Spiral waves and intracellular calcium signalling

JPhysiology (1992) 86, 123-128

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© Elsevier,Paris

Spiral waves and intracellular calcium signalling J D L e c h l e i t e r a, D E C l a p h a m b a Department of Neuroscience, Markey Center, University of Virginia HSC, Charlottesville, VA 22908; Department of Pharmacology, Mayo Foundation, Rochester, MN 55905, USA

Summary - Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intrac e l l u l a r C a 2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular C a 2÷ release in Xenopus oocytes, introduce the concept of C a 2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information. excitable media / IP3 / confocal microscopy / Xenopus oocytes

Introduction

Intracellular Ca 2+ release is the central point of convergence for a multitude of receptor-mediated signals (Berridge, 1987; Berridge and Irvine, 1989). How the Ca 2+ signal is generated and controlled, as well as how information is encoded, have become central questions in cell biology. The final Ca 2+ signal produced by a hormone is often very complex in both its spatial and temporal components (Berridge et al, 1988; Berridge, 1990; Petersen and Wakui, 1990; Tsien and Tsien, 1990; Berridge and Moreton, 1991; Cuthbertson and Chay, 1991; Meyer, 1991). Frequently, fluctuations in the amplitude of the Ca 2+ response are observed and it has been suggested that specific cellular instructions are encoded in periodic Ca 2÷ changes (oscillations) (Rapp and Berridge, 1981; Rapp, 1987). Consequently, much work has focused on defining the underlying basis of these oscillations with the hope of gaining insight into the possible function of Ca 2+ fluctuations in cell biology. We have focused our experiments on Ca 2+ signalling in the X e n o p u s oocytes. Our data indicate that intracellular Ca 2+ release can be viewed as an excitable process, in a manner analogous to electrical excitability in neural and cardiac tissue.

Results

P a t t e r n s o f Ca 2+ release

The process of receptor-induced Ca 2+ release in X e n o p u s laevis oocytes, as observed with confo-

cal imaging, is described in earlier reports (Lechleiter et al, 1991a,b). We find significant differences in the spatio-temporal patterns of Ca 2÷ release tranduced by m2 and m3 muscarinic acetylcholine receptors (mAChRs). The Ca 2+ response of an oocyte expressing m2 mAChRs is characteristically slow (> 20 s to onset), oscillatory and focal (fig la). Ca 2+ release from individual foci propagates irregularly for short distances ( - 20-200 gin), but inevitably stops and dissipates. On the other hand, the ACh-induced response of m3 expressing oocytes is large, rapid and usually transient. The characteristic spatial pattern of maximal Ca 2+ release is invariably a propagating wave which envelopes the entire oocyte (fig lb). In m a n y oocytes, a more complex pattern of Ca 2÷ release is also observed. Specifically, the occurrence of pulsatile foci that produce circularly propagating Ca 2÷ waves, on occasion creating spiral waves of Ca 2÷ release (fig lc). An important observation of the Ca 2+ release patterns

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Fig 1. ACh-induced Ca2+ patterns in oocytes expressing m2 and m3 mAChRs. (top) m2 mAChR (middle) m3 mAChR (bottom). Spiral Ca2+ waves induced by m3 mAChRs. This spatiotemporal pattern of Ca2+ activity (normal projection of five images collected over 10 s) is characteristic of a regenerative excitable medium and suggests that the Ca2+ mobilizing machinery behaves as such. (Adapted from Lechleiter et al, 1991a,b.) is the m u t u a l a n n i h i l a t i o n o f c o l l i d i n g w a v e f r o n t s w h i c h suggests an u n d e r l y i n g r e f r a c t o r y p h e n o m e n o m . The i n a b i l i t y to release Ca 2÷ is o n l y temp o r a r y , since the s a m e r e g i o n o f an o o c y t e s u p p o r t s r e p e t i t i v e wave p r o p a g a t i o n . A d d i t i o n ally, waves propagate with undiminished amplitude. T h e c o m p l e x s p a t i o - t e m p o r a l patterns o f Ca 2÷ release that are o b s e r v e d for h o r m o n e receptorm e d i a t e d signalling are also o b s e r v e d when intrac e l l u l a r Ca L÷ is r e l e a s e d by d i r e c t l y a c t i v a t i n g G - p r o t e i n m e d i a t e d signal transduction with unc a g e d GTP-7-S or by bolus i n j e c t i o n s o f IP3. G e n e r a l l y , an initial w a v e o f Ca 2÷ e n v e l o p e s the

Fig 2. Expanding circular wave pattern induced by GTP-7-S. Wavefront edges have been highlighted. Images were sequential!~ subtracted to define the wavefront edge above resting Ca2 levels (adapted from Lechleiter and Clapham, 1992b).

