Cdl cddum (1993) 14. w-%97 CDLmgman GroupUK Lid 1993
Localized calcium spikes and propagating calcium waves N.L. ALLBRITTON’ and T. MEYER2 ’ Depattment of Neurobiology, Stanford University,Stanford, CA; and 2Departments of Cell Biolagy and Pharmacology, Duke UniversityMadkal Center, Durham, NC, USA Abstract - Ca2+ signals control or modulate diverse cellular processes such as cell growth, muscle contraction, hormone secretion, and neuronal plasticity [i-3]. Elevations in intracellular Ca2+ concentrations can be hlghly localized to micron and submlcron domains [4, 51 or propagated as intra- and intercellular waves over distances as large as 1 mm [S-s]. Localized, subcellular Ca2+ spikes are thought to selectively activate effector systems such as Ca*+ activated chloride currents in pancreatic acinar cells , neurotransmitter release in synaptic nerve terminals [4, 10, 111, and morphological changes in neural growth cones [5, 12, 131. In contrast, long-ranged Ca2+ waves synchronize the activities of different cytoplasmic regions of a single cell, such as cortical granule exocytosis after egg fertilization [6, 7j or coordinate the activities of many cells, such as ciliary beating in pulmonary epithelium [8, 14, 151. The purpose of this review is to delineate the role of Ca2’ in the generation of localized, subcellular Ca2+ spikes and long-ranged intracellular and intercellular Ca2+ waves .
Can Ca” be a local second messenger and a global second messenger in the same cell? To answer this question we must understand the parameters that affect the times and distances over which second messengers a~ active. These spatio-temporal parameters are determined by the diffusion coefficient of the second messenger, and by the rates of generation and removal of the second messenger. In turn, the diffusion coefficient is determined by the messenger’s size and the properties of its buffers. The rate of increase in messenger concentration is dependent on the properties of the relevant channel or enzyme, and the rate of its removal is determined by
the time required for degradation, buffering, or sequestration.
Generation and amplification Ca2+ signals
Au initial Ca2’ infIux into the cytosol can be produced by ogning of voltage-gated and receptoroperated Ca channels in the plasma membrane. Alternatively, receptor-triggered activation of the phosphoinositide cascade at the plasma membrane can lead to production of inositol 1,4,Mrisphosphate 691
692 (lP3) and consequentopening of channels on intracellular Ca2+ stoics [l]. Following this externally induced Ca2’ rise, most cells trigger an internal Ca2+ spike by a cooperative positive feedback mechanism (Ca2’ feedback onto Ca2’ gated channels or phospholipase C). This type of feedback enables cells to impose a threshold on activation: an initial Ca2+stimulus is either suppressed or it leads to a maximal Ca2’ response. A key function of such a bistable switch is to strongly amplify a small increase in an extracellular stimulus . Ryanodine-receptors [161 and liganded P3-receptors [17, 181 are both Ca2’ channels which can be opened in response to an increase in Ca2’ concentration. Thus, the initial opening of a Ca2’ channel can trigger the opening of adjacent Ca2’ chaunels and thereby amplify Ca2’ signals. Ca2’ feedback processes are tissue specific and Ca2’-gated opening of ryanodine channels is importaut in cardiac muscle, to some extent in skeletal muscle, in many smooth muscle cell types and in a number of neuronal and neurosecretory cells . Positive Ca2’ feedback onto the IPs-receptor has been shown to exist in brain microsome preparations , smooth muscle cells [201 and others . Both types of positive feedback are cooperative with Ca2’
concentration. Though the Ca2’ binding sites on the ryanodine receptor and the IP3 receptor have not yet been identified, Ca2’ is thought to directly bind to these Ca2’ channels and rapidly open them [16. 17, 221. Hence, Ca2’-sated opeyf of Ca2’ channels can generate rapid and local Ca increases (Fip 1). In addition to opening Ca2’ channels, Ca2 acts as a cofactor for different PLC isoforms . Ca2’ increases the efficiency of PLC-B activated by the GTP-bound cc-subunit of its G-protein  and thereby further increases the IP3 concentration. The generated P3 then cooperatively opens more Ca2+ channels. This indirect Ca2’ amplification loop is much slower than Ca2’ activated channel openin [31. These contrasting time scales indicate that Ca2f amplification processes can be ouped into the fast, local Ca2’-gated opening of Ca% channels and into the slower, global coupling of IP3 and Ca2’ concentrations by Ca2’ sensitization of PLC (Fig. 1).
