11 thoughts on “Journal club presentation for Sept. 6 MUSTAFA

  1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    The authors tested the hypothesis that endothelium-derived relaxing factor (EDRF), an endogenous substance released in the circulation that contributes to vasodilation, is in fact nitric oxide (NO).
    Results: By using spectrophotometry to analyze hemoglobin reactions with NO and EDRF and the compounds’ coupling with N-(1-naphthyl)-ethylenediamine, conducting biochemical assays on bovine tissue samples (i.e. perfused veins and arteries), and assessing levels of cGMP, the authors determined that NO and EDRF had strikingly similar pharmacological profiles, i.e. short half-life, enhancement of arterial cGMP, formation of nitrosylhemoglobin upon binding to hemoglobin, etc.
    Conclusion: Due to the compounds’ chemical and pharmacological similarities, the authors concluded that EDRF is either NO or a nitroso substance capable of releasing it.

    Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    • In order to verify whether EDRF could truly be NO, the authors needed to conduct several different assays to obtain a fuller understanding of their pharmacological profiles. They made a commendable effort, conducting in-vitro experiments on bovine veins and arteries, evaluating hemoglobin reactions, etc.
    • Since I’m unfamiliar with the technique, I would have appreciated a diagram illustrating the superfusion cascade in order to better visualize it.
    • The authors varied the number of vascular strips they superfused (i.e. three in Figure 1, two in Figure 2, and three in Figure 3) in different experiments. However, for the sake of consistency, wouldn’t it be better to use the same number throughout?
    • In general, the authors used adequate controls.
    • There isn’t much mention of any statistical tests that might have been performed.
    • Figures are generally clear, though it might have been helpful to label the x-axes of the bar graphs
    • The data provided validate the stated results

    Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    • Introduction: Authors provide a concise overview of EDRF’s action in the circulatory system and the properties it has in common with NO, thus justifying their present experiments, which seek to confirm that the compounds are likely the same. However, further rationale could have been provided for why determining that EDRF is NO is such a vital research question. Also, the authors briefly reference another group that published the same finding while their manuscript was being prepared, which lends additional credence to their own results.
    • Discussion: Within the context of the literature, the authors provide a thorough discussion of why their results strongly suggest that NO and EDRF are the same. They also discuss alternate ways in which the results could be interpreted (i.e. their methods couldn’t have distinguished NO from a labile nitroso compound). However, the authors focus very heavily on how their findings relate to basic science, only briefly touching on the “physiological implications.” It would have been of interest to see further exploration of what import this finding could have in the clinic (the “big picture” beyond merely adding to scientific knowledge).
    • Conclusion: Given the results provided, the conclusion that EDRF is likely NO is valid.

  2. • The purpose of this research was to determine if nitric oxide (NO) is responsible for the vasodilation and smooth muscle relaxation mediated by endothelium-derived relaxing factor (EDRF). In this study, bovine intrapulmonary arteries and veins were perfused and the effects of EDRF released were analyzed. NO was delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade and then the results were compared to the EDRF data. The half-life of both EDRF and NO were found to be 3-5sec and for both EDRF and NO, pyrogallol inhibited the relaxation effects while superoxide dismutase enhanced and prolonged the relaxation effects. Veins and arteries were perfused with various concentrations of NO and their relaxation effects and the amounts of NO recovered were recorded. NO recovered and cGMP levels were similar in both NO perfused veins and arteries as well as A23187 perfused vasculature. The EDRF released from veins and arteries that were perfused with A23187, a known smooth muscle relaxant, was recorded and analyzed chemically for its similarities to NO. The EDRF was detected chemically as NO. Spectral analysis was also performed to visualize a shift caused by NO reacting with hemoglobin. This shift was also seen with a substance released after A23187 reacted with bovine vascular endothelial cells.
    • In this study, bovine intrapulmonary arteries and veins were perfused and the effects of EDRF released were analyzed while NO was delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. My primary question is why there were not both either perfused or superfused in a cascade so provide for more standardization. Also, A23187 was used to study EDRF release and it appeared to be appropriate given the results but it was no explained well as to why A23187 specifically was chosen. They also put a great deal of work into chemically identifying NO, which was well thought out. To my knowledge though, they failed to include how/if they tested all chemicals prior to experimentation. For example, they received A23187, pyrogallol, and various other reagents from Sigma but they never said whether they tested them all themselves upon receiving them to confirm whether the reagents were indeed pure and what they said they were.
    • Towards the beginning of the paper, it was stated that another manuscript was released which confirmed the biological and chemical identification of EDRF released from cultured endothelial cells as NO. However, later in the paper, it is stated that this manuscript actually showed that EDRF was either NO or some other labile nitroso compound that releases NO. Ultimately, the experiments and chemical tests carried out in this study or previous studied cannot definitively prove that EDRF is in fact the same compound as NO, though it does appear to be highly probable that the compounds are identical. Regardless of the probability, definite conclusions cannot be made and further experimentation needs to be carried out. Perhaps further experimentation should involve finding a way to isolate and stabilize EDRF and NO from these perfusion/superfusion tests and then try to determine their composition via spectroscopy.

