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CLASSICAL PERSPECTIVES |
Institute of Biomedical and Life Sciences, University of Glasgow G12 8QQ, UK
Email: d.j.miller{at}bio.gla.ac.uk
In the field of observation, chance favours only the prepared mind. Louis Pasteur
Sydney Ringer's four papers published in The Journal of Physiology in the early 1880s (1882a,b, 1883a,b) are rightly acknowledged as the starting point for the development of the modern understanding of the role of calcium in the contraction of the heart. In them, Ringer established the relative importance, both qualitative and quantitative, of sodium, potassium and calcium ions, as well the longer-term necessity to prevent or reverse the acidification associated with contraction. What emerges is the precise composition of a ‘saline’ able to maintain the normal heart beat form and frequency for several hours. This ‘saline’ is ‘Ringer's solution’. It is the precursor of all subsequent ‘physiological salines’ and related media that have enabled normal cell function to be sustained and studied in vitro. It provided the basis for salines infused into animals and humans, both experimentally and clinically, the latter a critical prerequisite for much modern medicine and surgery, both specifically in cardioplegia, but far more generally too. As we now understand more clearly, the heart provides a bioassay responsive to both electrophysiological (spontaneous beating) and contractile (calcium-entry dependent) consequences of the composition of the bathing solution. By focussing on the heart, Ringer's studies enjoyed an exquisite sensitivity to subtle variations of the ionic composition of bathing solutions. The fortuitous choice of the heart ensured that his conclusions apply to all cells and tissues.
Ringer's definitive final paper in the ‘calcium’ series (1883a) has been cited – mostly correctly for pagination, etc. – over 600 times in the last 20 years. However, citation analysis reveals (Simkin & Roychowdhury, 2003) that we can be confident that far fewer than these 600 ‘citers’ will actually have read the papers. As we pass the 120th anniversary of the publication of Ringer's papers, and approach the centenary of his death in 2010, what can be made of them, especially with the rose-tint of hindsight removed?
First, some pointers on features unusual for the modern reader. There are superficial distractions encountered when reading papers from the 1880s. In 2004, one is surprised to find that the word ‘frog’ is not to be found in the main calcium papers at all. However, the animal used is clear, for example, in Ringer's first paper in volume 3 (1882c, on the influence of season and temperature on drug actions). Rana temporaria is only cited explicitly in Ringer & Sainsbury, (1883). In this lack of detail, and many other methodological points, the reader is clearly assumed to be fluent with the scientific literature, the techniques, the fashionable questions and leading scientific personalities of the day. There are few recognizable references but a rather more frequent name dropping. It helps to recall that Ringer had already demonstrated the ‘actions of lime and potash salts on the ventricle’ to members of the Physiological Society at the University College London meeting in December 1882 (he became a member in 1884). His papers, and many of the others in these early volumes, indeed read more like house journals circulating amongst colleagues than the stylised, detached tone that we encounter in the modern literature. The text of papers of the period is not broken into Methods, Results, etc., but – in Ringer's papers in particular – read more like an experimental diary and discourse upon experiments done. Some authors did provide overall conclusions, to help their audience, but not Ringer. There are few graphical representations (other than anatomical) within contemporaneous papers, but engravings of kymograph smoked-drum traces are presented as fold-out ‘plates’ at the end of the volume. (Those familiar with such methods, still standard for undergraduates into the 1970s, will be impressed by the evident skill involved. The variable use of Roman and Arabic numerals between the plates and the main text increases the challenge of interpretation). Results tables are also rare and there is nothing statistical, of course, except in one paper where Ringer (1882c) does report ‘average responses’. (Pearson, one groundbreaker of statistics as applied to biological and experimental questions, did not start this work until the 1890s; others such as Spearman and Fisher came later still. Incidentally, all three, like Ringer, worked at University College London). Ringer also comments elsewhere that a few repeated observations were insufficient to permit him to draw firm conclusions; n numbers were evidently a matter of concern even in 1882–3.
