Harmit BindraCritical Appraisal: Impairment of Nitric Oxide Synthase but Not Heme Oxygenase Accounts for Baroreflex Dysfunction Caused by Chronic Nicotine in Female RatsLay AbstractIntroduction: The baroreflex or baroreceptor sensitivity is a physiological parameter that regulates changes in blood pressure. Baroreflex dysfunction is thought to contribute to many of the cardiovascular changes caused by chronic intake of nicotine. Nitric oxide (NO) and carbon monoxide (CO) can be synthesised in the endothelial cells by the action of nitric oxide synthase (NOS) and hemeoxygenase (HO), respectively. Inhibition of NOS and HO mediated pathways have been thought to cause reduction in baroreflex sensitivity similar to that of nicotine. This study targets these two pathways and their possible interactions in an attempt to reverse the deteriorating cardiovascular effects caused by nicotine.Methods: The sensitivity of baroreflex was determined by measuring changes in heart rate in response to changes in mean arterial pressure induced by sodium nitroprusside (SNP) and phenylephrine (PE). SNP and PE exert these cardiovascular changes by affecting the diameter of blood vessels.Six groups of conscious female rats were used (6-8 rats/group) to study the effect of NOS on the baroreflex dysfunction caused by nicotine. Rats were treated either with nicotine or saline solution for 2 weeks. Baroreflex curves using random doses of SNP and PE were obtained in conscious rats on day 14 after treating these rats with L-NAME (inhibitor of NOS), L-arginine (substrate of NOS) or saline solution for 15 minutes.In a second study, another group of 7 rats treated with nicotine was used to find out whether HO inhibition by zinc protoporphyrin (ZnPP) abolishes the baroreflex response provoked by L-arginine. Baroreflex sensitivity was measured after treating rats with L-arginine and ZnPP for 15 minutes. Finally, the effects of the inducer and inhibitor of HO, hemin and ZnPP respectively, were investigated on the baroreflex dysfunction.Results: Inhibition of NOS using L-NAME caused a similar reduction in the baroreflex response as nicotine. This effect could be reversed with L-arginine. No further reduction in baroreflex response was evident in rats treated with both nicotine and L-NAME. Interestingly, HO inhibitor led to no reduction in baroreflex response and did not reverse any changes in baroreflex activity caused by nicotine. This implies that there is no direct role of HO mediated pathways in the nicotinic-baroreflex activity. On the contrary, there was an increase in baroreflex activity when HO activity was facilitated.In conclusion, inhibition of NOS is responsible for reduction in baroreflex sensitivity caused by nicotine.Background information and rationale for carrying out the workSmoking cigarettes is one of the most well established causes of mortality in the world and it is well known for its devastating effects on the quality of life and the impact it has on the families, including their psychological, social and physical well being. The majority of the harmful cardiovascular effects of smoking arise from the use of nicotine. Chronic intake of nicotine has been shown to reduce baroreceptor reflexes by decreasing the responsiveness of stretch receptors in the carotid sinus together with arterial compliance (Ashworth-Preece et al., 1998; Giannattasio et al., 1994).Nitric oxide (NO) is highly reactive gas, synthesised via three isoforms of nitric oxide synthase, including endothelial nitric oxide (eNOS), neuronal nitric oxide (nNOS) and inducible nitric oxide (iNOS). NO has been involved in various physiological pathways. For instance, eNOS results in arterial vasodilation by causing relaxation of vascular smooth muscles (Prado et al., 2011). nNOS plays an important role in neuronal activity by serving as a neurotransmitter. iNOS is generated by the phagocytes to invade the bacteria as part of immune response. NO has an ability to diffuse through and act as an intracellular messenger. It has been implicated in strengthening the synapses (long term potentiation) in learning and cause NMDA induced neurotoxicity in Parkinson’s disease (Taqatqeh et al., 2009). In a study carried out using brainstem nuclei of rats, it was found that inhibiting NOS in the central nervous system reduced baroreflex activation (Lo et al., 1996).Carbon monoxide (CO) has long been considered to be a toxic gas due to its high affinity for haemoglobin over oxygen. Contrary to popular belief, our body cells can also synthesise CO via heme oxygenase (HO) – an enzyme that results in the generation of CO by catalysing the conversion of heme to biliverdin (Abraham & Kappas., 2008). It has been established that inhibition of CO formed by HO reduces reflex activity as well as bradycardic response provoked by glutamate in the nucleus of the solitary tract (Lin et al., 2004). Other studies have independently found that inhibition of HO induced CO increases blood pressure systemically (Zhang et al., 2001).Interestingly, there seem to be some sort of interaction going on between NO/NOS and CO/HO pathways (Li et al., 2009). Indeed, the endogenous effects of these two molecules are provoked by the activation of soluble guanylate cyclase and a further increase in the levels of cGMP (Tzeng., 2009). Although there is a crosslink between these pathways, it has not been researched whether interruption of these mediators alone or disruption in their mutual interaction is responsible for the baroreceptor dysfunction mediated by nicotine.Approaches to the questionThe study was split into two groups to evaluate the role of NO/NOS and CO/HO pathways in nicotine induced baroreflex depression.In a first study, six small groups of female rats, ranging from 6-8 in each group, were used to study the effect of NOS on the baroreflex dysfunction. Three of these groups were given intraperitoneal nicotine for 2 weeks using a dosage of 2mg/kg/day, whereas the remaining groups were treated with saline solution. These rats were cannulated intravascularly on day 12. Baroreflex curves using SNP and PE were obtained in conscious rats on day 14 after treating these rats with L-NAME, L-arginine or saline solution for 15 minutes.In a second study, another group of 7 rats treated with nicotine were used to find out whether HO inhibition by ZnPP abolishes the baroreflex response provoked by L-arginine. Baroreceptor sensitivity was measured after treating rats with L-arginine and ZnPP for 15 minutes. The sensitivity