  3. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    The hypothesis of Ignarro et.al. stated that endothelium-derived relaxing factor (EDRF) produced and released from arteries and veins was chemically the same substance as nitric oxide (NO). Ignarro et.al. concluded that EDRF is in fact NO. The major result that the author(s) present through enough pharmacological and chemical evidence is that EDRF released from arteries and veins is NO. They concluded this by working different methodologies such as identifying similarities of both as labile substances through the bioassay cascade technique, cGMP and vascular relaxation activity, and the ability of both to react with hemoglobin.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    – First they show the similarities between EDRF and NO of relaxant responses utilizing the bioassay cascade/super fusion technique (fig. 1) and stated this as their pharmacological evidence. They are basing that statement on their use of acetylcholine, A23, phenylephrine, and U46 but as a reader I would have preferred for the author(s) to A) explain what A23 and U46 are and B) provide a better understanding of their pharmacological manipulations/rationale. For this results/methods/figure I would have preferred to have a different type of tissue other than artery/veins as another form of a control. Also, is glyceryl trinitrate their control in this assay?
    – Second, they show that EDRF and NO are able to stimulate cGMP accumulation (fig. 2). Third, the author(s) show the amount of NO recovered when the tissue(s) where superfused with differing concentrations of NO (fig. 3). For both of these methods/results the main issue I identify is their use of phrases such as “comparable magnitudes” with no statistical information/data/analysis provided whatsoever.
    – Fourth, Ignarro et.al. claim that this accumulation of cGMP is inhibited by four different factors (data not shown). The fact that this data is not shown but it is introduced and mentioned throughout the article was disappointing. Was it supplemental data? The last result Ignarro et.al. show is chemical evidence that both EDRF and NO are able to react with hemoglobin (fig. 4). This method/result was a good way to bring home the message for the reader to understand and their use of deoxyhemoglobin and deoxyhemoglobin plus A23 was a great way to show comparisons.
    Overall for the methods section some controls were missing but the author(s) did a good job to break down the methods as thoroughly as possible. With the results section the fact that there are bar graphs and statements are being made about significances without statistical analysis provided hurts their conclusions.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The introduction of the article was very narrow, but the author(s) were able to explain the importance of their rationale. They made a good point in these sections to include findings in the field as they related to their premise. They also state multiple times that while their manuscript was being prepared a separate report was released that seemed to confirm their ideas. The discussion did a good job at digging deeper into their rationale and their findings and in this section they also do a nice job at pointing out to the reader with results from different articles that essentially acts as a second support to their findings. Again, a main issue I identify is their mentioning(results/discussion) of the ability of substances such as methylene blue to inhibit relaxation without showing data to support it. At the end of the discussion section where the authors mention work proposed by Dale and provide a further explanation to that idea was a good reference point. Although I did fell that the article did not have a “solid” conclusion statement and/or future directions.