Despite these superficial challenges, one soon gains an impression of a confident, meticulous, observant and insightful experimentalist at work. The (physio)logic of Ringer's approach chimes completely with that of the 21st century. As you read, you find yourself barely ahead of Ringer, despite the 120-year scientific advantage, our easy access to fine chemicals and extraordinary technological advantages. For example, at one stage (1882b, p. 383) he focuses quickly on potassium itself, rather than the attendant anions in the salts he was testing, as one key ingredient for a saline to maintain, or restore, normal function. Thus, he reports on potassium chloride, -phosphate, -sulphate, -citrate, -carbonate and -bicarbonate, -chlorate, -nitrate and -acetate in a few pithy sentences. In the same paper, the positive actions of serum albumin were carefully observed and understood; he had dialysed lambs' serum for 4 days, rendering it ineffective once shorn of its attendant ions – predominantly potassium, as he knew. Ponder doing this study yourself without a shelf of catalogues or telephone ordering; nor with a pH-meter, digital balance or modern pipette in sight. (Ringer (1883a) refers to chemicals prepared by a Mr Lawes and Mr Page or, commercially, from the firm of Hopkins & Williams, which provided the distilled water which eventually proved so crucial).
The amphibian heart is well suited to Ringer's technique because it has no coronary vascular supply; the extracellular space of the myocytes is freely contiguous with the contents of the ventricular lumen. The spongiform myocardial matrix within a gross trabecular structure thus allows for rapid extracellular exchange, even with a mechanically robust structure the size of a frog's ventricle. Indeed, even 80 years later, the superfused half-ventricle in skilled hands (see, e.g. Chapman & Niedergerke, 1970) would provide valuable new insights into the respective roles of extracellular and intracellular calcium ‘pools’, with exchange times hardly bettered until the advent of isolated single myocyte techniques (with their own, different attendant shortcomings).
Ringer (cf. 1882c) was also aware of the seasonal variation associated with much of the physiology and pharmacology of frog heart, turning these potentially distracting vagaries to his experimental and analytical advantage. The slow contraction time of the frog's heart at room temperature (cold!) allows for faithful recording of the movements via a lever on the smoked drum and allows visual observations to aid interpretation. (Temperature was rarely specified, even when it was an experimental variable. However, lab temperature was evidently hardly different from air temperatures outside, even in winter in London).
What did Ringer actually measure? His index of cardiac function was principally ventricular volume changes of the excised heart. These were recorded via Roy's tonometer, a closed vessel and lever arrangement allowing accurate reporting of volume transmitted via an oil reservoir. A double cannulation tube inserted through the atrium into the ventricle provided a saline flow. (A paper by Charles Roy on arterial compliance, also to be found in volume 3 of the Journal, gives some insight into that author's technical panache for recording mechanical events, as well as his clear grasp of the physics of his day. The chamber shown in Roy's PlatesV and 1 (loc. cit.) gives the general idea of his ‘tonometer’. The device applied to frog heart by Ringer is described in Roy's 1878 paper). Ringer had refined his use of this method in his earlier papers, ligating the heart to the cannula across the atrio-ventricular groove so that only the mechanical response of the ventricle itself was recorded. It is clear from many comments that he also closely observed the ventricle itself during his interventions. For the most part, the hearts would beat spontaneously, but were also challenged with electrical stimuli (‘faradaic discharges’). He was thus able to observe changes to spontaneous rate as well as the all-important form of the beat itself. In this entire group of papers, stress is laid on any ‘rounding’ of the peak of the beat which we would now describe as prolonged contraction or delayed onset of repolarization and slowed relaxation. Comparison is always made with the contraction ‘shape’, observed initially and repeatedly during the experiments, as seen upon exposure to ‘blood replacement’ fluid. This ‘shape’ forms the standard, control response.