o o c y t e , i n c r e a s i n g the Ca 2÷ c o n c e n t r a t i o n unif o r m l y in its wake. This is f o l l o w e d by a reduction in Ca 2+ levels during which time p u l s a t i l e focal sites o f C a 2+ release b e g i n to appear. We refer to this p u l s a t i l e C a 2+ activity as the reg e n e r a t i v e phase o f i n t r a c e l l u l a r Ca 2+ release. The s i m p l e s t pattern o b s e r v e d during this phase is an e x p a n d i n g , c i r c u l a r w a v e f r o n t (fig 2). The most c o m p l e x is a spiral w a v e (fig 3). As for h o r m o n e i n d u c e d Ca 2+ release, c o l l i d i n g w a v e f r o n t s annihilate one another. In c o n c l u s i o n , our d a t a indicate

125

C

,ol /

Vel°city (gm:)V. i <-planewave f 20 r~,=9gT/''*~/ -.16

,o

-.08 0 .08 .16 Curvature(gm1)

Fig 4. Curvature-velocity relationship for ACh-induced Ca2+ release (from Lechleiter et al, 1991b).

Fig 3. Spiral wave pattern of Ca 2+ activity induced by IP3 (from Lechleiter and Clapham, 1992a).

that regenerative Ca 2+ activity is mediated by the well established receptor-G protein coupled pathway which stimulates IP3 production (Berridge and Irvine, 1989). Ca 2+ release as an excitable m e d i u m

The wave patterns described in the preceding section are characteristic of an intracellular milieu that behaves as a regenerative excitable medium and suggest that the Ca 2÷ release process behaves as such. An excitable medium is made up of a coupled population of individual excitatory processes (Win free, 1990). Each excitatory process can be excited, in an all-or-none fashion, by a suprathreshold stimulus. Waves of excitation are then propagated from one excitatory process to the neighboring excitatory processes by a common coupling signal. Significantly, the coupling signal must build to a threshold concentration before stimulating the neighboring excitatory process. This creates a time dependence for stimulation on the geometry of the propagating wavefront (Zykov, 1980; Keener, 1986). Thus, a circular wavefront propagates more slowly than a planar wave, since each successive ring of excitation occurs in a larger area and requires more time for the concentration of stimulus to reach threshold level. This is in contrast to planar wave propagation where the next area of excitation is equivalent to the last and requires a constant amount of time for stimulation. The fastest propagation speed occurs when the wavefront

geometry is positive and the subsequent area of excitation is smaller than the last. A linear relationship between curvature and velocity is predicted for small curvatures and is experimentally observed for ACh-induced Ca 2+ activity (fig 4). The slope of this line represents the effective diffusion constant of the excitatory coupling signal, while the y intercept represents the planar (zero curvature) wave velocity and the x intercept represents a negative curvature (critical radius) below which propagation is not possible or conversely stated; the critical radius represents the minimal area necessary to initiate wave propagation. Treating intracellular Ca z+ release as an excitable process, we have estimated the mean planar velocity, critical radius and apparent diffusion constant for Ca z+ activity stimulated by ACh application, GTP-~t-S release and IP3 injections (table I). For comparison, the estimated coefficients of diffusion for Ca 2+ and IP3 within cells are respectively, 4 x 104 and 3.3 × 10-7 cmZ/s (Whitaker and Irvine, 1984; Backx et al, 1989). Thus, our diffusion constant estimates for the coupling excitatory signal suggest that Ca 2+ and not IP3, is the coupling stimulatory signal for receptor-induced Ca 2+ release in X e n o p u s oocytes

Table I. Curvature-velocity parameters for Caa+ activity

Stimulus

ACha GTPTSb IP3S3 b

Planar velocity (gmls)

Critical radius (gm)

Diffusion (× 10-6 cm2/s)

29 28 29.5

10.4 14.2 9.8

2.2 3.97 2.94

a From Lechleiter et al (1991b). b From Lechleiter and Clapham (1992).

126 (Lechteiter et al, 199!b; Lechleiter and Clapham, 1992b). Contribution of Ca 2+ to Ca 2+ excitability

The data discussed above indicate that Ca 2+ is important in coupling the individual excitatory processes. However, when we directly inject Ca 2+ into oocytes, we find that it is very inefficient at inducing regenerative Ca 2÷ activity when compared to IP3 injected oocytes (Lechleiter and Clapharn, 1992b). We also find that other compounds which raise intracellular Ca 2+, specifically caffeine, Ca- lonophore, ryanodlne or thapslgargin, are all unable to initiate regenerative Ca ,'+ activity. In fact, the uniform increase in intracellular Ca 2+ generated by these compounds inhibits regenerative activity that has been initiated by IP3 injections. We suggest that Ca 2+ is important for wave propagation and for the refractory states of the individual Ca 2+ release sites, but that Ca 2+ must act in concert with another molecular element, IP3, to excite the individual Ca 2+ release sites. 9+

•

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is turned by a region with a stronger ability to release Ca 2+, the curvature-velocity relationship cements the new shape since the turned end, the smallest wavefront curvature, propagates more slowly than the trailing wavefront with larger curvature. In this way, spiral wave patterns are produced. Since Ca 2+ waves and subsequent refractory periods are due to excitation, regenerative foci ultimately control the patterns of Ca 2+ signalling within the cell. When only one focus is active, the pulsating focus produces circular waves of Ca 2+ release. In other cases, broken arc and/or spiral waves patterns are created.