  4. Student: Lois Akinola Instructor: Dr. Ritter Date: 09/06/2019
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    The authors hypothesized that the endothelium-derived relaxing factor (EDRF) released from arteries and veins, was Nitric Oxide (NO). They reported that both molecules were equal in their lability, half-lives, susceptibility to inactivation and stability by various compounds. They both also produced similar effects in relaxation and stimulation of cyclic GMP accumulation as well as its inhibition by other compounds. Moreover, EDRF was also identified chemically as NO by two procedures, so that overall, they concluded that since EDRF and NO possessed virtually indistinguishable properties, biologically and chemically, they were one and the same molecule.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    Isolation of Aortic Endothelial Cells. Bovine aortic endothelial cells were isolated by a collagenase digestion procedure.
    Bioassay Cascade Superfusion Technique. This method measures changes in tension within endothelium-denuded artery and vein after superfusion and treatment with perfusate obtained from perfusing segments of main bovine intrapulmonary artery and vein with oxygenated Krebs bicarbonate solution at 37°C. This was used to determine the half-lives of both EDRF and NO, and to determine the chemical stability of both molecules when treated with pyrogallol or superoxide dismutase.
    Results: They found the approximate half-life of arterial and venous EDRF and NO to be similar (3-5 sec). Furthermore, they found that pyrogallol inhibited the relaxant actions of both EDRF and NO, and superoxide dismutase enhanced and prolonged their relaxant responses as reflected by the elongation of EDRF and NO half-lives to about 30-40 sec.
    Determination of Cyclic GMP Levels. Cyclic GMP levels were measured in arterial and venous strips that had been with perfusion media from intact artery or vein, and also from arterial and venous strips that had been superfused with NO.
    Results: Superfusion of endothelium-denuded strips of artery or vein with perfusion media from intact artery or vein and superfusion with NO caused similar responses in the relaxant effects as well as similar increases in cyclic GMP levels in the superfused strips. Furthermore, these effects were significantly inhibited by methylene blue, oxyhemoglobin, pyrogallol, and KCI (data not shown) in both EDRF and NO.
    Chemical Determination of NO. The concentration of NO in the superfusion media was measured during superfusion of vascular strips with either NO or perfusion media from intact artery or vein. The authors used a method of spectrophotometry where sulfanilic acid is diazotized by NO at acidic pH and coupled with N-(1-naphthyl)-ethylenediamine thus yielding a colored product which is measured using a spectrophotometer.
    Reactions Between Hemoglobin and EDRF or NO. Hemoglobin is an inhibitor of the relaxant effects induced by both EDRF and NO. hemoglobin reportedly binds to EDRF and NO to yield nitrosylhemoglobin.
    Results: EDRF released from both artery and vein was chemically identified as NO by two procedures. In the first assay, the amount of NO released from artery and vein by A23187 were closely similar to the quantities of NO recovered from superfusion of the strips with NO. Control strips without endothelium failed to release appreciable quantities of NO.
    Secondly, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The overall conclusion was that that the EDRF secreted from arterial and venous was nitric oxide. I think the author’s methods of investigation were very clear and thoroughly thought-out, and they used appropriate methods to investigate their hypothesis as well as appropriate negative controls and positive controls in their experiments. Their work resulted in clear conclusive results and are substantiated by the data provided. The only critique I have is that from a visualization point of view, they may have looked into utilizing a schematic of the mechanisms by which EDRF/NO was potentiating its effects.

  5. 1) Knowing that endothelium-independent vasodilators such as nitroprusside and glyceryl trinitrate elicit their physiological effects by releasing NO and that these effects closely resemble vasodilation mediated by endothelium derived relaxation factor (EDRF), the investigators hypothesized that EDRF is biologically and chemically equivalent to NO. They employed a bioassay that involved an apparatus in which isolated strips of artery or vein could be mounted and perfused and superfused with solutions to deliver or measure vasoactive compounds and measure tension. The overall conclusion to the study was that EDRF was in fact, NO based on the physiological response measured in vasculature and the chemical data supporting that EDRF is NO.

    2) I think the methods section was well described enough that I could get a sense of how this experiment was carried out however there were a few points where information was left out and only a citation given. For example under the section “Determination of Cyclic GMP levels it reads: “A modification (9) of the procedure described previously (18) was employed.” To understand how they measured cGMP levels I have to read the procedure in one paper, and read another paper to know what modification was made. They did employ controls in their experiments but I am unsure about the statistics. They didn’t mention how many measurements were done for cGMP and so although their graphs show error bars, they don’t have any context. I did not see any P values or discussion of significance. Overall I think the data does substantiate their hypothesis and the methods employed were appropriate to do so, (I think the spectrophotometric analysis clearly shows that EDRF is chemically identical to NO) however they should have included more in the methods about statistical analysis.

    3) I think the introduction is short and sweet and the rationale behind the study is pretty clear. I think the introduction could have done more to maybe explain some history behind EDRF, how it was first identified, isolated, etc. and why in previous years leading up to the study has EDRF not be identified chemically as NO? The rationale for the study is quite clear, but it helps to explain why it had not been investigated any earlier. I think the discussion rationally interprets the results to come to a logical conclusion. They acknowledge the possibility that EDRF could be a labile nitroso substance and not pure NO but went on to show that the pharmacological (same inhibitors for EDRF and NO signaling) and chemical data (both inactivated by superoxide anion and stabilized by superoxide mutase) strongly suggest that EDRF is NO.