Diffbsion and removal of Ca2’ and inositol 1,4,5t&phosphate (IPs) Our recent measurements of diffusion coefficients (D) of Ca2’ and IPs shed light onto the local and
Ca - pump
Ca - pump
Ca - pump
Calcium-Induced-Calcium-Release (CICR) Fig.
of the mechanisms
IPa-Calcium-Coupling (lCC) amplify
calcium-release (CICR) is defined as the feedback of Ca*’ onto the ryanodine receptor (RyR) or the W mceptor m-R). feedback is a fast (-10 ms) mechanism
to release Ca2’.
phospholipase C (PLC) which in tum generates slower (-1 8).
In these schemes. Cacy quesent.9
(ICC) is de&d
as the feedback of Ca2’ onto This positive feedback is much
the cytosolic Ca2+ concenttation and Carr the stomd calchan. (C&pump) refills the depleted calcium stores after channel closure.
A Ca2’ pump
LOCALIZED CALCIUM SPIKES AND PROPAGATING CALCIUM WAVES
global roles of Ca2+ . In a cytosolic extract from frog oocytes, Ca2+was found to diffuse slowly due to an extensive Ca2’ buffering capacity of the cytosol. D was found to be 13 pm2/s at 100 nM free Ca” and increased to 65 pm2/s at 1 pM free Ca2’. In contrast, D for unbuffered Ca2’ in cytoplasm would be -400 pm2/s. Ca2’ buffering can be described by a buffering ratio f = [Ca ‘bound]/ [Ca2+n& with [Ca2’bo~l being the concentration of Ca2’ that is bound to fast exchanging Ca2’ binding sites. Buffering ratios of Ca2’ in chromaffii cells 1261,neutrophils  and smooth muscle cells (personal communication, F. Fay, Worcester, USA) appear to be NO-fold or larger, indicating that the diffusion of Ca2+in those cells is even slower than in Xenopus oocytes for which f = -10-20 . The slow diffusion of Ca2’ limits the characteristic dimension, , of a local Ca2’ spike : = a (DT)‘~ where T is the duration of the Ca2’ spike and o. is 1.4-2.4, for one- to three-dimensional diffusion, 20pm
Ca2+. A transient, local Ipj or Ca” pulse is represented by the solid black circle in the lower left comer of a model cell. 10 ms and 1 s after a pulse of Ca”
Ca2’ would travel < 1 and -5 pm
(heavily dotted area), xqec~vely. and 25 pm (lightly
waves and spikes?
Fig. 2 Schematic representations of the range of action of
respectively. Buffering is a rapid mechanism for removal of a second messenger since on-rates of Ca2’ buffers are typically lo*-10’ IK’s-~ [29, 301, implying that calcium diffusion prior to buffering is in the sub-millisecond timescale and travels less than 1 pm from its origin. The free calcium concentration then drops from -100 pM to the [email protected] range and calcium travels with a slower diffusion coefficient. For example, a 10 ms long Ca2’ pulse would spread less than 1 pm from its origin (Fig. 2), thus, calcium can act as a local messenger. In contrast, the messenger IP3, which releases Ca2+ from intracellular Ca2 stores, was nearly unbuffered and its diffusion was 5-30 times faster than that of a2+ @ = 280 pm2/s) . In addition, the turnover rate (7) of IPs is relatively slow and ranges from 100 ms in olfactory neurons  to 30 s in myocytes 1321,defming a spatial range, or>, for IP3 in the 10-100 pm range (Fig. 2). Similar to the second messenger CAMP [33-35],lPs can therefore be considered to be a global messenger molecule, whereas the limited diffusion and potentially rapid removal of Ca2+categorize it as a fast, local messenger.
