of asthmatic disease. More recently, it has been shown that in childhood asthma such changes occur early.
Previous studies of the endothelin system have led to differing results. We found bronchial biopsies from patients with asthma and chronic airway obstruction to show significantly higher levels of endothelin ETB receptor mRNA than of endothelin ETA receptor mRNA . Inflammation induced in the human temporal by the pro-inflammatory cytokine interleukin-1beta (IL-1beta) artery has been found to result in an increase in maximal contraction and potency in response to S6c but not to any change in the ETA/ETB receptor mRNA ratio . The explanation suggested was that IL- 1beta may further stimulate translation of the mRNA to active receptors. The hyperreactivity could also be a function of the length of exposure to the pro-inflammatory stimuli. Culturing murine tracheal segments in the presence of IL-1β has been found to attenuate the maximal contraction produced by ETB receptors and downregulated expressions of the ETB receptor mRNA. This was first observed after 2 days of culture, reduction being maximal at 4 days . Bronchial smooth muscle from rats exposed to cigarette smoke for two weeks was found to show hyperreactivity to acetylcholine but no depolarization due to high K+ level, this being explained in terms of increased expression of the RhoA-protein .
Another well-known feature of pulmonary inflammation is epithelial damage. Early asthmatic inflammation has been found to cause disruption of the epithelium . In smoking, the respiratory epithelium is damaged or missing. In the presence of DSP, arterial endothelium lost its attachment and functional tests showed reduced dilation . The clearance of ET-1 in the airways through the ETB receptors on the epithelial cells is probably impaired, leading to enhanced access of ET-1 to underlying bronchial smooth muscle cells . The production of relaxant factors such as nitric oxide through ET-1 binding to receptors on the epithelial cells is also compromised .
In the present study, involving use of rat bronchi, we found the maximal contractile response to ETA and ETB receptors to be significantly augmented by the presence of DSP but not by nicotine incubation. In preliminary tests in rat mesentery artery, the water-soluble part of the smoking particles was not found to alter ETB receptor expression (data not shown). The identity and the characteristics of the receptors have been analyzed in detail previously . Further support for the hypothesis of an endothelin receptor protein increase was obtained by immunohistochemistry. This revealed a slight increase in ETA and a strong and significant increase in ETB receptors after culturing together with DSP. These up-regulated receptors were located in the smooth muscle cell layer. We have shown previously that eosinophilic inflammation induced in vivo by Sephadex results in ETB receptor up regulation, both functionally and at the mRNA level . We did not observe any further change in mRNA expression for ETA or ETB. However, there have been earlier reports of a decrease in ETB receptor mRNA expression in mouse airways after long-term treatment by IL-1β . To explain our own results further, we used confocal microscopy to indicate whether the amount and the localization of receptor protein were changed. This revealed an up-regulation of ETA and ETB receptors in the smooth muscle cells. De novo transcription and translation via protein kinase C  and mitogen-activated protein kinases [32, 33] have been shown to occur after organ culture of arterial segments.
In vascular studies, cytokines derived from inflammatory cells during airway inflammation, could lead both to increase in the level of ET-1 and an up regulation of endothelin receptors that synergistically enhance the contractility of the smooth muscle of the airways. Use of an endothelin antagonist is already an established treatment for pulmonary hypertension.
Most ET-1 is secreted abluminally towards the smooth muscle cells. A small part of ET-1 can be measured in plasma and smoking leads to raised level of plasma ET-1 . In our study we did not measure any ET-1 level but the mRNA expression of ET-1 was not altered. Thus, if up-regulation of ET-1 was present in our study, it must be explained as due to a translational mechanism, since the mRNA was unaltered. Raised levels of ET-1 may cause a raised consumption of ETB receptors, i.e. an increased turnover, explaining varying results in different studies
In order to test whether nicotine per se could be involved in this process, it was included in our study. We found no increase in ETA or ETB receptor expression after nicotine exposure. This is in agreement with the finding elsewhere that 24 hours of nicotine incubation of human coronary artery endothelial cells had no effect on the expression of ET-1 .