Decisions, directions, defects and fellow disciples (A rückblick of 50 years of basic medical research)

One of my favourite stories is from the Old Testament Book of Ruth. The widowed Ruth decided to leave her own country and return with her Jewish mother-in-law to her homeland. As an immigrant, she needed to work in this foreign land, and she soon found herself working in a field that belonged to the man who later became her husband. The phrase that is used about this seemingly chance situation is, ‘It so happened’. My life in science has been full of such ‘so happenings’; it has been a thread in my life thus far. I prefer ‘happenings’ to chance!

It began as an undergraduate; I was unsure of my next step after graduation. ‘It so happened’ that one day, in a place restricted to members of the male sex, the esteemed head of the Department of Pharmacology suggested that I consider staying on to study for a PhD. Decision number 1! After completing my PhD, I was asked to consider returning as a member of staff, but my wife Pam and I decided to commit ‘intellectual suicide’, as my PhD supervisor described it, and accept a position in a new Department of Pharmacology in Nigeria (Africa), then a British colony (decision number 2!). This meant being responsible for designing, equipping, teaching and initiating research. All great experiences for a young man! However, after three years, a position became available in the Department of Physiology at the university medical school across the campus. This department gave me ample time and had better facilities for research; it was funded by the Medical Research Council of West Africa. ‘It so happened’ that John Grayson, the departmental head, had a particular interest in blood flow measurement using the heated thermocouple technique and suggested that we get involved in measuring blood flow in local regions of the myocardium in dogs and primates, especially following acute myocardial ischemia. Thus began a continuing interest in myocardial blood flow and its regulation. One of the early articles (1) was published in the newly inaugurated journal, Cardiovascular Research. The University College Ibadan Medical School (Nigeria) (2) was a faculty of one of three universities in Africa that were set up after World War II to initiate and develop higher level education in the British colonies; the others were in Makerere, Uganda and the West Indies. In Ibadan, the idea was that non-Nigerians, mainly University of London (United Kingdom [UK]) graduates, would remain until Nigerians themselves could be suitably trained. We were there as ‘stop gaps’, although some of these ‘gaps’ remained for many years.

After nine years in Nigeria (a life-changing time for us) and with our children’s education to consider, it was time for us to return to the United Kingdom. It was then that ‘it so happened’ that ‘out of the blue’ (and on a blue airmail letter form!), I received an invitation to join a newly established Department of Physiology and Pharmacology in Glasgow (UK). How we came to Scotland, with no interview for the position, found a home and why we have remained very happily here for well over 40 years, is a story in itself!

Then came another of these ‘it so happened’ moments! Soon after arriving, I attended a meeting in Glasgow on coronary blood flow measurement. At that time, I think Glasgow was the only place in the UK where a research group developed with a particular interest in this area. It came to dominate my research life for the next 40 years. This particular meeting was in honour of Bill Fulton, a consultant in cardiology at Glasgow’s Stobhill Hospital (UK) who had done pioneer work on the human coronary collateral circulation (3). It was at this meeting that I first met Wolfgang Schaper, who became a long-term friend and who has since also worked extensively on collateral vessels in the heart (4,5). Now, ‘it so happened’ that at this highly significant meeting (for me, at least), I sat next to Iain Ledingham, a surgeon at Glasgow’s Western Infirmary (UK) who was just beginning basic physiological studies in the coronary circulation and who invited me to join him. I was there for the next 10 years or so as an Honorary Research Fellow. Besides examining the effects of altering oxygen and carbon dioxide concentrations in inspired air on coronary blood flow (Figure 1 [6,7]), by using the xenon clearance technique in closed-chest greyhound dogs, we also became interested in the effects of hyperbaric oxygen (8,9). ‘It so happened’ that there were two such hyperbaric chambers in the Department of Surgery that were mainly used to treat miners with coal gas poisoning. These chambers were large enough to hold an operating table, ancillary equipment and four or five people. The rationale for our own studies was that if we could bring patients who had suffered an acute heart attack into the chamber and increase their (and our) atmospheric pressure to twice the normal levels, and then give them oxygen to breathe, we might ‘drive’ oxygen into the ischemic myocardium and ‘salvage’ some of the dying cells; breathing oxygen at twice the atmospheric pressure would give an arterial oxygen tension of more than 1000 mmHg, compared with approximately 400 mmHg at normal atmospheric pressure. As we should have expected (Table 1), this approach failed because of the marked vasoconstrictor effect of oxygen; oxygen availability to the ischemic myocardium was actually decreased. Attempts to overcome the vasoconstriction with coronary vasodilator drugs, such as dipyridamole and carbocromen, also failed.

Figure 1).

Changes in myocardial blood flow (mL/100 g/min), measured using the xenon clearance technique, after alterations in arterial oxygen and carbon dioxide tensions (mmHg). Pa Partial pressure

TABLE 1.