  6. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    – hypothesis: that EDRF and NO are the same substance
    – they found the half life of EDRF to be 3-5s, which is the same as NO’s half-life, by measuring relaxation of an endothelium-intact segment of either artery or vein
    – strips of artery or vein had both relaxant responses and an increase in cyclic GMP when they were profused with NO or profusion media; these responses were inhibited by methylene blue, oxyhemoglobin, pyrogallol, and KCl
    – a substance isolated from bovine aortic endothelial cells (after reacting to A23187) caused an identical spectral shift to NO

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – the bioassay cascade superfusion was used to measure the half-life of EDRF, which was the same as the half-life of NO
    – both EDRF and NO were both inhibited or enhanced by the same substances (for example: pyrogallol inhibits their relaxant actions and superoxide dismutase enhanced their relaxant responses)
    – they used glyceryl trinitrate to “standardize the preparations” (9266)
    – figure 2 shows the similarity between the accumulation of cyclic GMP between EDRF released from perfused arteries and veins and superfused NO
    – in the results, the investigators noted multiple inhibitors of EDRF and NO (9267); they mentioned KCl, but said the data was not shown, so they do not have any data or anyone else’s research to back up that point
    – the investigators also said that perfusion media caused “small but significant relaxant responses” in cyclic GMP levels in strips of artery or vein, but did not mention any statistical analysis to indicate significance or any other method they might have used to determine significance

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – the introduction gives an overview of EDRF and NO, as well as their known similarities, such as the fact that both directly activate purified soluble guanylate cyclase and are both inhibited by methylene blue
    – the discussion repeats findings found in the results, such as the short half life of both EDRF and NO, that superoxide anion (such as pyrogallol) accelerates their inactivation and superoxide dismutase decelerates it
    – they include results from other studies that support their hypothesis that NO and EDRF are the same compound
    – arterial EDRF and hemoglobin and NO and hemoglobin both had very similar reactions, which the investigators said supported the hypothesis that EDRF is NO or some labile nitroso compound
    – they included the note that the chemical procedures they used are not specific for NO, so it is possible that “EDRF is a labile nitroso substance that spontaneously releases NO” (9268); they say that EDRF can’t be NO2-, because it is relatively stable and requires high concentrations to cause smooth muscle relaxation
    – while the authors were thorough in what they did, there was minimal, if any, discussion of the impact that their findings might have or future directions they might take

  7. Student Aminatta Tejan-Kamara Instructor Dr. Ritter Date 09/04/19
    Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Authors hypothesized that EDRF (released from both the artery and the vein) is biologically and chemically: nitric oxide (or potentially a labile nitroso compound.) The authors’ major results reported in support of this hypothesis include the following: the inhibition of both EDRF and NO by pyrogallol generated superoxide anion in the bioassay cascade superfusion experiments, and the accumulation of cGMP levels in EDRF and NO respectively, as well as the results from the spectrophotometric data (that illustrated the reaction between either EDRF or NO and the reduced hemoglobin molecule). The authors’ overarching conclusion from the study is that EDRF is NO (or possibly a labile nitroso compound).

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    The authors’ sought to find data in support of their hypothesis through various techniques including the bioassay cascade superfusion experiments, which appeared to be an appropriate method given their aim to demonstrate the EDRF released in the vasculature was biochemically and chemically the same as NO. Their reported data from the bioassay cascade superfusion experiments was well explained in the figure 1 data analysis section; the results are supported with the experiments conducted given that they offered a quantitative measure that suggested that EDRF is likely similar to NO. The quantitative measure was the EDRF half life, the authors explicitly explain how they quantified the similarities from the superfusion experiments, stating the approximate half life of EDRF released from the artery and vein, was “identical” to NO, thus providing corroborating evidence that the NO molecule may be similar in structure to the EDRF. Additionally the authors thought well to do subsequent studies in order to determine the chemical structure of NO and assess cyclic GMP accumulation, given that both experiments would allow for further information that would hopefully further support their overall hypothesis. It is not fully clear why they did not seek to remain consistent in their methodology, only using one instead of three vascular strips for superfusion in the Chemical identification of NO studies and two vascular strips in the cyclic GMP accumulation studies; the experimenters had previously used three vascular strips in the bioassay cascade superfusion. With respect to the experimenters’ use of controls, both “Cyclic GMP Accumulation in Target tissues” and “Chemical Identification of NO” had good use of controls that offered a necessary distinction to the other conditions that showed varying amounts of NO released. In terms of the data analysis under figures 2 and 3, it unclear as to the author’s reasoning for using the cyclic GMP data and the NO assay data to represent the respective mean +/- SE, without reporting the actual numeric values.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The authors provide a reasonable literature review in the introduction that offers a strong rationale for why EDRF is hypothesized to be NO. The experimenters also effectively acknowledge previous studies in the introduction that aided them in the direction of their research. In the discussion and conclusion sections of the paper, their careful use of language when stating their findings is appreciated, it is noted that they often qualified their statements that EDRF is NO “or possibly a labile nitroso compound,” in order to demonstrate their inability to be unequivocally certain given the constraints of their study designs. Overall, the experimenters completed experiments with effective data analysis that well expressed the results that support the overall conclusion.

