ITP

Abstract

I describe how we came to microinject inositol trisphosphate into sea urchin eggs and found that is was a very potent activator of calcium release.

When I went back to my lab books I was struck by their quaintness, a quaintness not unusual in things rediscovered after a couple of decades. My experimental summaries were made by hand on paper, not in a spreadsheet; figures for publication were drawn in ink; the manuscript corrections were cut and pasted in. 1984 did not in the event epitomise the continual war and totalitarian repression imagined by Orwell, but it was the year that Indira Ghandi was assassinated, that the Soviets (another quaint item) boycotted the Los Angeles Olympic Games and during which Apple launched the Macintosh. These facts I learned by typing a few words into an internet search engine, an act that will itself seem quaint, I am sure, in twenty years time.

1984 was for me not only the year in which I cut and pasted a manuscript physically for the last time and thereafter did it electronically. It was also the year that for me calcium signalling finally embraced lipid biochemistry. I want to set out briefly the circumstances that led me to microinject ITP into sea urchin eggs.

The calcium legacy-from Tripos to tripod

I owe my interest in calcium signalling to Peter Baker. Peter was a student of Alan Hodgkin’s and thus keen on the giant axon of the squid and also my Physiology supervisor in Cambridge. He wrote a startlingly good review summarising calcium transport and compartmentation [1] and I was hooked. At that time there was great interest in calcium and the control of neurotransmitter release [2]. I became interested in measuring the calcium-stimulated kinetics of vesicle-membrane fusion directly by using calcium-buffered solutions in broken cells. This led us eventually through Baker’s interest in sea urchin eggs [3] to Vacquier’s experiments on the sea urchin cortical granules [4, 5]. Steinhardt had worked with Baker and Hodgkin on the sodium-calcium exchange in squid axon [1] and had gone on to perform the classic experiments with calcium ionophore in sea urchin eggs that he describes in this volume. I was very much taken with fertilization, so it was natural that I spend a year with Steinhardt in Berkeley; at the time he was looking at both calcium and pH changes at fertilization [6-9]. During that year I learned microinjection and fluorescence methods and, most importantly, the logical tripod essential for demonstrating causal linkages, described by Steinhardt in his article in this volume.

Phorbol ester – an exchanger offers a clue

Back in London in the autumn of 1983, Peter Baker mentioned to me that he had found that phorbol ester (known then as TPA) stimulated protein synthesis in sea urchin eggs. The significance of this experiment was that TPA, a tumour promoter, had been recently shown by Nishizuka to stimulate protein kinase C [10, 11]. I knew from Steinhardt’s work that protein synthesis was stimulated after fertilization by an increase in pH and from Epel’s earlier studies that the pH increase at fertilization was due to a sodium-dependent extrusion of protons that could be detected as an acidification of the sea water in which eggs were fertilised [12]. So I conjectured that TPA was stimulating this sodium-hydrogen exchange. In early December I found that TPA caused an acidification of the sea water in which unfertilised eggs were suspended. TPA mimicked the natural stimulation of protein kinase C by diacylglycerols [10], products of the hydrolysis of phosphatidylinositol phospholipids. I had followed with great interest the idea that receptor mediated stimulation of phosphoinositide turnover was correlated with calcium mobilisation [13, 14] and had noted Berridge’s groundbreaking observation that phosphoinositide turnover lead to the rapid production of inositol 1,4,5-trisphosphate [15]. However, until Baker’s chance remarks led me to look at the effect of TPA on sodium-proton exchange, it had not occurred to me that there might be parallels between receptor-mediated calcium mobilisation and calcium mobilisation at fertilisation. I called Michael Berridge who introduced me to Robin Irvine, a very talented biochemist who had isolated and characterised the many inositol polyphosphates. He sent me a panel of inositol polyphosphates. I put them in the freezer.

A Christmas present

When thinking about experiments I am cautious and a sceptic. From experience I was very aware that unfertilised sea urchin eggs were easily activated – it could happen just by inserting an electrode or micropipette. I imagined the need for very many experiments and their controls. The inositol polyphosphates stayed in the freezer. It was Josh Zimmerberg who persuaded me to defrost them. Josh was visiting over the Christmas holidays to collaborate on the idea that exocytosis was driven by osmotic forces, something we later published [16]. Two days before Christmas, we carried out some preliminary experiments with ITP at 100 μM in the pipette and found a very rapid fertilization membrane elevation consistent with a substantial release of calcium. We left to celebrate the holidays in good spirits. My memory has always told me that when I came after Christmas to do controls, the euphoria evaporated. Going back to my lab book, I see that I was not mistaken (Figure 1). I had used the same eppendorf pipette that I used to fill the ITP micropipette to fill the control pipette and thus these control eggs activated when microinjected. What this showed was that ITP was very potent and that we had been injecting it far in excess. Once this was clear, I was content. ITP was so effective as an activator that it was straightforward to distinguish its effects from those of inadvertent activation by microinjection (Figure 2).

Figure 1.

Problems with the controls. A page from the lab book.

Figure 2.

Learning the nomenclature. Entries in the lab book use ITP as an abbrieviation for inositol 1,4,5-trisphosphate. (a) Summary of experimental data in a spreadsheet-free era; (b) An early draft of a figure (top left) uses ITP, not Ins 1 4 5 P₃; the later draft of the figure and some photographs of InsP₃-activated eggs are also shown.

Nineteen eighty four

The prize in 1984 goes to the Macintosh with its icon-based operating system. I doubt in the broad scheme of things that phosphoinositide signalling qualifies even as a runner-up. Nonetheless, in the small world of signalling, it was a fine year for phosphoinositides. It was the year in which Berridge clearly formulated phosphoinositide signalling as a bifurcating pathway with InsP₃ as a calcium-releasing agent and diacylglycerol as a protein kinase C agonist [17, 18]. In the context of fertilization, Berridge’s predictions of the control of the pH change by the diacylglycerol arm of the pathway were confirmed by our work with TPA, begun in 1983 [19]. It was also the year in which it was demonstrated that phosphoinositides turned over at fertilisation [20]. It was natural to think that the analogy between receptor activation and sperm-egg interaction at fertilization was exact [21]. It now seems more likely that the prediction made in 1984 that the sperm introduces InsP₃ or a phospholipase activity when it fuses with the egg [22] is closer to the mark [23, 24].

Epilogue

These experiments with ITP have always been important to me because they linked together those scientists who made the biggest impact on the way I think about things: Peter Baker, Richard Steinhardt and Joshua Zimmerberg.