The control solution was generally reconstituted from dried bullocks' blood, but the exact composition is hard to establish accurately. Blood was generally re-suspended in 3 or 4 parts of a 0.75% NaCl saline. In some papers, fresh blood was treated similarly. We could thus anticipate that physiologically significant amounts of Ca and K would be contributed from the whole (or dried) blood.
The papers report the clear-sighted development of a ‘recipe’ for a solution able to sustain the normal beat; the acceptable concentration ranges for sodium and potassium are carefully refined through 1882a,b and 1883a,b. Reading the experimental reports and viewing the traces allows anyone with 21st century knowledge of cardiac muscle physiology to ‘spot’ effects probably attributable to action potential duration changes, mild hyper- or depolarization or the Na-withdrawal contracture. The entire latter half of Ringer's (1882b) paper reports conditions that reverse the ‘zero sodium’ contracture to which we would now attribute his observed action of distilled water; his Fig. 4(B) ‘water rigor’ recognizably shows this phenomenon. However, the greatest single insight that led to the definitive 1883a paper is best described by Ringer's own opening paragraph:
‘After the publication of a paper ... (Ringer, 1882a) ... I discovered, that the saline solution which I had used had not been prepared with distilled water, but with pipe water supplied by the New River Water Company. As this water contains minute traces of various inorganic substances, I at once tested the action of saline solution made with distilled water and I found I did not get the effects described in the paper referred to. It is obvious therefore that the effects I had obtained are due to some of the inorganic constituents of the pipe water.’
Do you think Ringer's technician was fired, or perhaps promoted? (Ringer's obituary (1910) implies that his lab. assistant always had difficulty keeping up with his boss's demands during the frequent, but often brief visits that Ringer habitually squeezed between his early morning ward rounds and those to his consulting rooms in Cavendish Place). The chemical analysis of ‘pipe water’ that follows lists ‘38.3 parts per million’ of calcium in first place. This represents almost exactly 1 mM Ca2+, and explains why, for the most part, the earlier papers had been able to report that only K and Na need be added (to the ‘pipe water’, which itself contained ca 0.2 mM K and 1 mM Na) in order to sustain or restore the beat. The cliché that ‘the rest is history’ is, for once, appropriate.
The decisive conclusions (repeated with differing details at several points) are again best expressed in Ringer's own word: (e.g. 1883a, p. 32)
‘A mixture containing 100 c.c. saline [0.75% NaCl], 5 c.c. sodium bicarbonate [0.5%], 5 c.c. calcium chloride ...[1 in 1082, i.e. approximately 0.1% Ca], with 1 c.c. potassium chloride ...[1%] makes an excellent artificial circulating fluid, for with this mixture the heart will continue beating perfectly.’
[This solution equates to approximately (mM): total Na 133, KCl 1.34, NaHCO3 2.76 and CaCl2 1.25, a ‘recipe’ hardly bettered since.]
‘I conclude therefore that a lime salt is necessary for the maintenance of muscular contractility ... yet if unantagonized by potassium salts the beats ... become so broad ... that much fusion of the beats would occur and the ventricle would be thrown into a state of [pseudo]tetanus.’
In regard to this phenomenon of ‘tetanus’, a further paper in the same volume by Ringer (Ringer & Sainsbury, 1883) merits attention. It studies the cardiac refractory period as first described by Marey in 1876; the word ‘drugs’ in the title of the Ringer and Sainsbury paper actually refers to the action of KCl, NH4Cl and NaCl. Raising [K] (e.g. 1883a, p. 32) lowers excitability and prolongs the refractory period. Page 362 (loc. cit.) contains the statement:
‘The striking contrast between potassium and sodium. with respect to this modification [of refractoriness] is of the greatest interest ... because, from the chemical point of view, [it] would be quite unlooked for in two elements apparently so nearly akin.’