  8. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    H: EDRF released from both arteries and veins is NO.

    R/M: EDRF and NO displayed identical relaxant responses and half-lives (stability) when tested in the Bioassay Cascade Superfusion. Relaxant responses and cyclic GMP accumulation were comparable between EDRF and NO when tested in target tissues. EDRF was confirmed as NO in both the artery and veins through comparison of the quantities of NO released. The chemical identification of EDRF as NO was also validated through comparing similarities in spectral shifts from EDRF’s ability to yield nitrosyl when in contact with hemoglobin.

    C: As hypothesized, EDRF released from both arteries and veins was pharmacologically and chemically identified as NO or a labile nitroso compound that releases NO. This study also confirmed that both the artery and vein have the ability to release EDRF.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    The background and use of A23187 was not properly explained, although it was assumed it was used for comparison of NO to EDRF. Due to the known relaxation properties of EDRF in the veins and arteries; Bioassay Cascade Superfusion was used to compare NO’s ability to also cause vaso-relaxation. Pre-constriction in the Bioassay Cascade Superfusion was an effective way of ensuring an accurate measurement of relaxation. Perfusion of Ach was used as a control measure for this assay due to is vasodilatory properties. Glyceryl trinitrate was also used as validation of NO’s stability similar to GDRF. As stated, it was previously known that GDRF led to an accumulation of cyclic GMP; measuring the cyclic GMP accumulation in response to both NO and GDRF was an appropriate method of identifying NO as GDRF. Methylene Blue, oxyhemoglobin and pyrogallol were used as controls for the evaluation of cyclic GMP accumulation due to their abilities to inhibit this effect. GDRF was known to cause release of NO in the endothelial cells of veins and arteries. Therefore, measuring the quantities of NO released after treatment of either GDRF or NO is an accurate way of determining their biochemical similarities. When comparing quantities of NO released, strips that did not contain endothelium were used as valid controls. As GDRF has been shown to react with hemoglobin to produce nitrosyl; examining NO’s effects with hemoglobin seems fit. Finally, when comparing absorbance characteristics of hemoglobin; endothelial cells lacking A23187 treatment were used as control measures. Statistical analysis was not provided although EDRF and NO showed no difference in results for the bioassays conducted when compared to controls. A lack of differences between the effects of NO and GDRF in all the aforementioned measures confirm that GDRF and NO produce similar results chemically and biologically.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The introduction began by giving detailed background on the role EDRF plays in the cardio-vascular system through prior research findings. Moreover, adequate information was used to display that NO showed practically identical effects compared to EDRF in similar studies. Comparing the effects of both EDRF and NO on vascular smooth muscle relaxation and cyclic GMP accumulation gave enough support for their scientific rationale for further exploring their hypothesis. The final sentence of the introduction continued on with an additional study that was published while the current study was under investigation, stating biologically and chemically, EDRF and NO were one in the same, further strengthening their scientific premise. These previous studies further supported the findings of the current study that GDRF is in fact NO. The lack of differences in the results of both GDRF and NO in this studies’ assays supported this conclusion.

  9. Donald Jessup
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Hypothesis: Nitric Oxide (NO) is the chemical identity of Endothelium-derived releasing factor (EDRF) based on a comparison of their chemical and biological properties in insolated tissue.

