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Author Response: Conflicting Reports on the Outflow Effects of Bumetanide
Conflicting Reports on the Outflow Effects of Bumetanide
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Kristine Erickson Boston University School of Medicine
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kerick{at}bu.edu Kristine Erickson
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Dr. Clark's letter to the Editor was clearly written in response to a presumed critique of the organ culture methodology used in his laboratory. This was not at all the intent of our comments in the manuscript. Constant flow perfusion, the method developed by Johnson and adopted by Dr. Clark and others, is an invaluable method for increasing our understanding of the biology of the intact human trabecular meshwork. Indeed, it has several advantages over our system, not the least of which is that long term studies (i.e. several weeks) are possible. On the other hand, this methodology does not have a track record for use in short term pharmacologic studies. In fact, responsiveness to hormones/agonists which increase outflow facility (i.e. epinephrine, ANP, PGE2, cholinergics, endothelin, etc.) has not been reported. The only increases in facility that have been reported were by Johnson who demonstrated responsiveness to ethacrynic acid and cytochalasin D, both of which dramatically disrupt the cytoskeleton. It is clear that both methodologies offer advantages and disadvantages. It is also clear that both methodologies will, in their own ways, contribute to our understanding of the pathogenesis of glaucoma. Kristine A. Erickson |
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Abbot Clark, Senior Director, Glaucoma Research Alcon Research Ltd.
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abe.clark{at}alconlabs.com Abbot Clark
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There have been several reports that suggest that activity of the Na-K-Cl cotransporter in the trabecular meshwork is capable of altering of TM cell volume and thereby may regulate the aqueous humor outflow facility.1,2 The recent study of Al-Aswad and colleagues reports that regulators of the Na-K-Cl cotransport system, including bumetanide (a cotransporter antagonist), alter the outflow facility of in vitro perfused calf and human eyes.3 However, the results of an earlier published study did not detect any outflow facility or IOP effects of bumetanide in perfused monkey eyes in vivo, in perfusion cultured human eyes in vitro, or in trabecular meshwork and ciliary muscle tissue strips.4 Al-Aswad and colleagues offered several reasons for this apparent discrepancy in their discussion. Unfortunately, we take exception with several of their explanations, which we feel inaccurately represent our methods and our data. (1) Two different ocular perfusion culture systems were used in these two studies. In our study, we used a slight modification of the Johnson constant flow, variable pressure system with which we5 and others6-9 have had considerable experience over the past 10 years. In contrast, the Al- Aswad work was done using the Erickson constant pressure, variable flow perfusion system. As mentioned by Al-Aswad et al, there are a number of important differences between these two systems. Al-Aswad and colleagues make a theoretical argument that technical difficulties (such as pulsations in the syringe pump) can lead to artifactual variations in pressure in the constant flow system. In our years of experience with this model we have never encountered this problem. In addition, we have not seen the “pressure spikes” that Al-Aswad et al. claim are common in this method, which they say leads to instability and damage to the outflow tissue. In our system, intraocular pressure (IOP) is continuously monitored with very accurate pressure transducers. IOP readings are taken every minute and pressures are averaged every 10 minutes. Test compounds are introduced into the eye through an FPLC injector so that the flow to the eye remains constant, and pressure is not disturbed during the course of the experiment. Therefore, this system provides very stable baseline pressures. In each experiment, a vehicle control is run concomitantly with the test substance. In contrast, the constant pressure perfusion system of Erickson does not have a vehicle control. The system measures outflow volume every 15 minutes, and introduction of the test agent requires interruption of media flow, an exchange of media in the anterior segment, which is followed by periodic measurements of outflow volume. (2) Al-Aswad and colleagues suggest that another possible explanation for lack of effect of bumetanide in our study is due to the relative health of the ocular tissue used in our studies (“tissue vaibility appears to have been a major problem in the Gabelt study …”). We take exception with this statement. We use human donor eyes that are enucleated within 3 hours of death and placed on ice for shipment to our laboratories. The eyes are dissected within 20 hours of death (not up to 24 hours as claimed by Al-Aswad and colleagues) and perfusion cultured immediately after dissection. Each of the perfusion cultured eyes in our studies is carefully examined by light and electron microscopy at the conclusion of the study (not just by light microscopy as they claim). The health of tissue in all four quadrants of the outflow pathway is carefully examined, and those eyes that are unhealthy are excluded from the study. We are confident that if other groups were to use these strict criteria of morphologically examining each eye used in their studies, many more eyes would be classified as “unacceptable” and excluded from the study, which would provide more reliable and relevant data. (3) We evaluated 5 sets of eyes perfused with and without bumetanide and found no evidence for an effect of bumetanide on IOP and outflow facility using our stable perfusion culture system. We used the same concentration of bumetanide (10-5 M) that was reported in the Al-Aswad study. We felt that our perfusion culture data demonstrating no outflow effect of bumetanide, coupled with the lack of effect of bumetanide on perfused monkey eyes and the lack of effect on trabecular meshwork and ciliary muscle tissue strips, argued against using additional valuable human donor eyes to statistically prove a lack of effect. It is often the case that slightly different experimental techniques yield different results. We hope that these conflicting data can be resolved by further research including studies using conscious animals as well as clinical studies. Abbot F. Clark References 1. O’Donnell ME, Brandt JD, Curry FR. Na-K-Cl cotransport regulates
intracellular volume and monolayer permeability of trabecular meshwork
cells. Am J Physiol. 1995;268:C1067-1074.
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