This sharp, late 20th rather than late 19th century view of ‘selectivity’, as we might now describe it, is further developed by Ringer (1883c) in studies of the other alkali metals rubidium and caesium, as contrasted with potassium. But by then he had also found the near-equivalence of strontium and calcium for contractility:
‘We find that rubidium can largely replace potassium and I have shown in a previous paper [Practitioner, 31, 81–93, August 1883] that strontium can replace lime.’
The previous page (1883a, p. 31) reports how he came to the conclusion that the alkalinity of the blood ...
‘is no doubt only necessary indirectly for contractility, for muscular contractions develope [sic] acidity and this must give an acid reaction to a neutral fluid, and the heart cannot contract when supplied with an acid circulating fluid.’
This remarkable series of papers 1882–83 clearly laid out the thinking on ionic selectivity that did not come to full fruition until the studies of Hans-Christoph Lüttgau, Bertil Hille, George Eisenman and others 80 and 90 years later. Full understanding of cardiac refractoriness awaited studies by Silvio Weidmann, Denis Noble and many others. As for the role of calcium itself in cardiac contraction, an even longer time was needed to accumulate the definitive works of Rolf Niedergerke, Harald Reuter, Dick Tsien, Alex Fabiato, Mark Cannell, Jon Lederer, Gil Wier and many others, to bring us to the current paradigm of Ca2+ entry through sarcolemmal Ca2+ channels, of Ca2+-induced Ca2+ release from and ATP-fuelled re-uptake to the sarcoplasmic reticulum and the quintessential excitation–contraction coupling ‘link’ of the calcium ‘spark’.
The first spark on the calcium powder-trail was, to paraphrase Pasteur's aphorism, an apparently serendipitous observation being made by a prepared mind. An artefact, an oversight or error in technique, once understood, has so often explained a basic phenomenon of biology, in this case what makes the heart beat. That prepared mind was Sydney Ringer's. His preparedness can still be fully understood by studying this brief series of exemplary papers published within just two years, 1882 and 1883; they deserve to be on your reading list, even in 2004.
Supplementary material
The original papers by Ringer published in The Journal of Physiology can be accessed online at:
DOI: 10.1113/jphysiol.2004.060731. They can also be accessed at http://www.blackwellpublishing.com/products/journals/suppmat/tjp/tjp161/tjp161sm.htm
References
Chapman RA & Niedergerke R (1970). Effects of calcium on the contraction of the hypodynamic frog heart. J Physiol 211, 389–421.
Obituary Sydney Ringer (1910). Br Med J 2, 1384.
Ringer S (1882a). Regarding the action of hydrate of soda, hydrate of ammonia, and hydrate of potash on the ventricle of the frog's heart. J Physiol 3, 195–202.
Ringer S (1882b). Concerning the influence exerted by each of the constituents of the blood on the contraction of the ventricle. J Physiol 3, 380–393.
Ringer S (1882c). Concerning the influence of season and of temperature on the action and on the antagonism of drugs. J Physiol 3, 115–124. [A paper on atropine and aconitine, this paper follows up on work reported in The Journal, volumes 1 and 2].
Ringer S (1883a). A further contribution regarding the influence of the different constituents of the blood on the contraction of the heart. J Physiol 4, 29–42.
Ringer S (1883b). A third contribution regarding the influence of the inorganic constituents of the blood on the ventricular contraction. J Physiol 4, 222–225[A brief note relating of alkalinity and potassium antagonism of ‘lime’ effects; prolongation of beat, delay to diastole].
Ringer S (1883c). An investigation regarding the action of rubidium and caesium salts compared with the action of potassium salts on the ventricle of the frog's heart. J Physiol 4, 370.
Ringer S & Sainsbury H (1883). On the influence of certain drugs on the period of diminished excitability. J Physiol 4, 350–364.
Roy CS (1878). On the influences which modify the work of the heart. J Physiol 1, 452–496.
Simkin MV & Roychowdhury VP (2003). Read before you cite! Complex Systems 14 (3): (c) 269–274.
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