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Pattern formation in an excitable medium

Pulsatile release of Ca 2+ at multiple foci creates multiple propagating wavefronts. This creates a multitude of wavefront collisions and as a consequence of annihilation, a multitude of broken arcs of Ca 2+ waves. There are two underlying properties of an excitable medium that are central in determining the patterns that are formed by these wavefronts. The first is the curvature-velocity relationship discussed above for which wavefronts with increasing curvature propagate with increasing speeds. The second property is called the dispersion effect in which the speed of propagation is slowest in the most recently excited areas (Miller and Rinzel, 1981; Dockery et al, 1988). This is due to a time-dependent recovery in the refractory state of the Ca 2÷ release sites. We equate the refractory period to the time when Ca 2+ processes are unable to release Ca 2+. Refractory periods account for the annihilation of Ca 2+ wavefronts when Ca 2+ waves impinge on recently excited areas. However, a gradient of refractivity exists between no Ca 2÷ release and full Ca 2+ release. It is these differentially recovering regions, in concert with the curvature-velocity relationship, that direct the propagation of Ca 2÷ waves. For example, once the end of a Ca 2+ arc

Model f o r intracellular Ca 2+ excitability

We have proposed a model for regenerative Ca 2+ release based on the roles of IP3, the IP3 receptor and Ca 2+ itself (fig 5). We suggest that the IP3 concentration, and consequently the total number of bound IP3Rs, remains relatively constant during the regenerative phase of Ca 2÷ release. This is based on the similarity between Ca 2÷ ac-

R - ~ G;,-~ PIC

Fig 5. Molecular model for regenerative Ca2+ signalling. Ca2+ waves are generated by cyclical Ca2+ stimulation (grey active zone) and inhibition (refractory zone) of 1P3-gated (black spheres of inset) Ca2+ release (IP3Rs shown as tetramers) (from Leehleiter and Clapham, 1992b).

127 tivity produced by IP3 and the non-hydrolyzable analog IP3S3. We account for pulsatite Ca 2+ activity with cyclical stimulation and inhibition of the IP3 bound IP3 R by Ca 2+ (Bezprozvanny et al, 1991; Finch et al, 1991; Iino, 1990). At low Ca 2+ concentrations, Ca 2+ acts as a co-agonist with IP3 to release Ca 2+ from stores. This creates a positive feedback loop which stimulates more Ca 2÷ release until the cytoplasmic Ca 2÷ increases past an inhibitory concentration or until the Ca 2÷ stores are depleted of Ca 2÷. Ca 2+ release is now refractory until the Ca 2+ pumps lower the Ca 2+ concentration and/or refill the Ca 2÷ stores. We consider the positive feedback of Ca 2÷ on IP3 R channel activity (CICR) as the elementary excitatory process. With the IP3R channel as the central element of molecular excitability, Ca 2+ release becomes analogous to electrical excitability in neuronal and cardiac cells where voltage sensitive channels play similar roles. Propagation of Ca 2+ release is also analogous to action potential propagation in neuronal and cardiac cells. However, instead of waves of depolarization, Ca 2÷ waves are due to suprathreshold increases in the Ca 2+ concentration at the neighboring excitatory processes (the IP3 bound IP3 R channel), which in turn release Ca 2+ and excite their neighbors. Therefore, Ca 2+ itself couples the individual excitatory processes into an excitable medium. One final analogy to neuronal excitability is the refractory state of the Ca 2÷ release process. This property results in the annihilation of colliding wavefront and prevents propagation of a signal past the point of collision. This suggests that one of the encoding mechanisms for intracellular Ca 2÷ signalling is directionally dependent, since a spatial location can receive input from only one source at a time. Thus, in addition to frequency, amplitude and space, cellular information can also be encoded by the phase of the signal input.

Conclusions Intracellular Ca 2+ release is clearly important in many signal transduction pathways and it is worth mentioning the possible significance of Ca 2+ foci and spiral wave formation. Until recently, IP3 -induced Ca 2+ release was analyzed with little information in the spatial domain. The advent of video microscopy and suitable fluorophores initially changed this and allowed greater resolution in the spatial examination of Ca 2+ signalling. Our use of confocal microscopy has now yielded precise

spatial information concerning the nature of Ca 2+ release in X e n o p u s oocytes. This provides significant advantages for the analysis of Ca z+ signalling. First, the spatio-temporal pattern formation itself reveals information about the cellular machinery involved in Ca 2+ signalling (eg IP3 production and IP3 R activation). Second, changes in intracellular Ca 2+ can now be precisely determined in spatial and temporal coordinates. This provides a physical entity from which other Ca 2+ signalling effectors may be studied in the future. Thus, one can begin to bridge gaps in the signalling cascades, from hormonal receptor activation and pattern formation on one side, to specific patterns of Ca 2+ release and effector processes on the other side (eg cell growth, differentiation and secretion).

Acknowledgments We thank Dr Patricia Camacho for her careful critiques and help in preparing this manuscript,

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