The effects of hyperbaric oxygenation on myocardial blood flow, oxygen availability and consumption, and on peripheral coronary flow, in the ischemic region of the left ventricular wall 2 h after coronary artery occlusion in dogs

	Air	Oxygen at 2ATA
Myocardial blood flow (mL/100 g/min)	24±6	14±3*
Myocardial oxygen availability (mL/100 g/min)	7.1±1.9	4.4±1.0
Myocardial oxygen consumption (mL/100 g/min)	4.1±1.4	2.6±0.5
Peripheral coronary flow (mL/min)	2.0±0.5	1.5±0.4*

The arterial oxygen tension when breathing oxygen at twice the atmospheric pressure (2ATA) exceeded 1000 mmHg (compared with 100 mmHg when breathing room air).

^*P<0.05. Blood flow and oxygen consumption in the adjacent essentially normal region of the left ventricular wall were unaffected by hyperbaric oxygenation

One problem with such studies, in which coronary blood flow in the ischemic myocardium was assessed 2 h to 3 h after acute coronary artery occlusion, was the demise of many of the dogs in the immediate 30 min postocclusion period due to severe ventricular arrhythmias, culminating in ventricular fibrillation. Dick Marshall and I overcame this problem by gradually releasing the occlusion when the first ventricular arrhythmias occurred, and continuing to occlude and releasing the occlusion during this critical initial period. We never mentioned this ‘trick’ in any of our papers (9,10), but realized much later that what we were probably doing was inducing ‘ischemic preconditioning’ (11) – a subject in which we later became particularly involved (12,13). We learned the importance of describing every aspect of the techniques used; this ‘defect’ in our description of the methods used in our studies meant that it was only years later that the phenomenon was fully recognized. However, these studies led to something else! ‘It so happened’ that the only pharmaceutical company near Glasgow was a research institute belonging to the Dutch company Organon. Their head of pharmacology asked me whether we had a method for examining the possible effects of newly developed drugs against ischemia-induced ventricular arrhythmias. The one we first examined, designated ORG 2001, proved to be highly effective (14) and also active when given orally (15). This new interest in ventricular arrhythmias led to another highly significant ‘happening’. At a meeting of the British Pharmacological Society in 1970, I met a Hungarian pharmacologist studying at the University of Oxford (UK) called Gyula (Julius) Papp. He had a ‘message’ for me from his department head, Laszlo (Laci) Szekeres. Would I be willing to go to Hungary to discuss a possible collaboration with my cardiovascular research group at Glasgow’s University of Strathclyde (UK)? Thus, I visited Hungary for the first time. The agreed collaboration included visits from members of the Szeged group to Glasgow and a number of joint research publications. One of these visitors was a very young Agnes Vegh who, much later and after I had retired, became a close colleague and good friend. This particular collaboration with Agnes and Laszlo recommenced in 1989 with funding from the Royal Society (UK), the Leverhulme Trust (an Emeritus Fellowship) (UK) and the Szent-Györgyi Albert Fellowship award from the Hungarian Government. This series of experiments began with yet another ‘as it happened’. In Glasgow, I was asked to supervise a Japanese postdoctoral fellow, Dr S Komori, who suggested we examine whether ischemic preconditioning could reduce the severity of postocclusion arrhythmias (16). This study became the basis of a series of experiments regarding the possible mechanisms of this profound antiarrhythmic effect (17–20). We discovered that this protection existed in two phases, with a delayed phase 12 h to 24 h after the preconditioning stimulus (21). Furthermore, this delayed protection could be induced, not only by brief periods of coronary artery occlusion (or ‘classical preconditioning’), but also by cardiac pacing and exercise (22,23) (Figure 2).

Figure 2).

The incidence of ventricular fibrillation (VF) and survival from the combined ischemia-reperfusion insult in control dogs (SC1 and SC2) and in dogs subjected to a single-pacing stimulus (SC1; left hand columns) or to a repeat pacing stimulus (SC2); the dogs were then subjected to coronary artery occlusion 24 h, 48 h, 72 h or 96 h after cardiac pacing. A single period of pacing protects for 24 h after the pacing stimulus, but this protection is lost after 48 h. However, repeat pacing protects for at least 72 h. The filled columns show VF during occlusion and the shaded columns show VF during reperfusion. *P<0.05 versus controls. Adapted from reference 22

