Two questions

2. Two questions For wetheads • Can one design an experiment to tell if IP3 has to oscillate in order to get an observed calcium oscillation? • How do spatially distributed coupled oscillators behave? For nerds

3. Question 1: Dynamic Probing of Calcium Oscillations James Sneyd, Krasimira Tsaneva-Atanasova University of Auckland, NZ David Yule, Trevor Shuttleworth University of Rochester, USA Michael Sanderson University of Massachusetts Medical Center, USA wetheads

4. This is absolutely not a leak. Very naughty to call it a leak. It is a carefully controlled and modulated calcium influx. Calcium pressure Why? So cells can raise their internal Ca2+ quickly, and then decrease it quickly. Ca2+ (mM) PM pumps ICa RyR Ca2+ (50 nM) leak IPR ER Ca2+-B (buffering) serca Mitochondria

5. Really mean nM, not nm Typical Calcium Oscillations A: Hepatocytes B: Rat parotid gland C: Gonadotropes D: Hamster eggs (post-fertilisation) E, F: Insulinoma cells It is believed that the signal is carried by the frequency of the oscillation.

6. Summary of calcium homeostasis

7. Oscillations mediated by the IPR We know that the kinetics of Ca2+ activation and inactivation of the IPR is sufficient (theoretically) to cause Ca2+ oscillations. Result from sequential positive and negative feedback. But.......

8. IP3 and Ca2+ interactions

9. 1 2 So, what causes the oscillations? Two basic hypotheses 1 Oscillations caused by Ca2+ modulation of IP3 production and/or degradation IP3 and Ca2+ MUST oscillate together. Constant IP3 sets sensitivity Oscillations caused by Ca2+ modulation of the IPR. IP3 is NOT oscillating, or doesn’t have to. Agonist PLC IP3 IPR Ca2+

10. Does IP3 even oscillate? Hirose et al, Science, 1999. Measured flourescence of a GFP -labeled PH domain of PLCd1. This binds both membrane-bound PIP2 and diffusible IP3, but IP3 displaces PIP2. Hence, higher [IP3] causes translocation of the GFP to the cytoplasm, and an increase in cytoplasmic flourescence. So, yes. IP3 oscillates in Madin-Darby canine kidney epithelial cells. But this doesn’t mean it must.

11. Are IP3 oscillations necessary? I. Wakui et al, Nature, 1989. Tried to clamp [IP3] by using a whole-cell patch pipette filled with a nonhydrolysable analogue of IP3, IP3S3 (inositol trisphosphorothioate). Oscillations continued as normal. Concluded that IP3 oscillations are not necessary. (Oscillations were detected by measurement of the whole-cell Ca2+-sensitive Cl- current). This was in ACh-stimulated pancreatic acinar cells. We shall see these again in a minute.

12. Are IP3 oscillations necessary? II. Dupont et al, FEBS Letts., 2003. Tried to remove Ca2+ modulation of IP3 production by increasing the rate of the pathway that doesn’t depend on Ca2+. (In hepatocytes.) Ca2+-dependent IP4 3-kinase IP3 PLC control 5-phosphatase IP2 Addition of exogeneous 5-phosphatase increases the rate of the red pathway. Add more agonist, oscillations come back and look the same. Not Ca2+-dependent

13. Difficulties. • How do you tell if [IP3] is clamped? • How do you tell if most of the IP3 metabolism is via a Ca2+-independent pathway? • How can you tell what the situation is in vivo? • Basically, how can you tell if you’ve done what you think you’ve done, or if the cell does what you think it’s doing (if you haven’t done what you thought you did)? • Tricky. Hmmmmm...... Call in the math nerds and sound the trumpets.

14. cell membrane Jserca JIPR Jleak Jpm Constructing models This is a really crappy model which has since been changed. One really neat thing about our results is that it doesn’t seem to matter what expressions you use here.

15. Different flavours Each model can be constructed in two different flavours: Where Ca2+ oscillations occur for constant [IP3]. Where Ca2+ oscillations occur only when [IP3] oscillates also. So we can investigate the differences caused only by the different dynamic assumptions, not the other model details. But I won’t show the equations for each flavour. We’ve done this with a pile of different models and they all say the same thing.

16. 1 Dynamic behaviour constant IP3 Typical behaviour of such models oscillation period time (s) [IP3] Add a pulse of IP3 here, then let it decay away. Get a transient increase in frequency. An increase in IP3 decreases the oscillation period.

17. 2 Dynamic behaviour oscillating IP3 Typical behaviour of these other models phase delay IP3 pulse [IP3] time (s) limit cycle oscillation Add a pulse of IP3 here, then let it decay away. Get a phase shift. [Ca2+]

18. 1 2 Thus, we predict.... Oscillations caused by Ca2+ modulation of IP3 production and/or degradation Start oscillations by agonist application Photorelease pulse of IP3 Get an initial response to the pulse and then a phase delay, with the oscillations appearing with the same period as before. Constant IP3 sets sensitivity Start oscillations by agonist application. Photorelease pulse of IP3 Oscillations should speed up.