    Major Results: 1.) When perfused across isolated segments of artery or vein arranged in a superfusion cascade experiment, NO and EDRF behaved similarly when compared to other compounds and had their effects extinguished in a similar time dependent manor that was likely due to the very short half-life of NO. 2.) Target tissue for NO, and EDRF produced similar increases in cGMP levels in another superfusion experiment and both accumulating effects were inhibited by 1uM methylene blue, oxyhemoglobin, pyrogallol and KCL. 3.) Chemical Identification experiments with a single isolated strip of endothelial tissue was superfused with A23187 (a Ca+ ionophore used to increase intracellular Ca2+) and NO, nitric oxide released from both perfusions was similar but NO levels released by those preparations lacking endothelium as a control did not release NO. Further experiments were able to show that in the presence of hemoglobin EDRF produces Oxyhemoglobin detected by a change in the spectrophotographic absorbance of hemoglobin, a change that was not seen in the absence of A23187.

    Overall Conclusion: Based on the above experiments the authors concluded that EDRF was either NO or some liable nitroso compound that readily released NO, and that not only artery but also venous tissue could release NO. This was further supported by a paper the authors cite which was released while this manuscript was being prepared and found similar results.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    I found the experimental design of this study to be straightforward but still very probative. Using the stacked sections of intrapulmonary tissue isn’t without questions of conflicting cell types, unpredicted interactions, or various other issues of using an ex-vivo tissue preparation but that was well controlled for by the study choosing to approach the problem from both the release of EDRF and then the intracellular cGMP pathway and then demonstrating that both of those effects could be terminated by blockers and antagonists like methylene blue. Furthermore I thought using hemoglobin’s high affinity for NO vs oxygen and measuring that as a shift in the absorbance spectra after the exposure or absence of A23187, was a very clever experiment and added a lot of credit to the authors conclusion. Overall I felt the paper stays well within their reported results even when reporting such an absolutist conclusion that doesn’t leave a lot of room for alternative interpretations.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The introduction was probably the weakest component of this paper. The authors do an excellent job of laying out their rational for the study, especially as it relates to their already published works but it doesn’t do a good enough job of establishing what the discussion in this field is like around this topic. It could have done more than just saying that the exact identity of EDRF was unknown but potentially suspected to be NO. The Discussion by contrast was really good, it walks back through the experimental results but expands on them and adds context from additional studies and I felt that really helped me to see the knowledge gap this study was trying to fill (beyond the obvious that EDRF wasn’t precisely known). I also appreciated that despite the great amount of confidence the authors have for their results they still provide an alternative interpretation when discussing the idea of a liable NO releasing nitroso compound. Overall the othroganol lines of investigations used in this study converge together nicely and provide very strong evidence for that EDRF was NO.

  10. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    The authors hypothesized that nitric oxide (NO) is the mediator of the smooth muscle relaxation actions of endothelium-derived relaxing factor. They perfused bovine intrapulmonary artery and vein and compared the vascular effects of EDRF release with the effects of NO, which was delivered by superfusion over artery and vein strips with acetylcholine, arranged in a cascade. It was found that NO and EDRF were both easily broken down and inactivated by pyrogallol or superoxide anion; they were both stabilized by superoxide dismutase; they were also inhibited by oxyhemoglobin and potassium. Both compounds showed increases in cyclic GMP, in both artery and vein, which was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. Both NO and EDRF reacted with hemoglobin to produce nitrosylhemoglobin, and they also both produced the same product after reacting with N-(1-naphthyl)-ethylenediamine. Therefore, the authors concluded that EDRF was chemically and biologically identified as NO.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    • For the Bioassay Cascade Superfusion technique, segments of bovine artery and vein were mounted, and tension was monitored. This was an appropriate technique to use because it records smooth muscle contractions. They also made sure to pre-constrict the vessels, which was appropriate because they were treating with a vasodilator.
    • I appreciated that the authors considered the interaction of atmospheric oxygen on the stability of deoxyhemoglobin and nitrosylhemoglobin, in the reactions between Hemoglobin and EDRF and NO; this was an important point to prove as it could have affected the experiment’s results.
    • Additionally, it was important that they used propylbenzylcholine mustard to prevent the contractile effects of acetylcholine, since it could also potentially affect the nervous system in multiple ways. Blocking contraction was an effective was to show the relaxation effects alone.
    • I would have liked to see an analysis of the chemical structure of EDRF compared to NO, perhaps by using spectroscopy analysis. Their conclusion was reached through determining that both compounds reacted similarly and produced identical compounds in various reactions. An analysis of structure would have added value to this conclusion. However, the data obtained sufficiently substantiated the results.
    • I would have also liked to see an analysis of any differences between the artery and vein. Nitrates can dilate both arteries and veins, but venous dilation is affected more significantly, which causes a reduction in stress to the ventricle of the heart. I’m curious to know more about this distinction.
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    I thought this was an overall strong analysis and comparison of endothelium-derived relaxing factor and nitric oxide; the experiments conducted were strong and the results supported the conclusion. It had been previously reported that EDRF and NO caused relaxation in vessels and had similar properties; the rationale for this experiment was to closely compare the two substances to support that they were chemically the same. The conclusion also supported other literature in the field that was coming out at the same time, which made it even stronger and built upon it. The authors did discuss the possibility of another nitroso species, which they were not able to distinguish at the time. They could have considered further studies to rule out other nitroso species by more experimentation.