of baroreceptors was determined by measuring changes in heart rate in response to changes in mean arterial pressure induced by vasoactive drugs, such as sodium nitroprusside (SNP) and phenylephrine (PE). This was carried out using regression analysis. Randomised doses SNP and PE doses, ranging from 1 to 16µg/kg, were injected intravenously to obtain a baroreflex curve. An index of baroreflex activity was found by expressing the slope of the regression line as beats/min/mmHg.In the final part of the study, the effects of the inducer and inhibitor of HO, hemin and ZnPP respectively, were investigated on the baroreceptor dysfunction induced by nicotine. This was done using 5 different groups (5-8 female rats/group) for a 2 weeks period in which baroreflex testing was carried out using hemin, ZnPP, hemin + L-NAME, hemin + ODQ (guanylate cyclase inhibitor), and CORM-2 (CO releasing agent).Two further control groups were used in which rats received saline solution for 2 weeks and the baroreflex readings were then taken post-treatment with hemin or CORM-2. To measure the activity of NOS and HO, rats were treated with nicotine or saline for 2 weeks in the presence or absence of hemin and their brainstem was dissected and freezed at -80C.Key Results and analysisBoth nicotine and NOS/NO pathway inhibition produced a similar effect on baroreflex activity. Rats treated with nicotine showed reduced slopes in the baroreflex curves exhibited by PE and SNP in comparison to the saline treated rats, suggesting a reduced baroreflex response. In rats treated with nicotine, there was a decrease from 2.1±0.2 ms/mmHg to 1.1±0.2 ms/mmHg in the baroreflex sensitivity exhibited by the PE. A similar reduction from 0.9±0.1 ms/mmHg to 0.4±0.1 ms/mmHg was seen in the baroreflex sensitivity exhibited by SNP. These results were statistically significant (P<0.05). A decrease in baroreflex sensitivity was demonstrated in rats treated with L-NAME and such decrease was similar to that of nicotine treated rats. Any reduction in the baroreflex sensitivity was reversed with L-arginine and remained unaffected in rats treated with ZnPP. Moreover, no further reduction in baroreflex response was seen in rats treated with nicotine plus L-NAME.In short summary, the study was quite clear in explaining the involvement of NO/NOS pathway in the reduction of baroreflex activity caused by nicotine. First of all, inhibiting NOS using L-NAME caused similar reduction in baroreflex response as nicotine. Secondly, this effect could be reversed with the substrate of NOS (L-arginine). Thirdly, having both nicotine and L-NAME did not cause any further reduction in baroreflex response.The inhibition of HO by ZnPP had no effect on the baroreflex sensitivity in nicotine treated rats, implying that there is no direct role of HO pathway in the nicotinic-baroreflex activity. Any decrease in baroreflex sensitivity by nicotine could be reversed with hemin as the curve deviated more towards saline treated rats. Interestingly, when rats were treated with L-NAME or with ODQ, the protective effect of hemin to reverse the reduction in baroreflex sensitivity was no longer evident. This suggested that the initial reduction in baroreflex response was probably due to an increased activity of NOS that was no longer seen when L-NAME was used. Indeed, the activity of HO and NOS was found to increase in the brainstem tissue of rats treated with nicotine in the presence of hemin. Together, these findings imply that NOS is a downstream pathway responsible for changes in baroreflex sensitivity and hemin is somehow feeding into this pathway and activating it to facilitate baroreflex response. There was no reduction in baroreflex response caused by nicotine with carbon monoxide release agent (CORM-2). This is supporting the idea that reduced baroreflex response is possibly due to NOS activity and not related to CO.Likely impact of research outcomeThe results implicated NOS pathways to be responsible for the deteriorating effects of nicotine on baroreflex sensitivity. Although, the current study implicated NOS pathways as a downstream mechanism and HO acting at the upstream level, more work is needed to investigate the effects of CORM-2 and hemin and shed light on the cellular cascades responsible for bringing these changes on baroreflex sensitivity. Taking into consideration that the ultimate pathways involved in the baroreceptor dysfunction from this study is NOS related, activation of NOS could be an important therapeutic target in treating the deteriorating effects of nicotine on cardiovascular system, especially the baroreceptor dysfunction. However, it is too early to accept this claim as these results need to be replicated and clinical trials must be carried out before considering any changes in the clinical practice.Future work and conclusionAt present, this study is unlikely to have any major impact on the development of therapeutic drugs. Except the possible involvement of NOS, the signalling cascades responsible for baroreflex dysfunction still remain unclear. Contrary to these findings, the same author previously reported that CO formed by HO attenuated the baroreflex sensitivity in the nucleus tractus solitarii of rats (Lo et al., 2000; Lo et al., 2006). The author has attempted to justify the possible variation between the two studies with the use of conscious rats in the current study and anesthetised rats in the previous study. In addition, the inhibitor of HO was injected directly into the medullary nucleus in the previous study as opposed to an intravenous injection in the current study. Although these changes may contribute to the differences in terms of accuracy and reliability of the results, they are unlikely to fully account for the involvement of CO mediated changes in baroreflex sensitivity. Therefore these experiments need to be replicated before considering any clinical trials.The whole brainstem was dissected to measure the activity of NOS. This may not accurately reflect the levels of NOS in the cardiovascular nuclei of medulla therefore the study can be extended to investigate this. The use of animal models to test baroreflex sensitivity and the pharmacological agents to counteract such changes may not work similarly in humans. Baroreflex sensitivity is blunted to different degrees with increasing age (huang et al., 2007) and this may have an effect on the appropriate dose required to show any therapeutic benefits. 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