19. Pancreatic acinar cells Typical secretory epithelial cells are pancreatic acinar cells, parotid acinar cells, avian nasal gland cells, etc.

20. Mouse pancreatic acinar cells The methods slide • Blend mouse • Add a pinch of salt • Get pancreatic cells • Add photoreleasable IP3 • Measure whole-cell Ca2+-sensitive Cl- current by patch pipette. • This detects (essentially) the apical concentration of Ca2+.

21. Pancreatic acinar cell responses

22. Results IP3 pulses IP3 pulse gives a clear phase delay These oscillations rely on IP3 oscillations together with the Ca2+ oscillations Note how the period is long. This is consistent with the kinetics of IP3 metabolism. This is in conflict with current dogma on the mechanism of Ca2+ oscillations in pancreatic acinar cells.

23. Side Issue Note this funny initial behaviour upon agonist application. Oscillations on a decreasing baseline. Neato. Math nerds love this kind of thing. We also believe we know why this happens. That was going to be the topic of my talk. Basically, the cell is hosing out Ca2+ during this initial period, and thus slowly modulating the oscillation properties. Analysis involves a two time-scale bifurcation analysis.

24. Mouse pulmonary vascular smooth muscle To a math nerd, mice are the same as rats anyway Mouse • Cut mouse up. • Needle in trachea, blow up lungs. • Needle in pulmonary artery thingy from the heart. Fill pulmonary arteries up with jello. • Pump the airways full of agar. • Blow gently to get the agar out of the major pulmonary arteries, but leave it in all the alveoli. • Take out lung. Freeze and slice. • Melt away the jello. • Hey presto. A lung slice. Garnish with parsley and serve.

25. Calcium oscillations in smooth muscle: I (From Mike Sanderson’s lab.)

26. Calcium oscillations in smooth muscle: II (From Mike Sanderson’s lab.)

27. Results IP3 pulse gives a clear increase in frequency These oscillations occur at constant [IP3]. Or, at least, oscillations in [IP3] are unimportant and not necessary. Note how the period is short. This is consistent with the kinetics of Ca2+ modulation of the IP3 receptor. IP3 pulse here

28. Conclusions Well, they’re easy for me because I don’t do them. The wetheads say they aren’t all that easy, but I don’t believe them. • A relatively simple experiment can be used to determine the dynamic properties of Ca2+ oscillations in vivo. • This experiment doesn’t actually tell us that the specific mechanism is, it just tells us which class of oscillations we are looking at. • An easy way to resolve one of the current major questions in the field. • Couldn’t be done without mathematics. Even better.

29. More work needed • Have to do that GFP experiment thing in pancreatic acinar cells. We predict that IP3 has to be oscillating. This isn’t known. But that dye is bloody expensive and I ain’t paying for it. Well, actually, I am now because we just got an NIH grant. So a big thank you to George W. We love you. (Just kidding, we don’t really.) • Mike has some of that dye but we can’t load into his lung slices. It’s also difficult to get into pancreatic acinar cells. Not sure what to do, actually. Maybe a cultured cell line? • Do the full dose responses and controls with Ca2+ release. (Already done most of this.) • Does the oscillatory mechanism change with time? A fascinating possibility. • Most importantly, do the experiment in other cell types, especially hepatocytes.

30. Future directions • Interaction of cAMP and Ca2+ pathways. Effects of phosphorylation of the IPR. • Control of smooth muscle contraction by Ca2+ oscillations. • Control of saliva secretion in parotid acinar cells by Ca2+ oscillations. • How important are stochastic effects? • How important is the detailed model of the IPR? Or the RyR? And all of these questions will need detailed modeling (in conjunction with experimental work) to understand the answers. Many years of fun ahead.

32. Question 2: coupledcalcium oscillators Three spatially distributed coupled oscillators Real image FEM mesh Apical Region a b c Mitochondrial buffer Basal Region • Two dimensional model; no flux boundary conditions are applied on the external borders of each cell and the cells are connected by flux BC applied on the internal borders. • Question: How important is intercellular diffusion of Ca2+ and IP3 for the coordination (or lack thereof) of the intercellular waves?

33. A point model

34. Bifurcations of the point model

35. Typical solutions of the point model

36. But so what? The point model seems to be a crappy guide to the behaviour of the distributed model. So, what do we do now? The End

37. Identical cells Falls into the 2/1 pattern, where two go together with the third slightly out of phase. This seems to be a lot more stable.

38. Simulations of an 8-cell acinus coupled cells uncoupled cells Quite different behaviour. We now need to go back and inspect the data more thoroughly, at higher time resolution, to see which kind of behaviour is seen. Will we ever do this.......? I’m not sure.