  11. Student Brennen O’Rourke Instructor Ritter Date 9-6-19
    Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    – Hypothesis: EDRF is either identical to NO or another labile nitroso compound.
    – Major Results: EDRF and NO possess identical biological and chemical properties, including (1) vasodilatory/relaxant effects on vascular smooth muscle, (2) short half-life, (3) inactivation and activation by the same compounds, (4) resultant increases in cGMP levels (and inhibition of such cGMP accumulation by the compounds), (5) reaction to yield nitrosylhemoglobin, and (6) diazotization of sulfanilic acid.
    – Conclusion: EDRF and NO have identical biological and chemical properties and, from the observations reported in this article, are therefore either indistinguishable compounds or EDRF is a similar labile nitroso compound.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    – Methods: The methods selected are thorough and appropriate, given the available technologies in 1987. However, the methods section fails to address the source of the bovine tissue being tested, the number of animals from which the tissue was isolated (therefore limiting extrapolation or generalization of the results), and any statistical methods used in the analysis of results. The only mention of “n” or statistics is found inconsistently in the figure legends, and statistics are limited to “mean+SE” without tests of statistical significance. The experimental methods evaluate either the biological or chemical properties of EDRF and NO. Biological methods focus on perfusion and superfusion of bovine intrapulmonary vascular tissue and the resulting changes in smooth muscle tension (contraction) and comparisons between hemoglobin reaction products and between levels of cGMP production. These methods are standard measures of vascular smooth muscle cell activity and are therefore strong functional methods of comparing EDRF and NO activities. Chemical properties are examined via half-life calculation and, more telling, by the similar results of diazotization of sulfanilic acid- an assay specific for identifying NO (and NO-producing compounds). The authors do well to address the weakness in this assay: that it cannot be used to definitively distinguish NO from another labile nitroso compound. Controls were not obviously defined and were either (1) vascular strips that were not perfused/superfused with NO or (2) endothelium-denuded strips that were precontracted or relaxed with phenylephrine or Ach and other compounds for baseline comparison of function.
    – Results: Without considering the reproducibility of the results or lack of thorough statistical analysis, the data substantiate the results that EDNF and NO produce similar levels of vascular smooth muscle contraction and cGMP accumulation and that both are inhibited and activated to a similar extent by the same compounds (ex: superoxide dismutase). Chemical determination of the identity of EDNF as NO was elegantly performed through spectrophotometric measurement of diazotization products and via spectral shift upon reaction with hemoglobin. These two reactions are relatively specific for NO-producing compounds and are therefore convincing. More physical techniques, such as mass spectrometry, would have furthered the results, but would have been difficult due to the technologies available and the diminutive size of the molecules being tested.
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    – Introduction: Concise, yet provided a clear summary of the known similarities between NO and EDRF and the assays previously published to determine these similarities. The background on nitroso compounds causing vascular smooth muscle relaxation and cGMP accumulation was particularly helpful, given the focus on these two readouts in the results of this paper. The authors also did well explaining how the current paper builds upon their previous work and how it is corroborated by another report published within the same time frame.
    – Discussion: Very strong discussion in that it summarized the current results, placed these results into the framework of what was already known about NO and EDRF, the biological implications of these findings, and also identified weaknesses in the methods (particularly in how diazotization is not specific for NO alone).
    – Conclusions: Conclusion that EDRF and NO are the same or similar compounds is well-substantiated and was linked well to findings in the literature, particularly to multiple other reports published concurrently. I was impressed that the authors were transparent that their findings were not conclusive but were highly suggestive.

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