Alongside this major interest in coronary blood flow regulation and ventricular arrhythmias, I was also involved in a completely different area of basic cardiovascular research, namely bacterial sepsis (24,25). This had been triggered by membership, in the 1970s, within a ‘shock group’ based at the Western Infirmary. At that time, the intensive care unit was housed in the Department of Surgery under the direction of Iain Ledingham. One of the major problems regarding sepsis in patients is the loss of vascular reactivity and lack of responsiveness to sympathetic nerve stimulation and to the key vasoconstrictor mediator, noradrenaline. The mechanism for this loss of vascular responsiveness was unknown. It was time for another ‘as it happened’! This came in the form of a surprise letter from Professor Jean-Claude Stoclet, Head of the Department of Pharmacology at the Université Louis Pasteur in Strasbourg (France). He asked if one of his PhD students could spend time in Glasgow learning the cardiovascular in vivo techniques that we regularly used. Because we had never met, Jean-Claude invited me to lecture in his department and, perhaps surprisingly, I chose to talk about the problem outlined above – the loss of vascular responsiveness in bacterial sepsis. This led to a decision and an attempt to find the reason for this problem. With the help of a grant from the European Union and two enthusiastic students from Glasgow working in Strasbourg (who became known subsequently as the ‘shocking ladies’!), in a very short time, it was discovered that the culprit was nitric oxide (26–29) – a finding that led to a clinical trial of specific inhibitors of nitric oxide synthesis through the L-arginine pathway. This was of interest because we had also shown that nitric oxide had been involved in the antiarrhythmic effects of ischemic preconditioning (20). I argued that hearts removed from animals given bacterial endotoxin should be protected against the effects of coronary artery occlusion. Because there were two research groups in the same laboratory, I suggested that at the end of the experiments on sepsis, hearts should be removed and given to those working on ischemia-induced ventricular arrhythmias; these hearts, in which nitric oxide had been generated in large amounts by bacterial endotoxin, should be protected against ischemia. My PhD students working in these fields needed a good deal of persuasion (‘what a crazy idea’!), and it was left to the arrival of a postgraduate student from China to take on this idea, with quite striking positive results (30,31).

I now come to my introduction to the Prague group. We had been asked by the European section of the International Society for Heart Research to organize a meeting in Glasgow in 1990. Because I had often travelled ‘behind the iron curtain’, particularly to Poland, East Germany and, of course, Hungary, I knew the difficulty scientists in those countries had regarding meeting fellow scientists in Western Europe; thus, we included funds in our budget to bring scientists from Eastern Europe to Glasgow for the meeting. This was in 1988, when the possibility of a large number of people from these countries being allowed to travel was somewhat remote! Then 1989 happened – the fall of communism and the relative freedom to travel to the West. All the bursaries were accepted, including one by Frank Kolar and his family who came all the way from Prague to Glasgow by car! A consequence of this meeting was the setting up of a joint research program (funded by the European Union) among several laboratories in Western Europe (France, Germany, Holland, Spain and Scotland) and relevant departments in former communist countries (Czechoslovakia, Hungary and Poland). One of the first to come to work in Glasgow was Frank Kolar (32) and this led to a friendship with his department head – Boja Ostadal – whose special birthday we are celebrating. Perhaps because of this relationship, I was asked to join the committee responsible for organizing the international meeting of the International Society for Heart Research in Prague in May 1995. I was privileged to help in what turned out to be a quite wonderful and significant occasion (Figure 3).

Figure 3).

The organizing committee for the 1995 meeting of the International Society for Heart Research (the ‘Prague group’). Photograph taken after the meeting. The identity of the ‘ghost’ at the rear of the picture is unknown!

So far I have outlined how ‘happenings’ have influenced my life in scientific research, its decisions and directions. But what about the missed opportunities or ‘defects’ – the failures to see the implications of some research findings and dismissed because of that lack of flair or imagination that separates the moderately good research scientist from the ‘genius’? One such instance for me was a finding that was not easy to interpret – the significance of which became clear much later. In the early 1970s, I suggested to a young PhD student from Ghana (Africa) that he study receptors in isolated coronary arteries. Twice a week, we obtained pig hearts from the local abattoir, and Alfred Abaitey dissected the left coronary arteries, set them up in an isolated organ bath and examined the effects of various agonists and antagonists. Because he obtained several artery strips from each heart, too many to use on the day the hearts were obtained, he stored some overnight in Krebs solution in the refrigerator. He found that, whereas in fresh specimens noradrenaline relaxed precontracted vessels, in stored vessels, there was usually a constrictor effect. We thought this might result from a change in receptor type from beta-adrenoreceptors (inducing relaxation) to alpha-adrenoceptors (inducing contraction). We joked about looking for beta-receptors ‘floating about’ in the Krebs solution. We now realize that what was probably happening was a loss of endothelial cells from the vessel wall on storage and that the relaxation was due to the release of some dilator substance from these cells. Later, of course, this was found to be nitric oxide and resulted in the Nobel Prize being shared by, among others, Bob Furchgott following his beautiful studies on vessels, albeit not coronary vessels, denuded of their endothelial cells. Later, we became interested in how endothelial denudation, using Triton X-100, modifies vascular responsiveness and arrhythmia severity (33). Interestingly, such endothelial denudation increases arrhythmia severity in normal isolated hearts, but not in hearts removed from hypertensive animals (34).

One of the sad things about scientific research is that it is rare to be allowed to follow interesting observations to a satisfying conclusion, particularly when it is time to retire! However, one great joy of such research comes from the friends (fellow ‘disciples’) one makes all over the world and who remain friends long after active retirement. I have made many such friendships, one to whom this contribution is dedicated and whose special birthday we celebrate this year – Boja Ostadal.

Many colleagues and friends have collaborated in the studies summarized above. Of these I would especially thank Dick Marshall, Kathy Kane, Brian Furman and Cherry Wainwright in Glasgow, Jean-Claude Stoclet in Strasbourg and particularly Laszlo Szekeres and Agnes Vegh in Szeged, as well as a long list of PhD students in all three departments.