Which second messengers
In contrast, II?, would travel
dotted area) in 10 ms and
The values for D of Ca2’ and
Positive feedback in the Ca2’ cascade is the basis for the generation of repetitive Ca2’ spikes and for the propagation of Ca2’ waves [l-3]. A Ca2’ wave is likely to be a Ca2+ spike propagating in space, indicating that spikes and waves am generated by simiIar mechanisms. ‘Ihe role of Ca2’ waves is to transmit information within cells and between cells. Well studied ex Y.esw;e, Y$..i; eggs [361 and Ca connected glial 1371and epithelial cells . These waves are generated by the diffusion and subsequent amplification of IPs or Ca2+ (Fig. 3). Hence, the diffusion coefficient of the second messenger and the properties of the messenger’s amplifier determine the velocity and shape of the wave. Three models have been presented relating the diffusion coefficient of the messenger that propagates Ca2’ waves to measurable wave parameters [36, 38, 391. They predict a D for the diff&ive
messenger of Ca2+ waves of -300-600 prn2/s. however, have very slow rise times but a velocity However, the models make simplifying assumptions that is nearly the same as in unfertilized Xenopus regarding the nature of the messenger amplification oocytes, and several fold slower than that of process and the diffusive properties of the mess- myocyte Ca2’ waves. For example, the fertilization enger. For example, the diffusion coefficient of the wave of Xenopus oocytes has a t of >5 s and a velsecond messenger is assumed to be constant in all ocity of -10 pm/s [6,7,42]. This fertilization wave models. While this appears to be the case for inosi- is likely to occur by a different mechanism than the to1 1.4.5trisphosphate (IPs), it is clearly not the case waves described in unfertilized oocytes. Additionfor Ca2+. D for Ca2’ diffusion depends on the con- ally many mammalian cells such as hepatocytes, centration of Ca2’ 1251. In addition, the magnitude epithelial cells and glial cells have Ca2’ waves with of the constant y in the dispersion equation D = “/v2t reported rise times of -1-2 s and velocities of -20 (with v the velocity of the Ca2+ wave, and t, the pm/s [15,43,44]. These slower rise times suggest local messenger rise time) depends on the nature of that Ca2’ waves in fertilized Xenopus eggs, hepatothe amplification process 136, 381. This number is cytes, epithelial cells and glial cells are IP3 driven known only for a few simple model amplifiers and could vary in different cell types. For these reasons, current models predicting the D of the messenger for Ca2’ waves can be used to identify a likely candidate for Ca2+waves but cannot be used to unambiguously identify the diffusing messenger. The two candidate messengers for Ca2+ wave Cap- wa\ propagation are IF’3 and Ca2’ itself. IP3 driven Ca2’ waves can result from the lo;tl production of IP3, diffusion of IP3 to $stant Ca stores, and amplification of IPity Ca ac$ation of phospholipase C (Fig. 31; Ca driven Ca waves can be propagated by Ca diffusion to Ca2’ activated ryanodine receptors or IPs receptors [l (Fig. 3). If we use the dispersion relation, D = p 4t., as a guide, we expect that at an identical v and y, waves propagated by a more slowly diffusing second messenger will have a IPs-wavl faster rise time than waves propagated by a more rapidly diffusing messenger. For example, D for I Ca2’ below 1 pM is 165 pm2/s, and D for IP3 is -300 pm2/s; we therefore expect the rise time of calcium for IP3 propagated waves to be slower than u~rfuslon-+AmplllcaUon~Dl~lon~AmpllRcstlon-,Dmudon the rise time forCa2+ propagated waves. It appears that both Ca2+ and IP3 can be the Fig. 3 Schematic representation of three mechanisms of calcium driving messenger for Ca2+ spikes and waves given wave propagation. Ca2+ driven Ca2+ waves may be generated by the right set of circumstances. Cardiac myocyte the feedback of cytosolic Ca2’ (C~C$ onto the ryanodine receptor Ca2+waves have a fast local rise time (-100 ms), a (RyR) or IP3 receptor W-R). In these waves, calcium is the steep concentration gradient and a relatively fast diffusing messengex and sequential ca2’ release is triggered by IP3 velocity of up to 100 pm/s . Waves in unfer- Ca’+-gated release of Ca” from intracelh~lar stores (w). driven Ca2+ waves result from the local production of IP3 by tilized Xenopus oocytes have velocities of -20 pmk receptor (R) stimulated phospholipase C (PLC), diffusion of IPJ to  and fast rise times of -50 ms . The rapid distant Ca2’ stores, and amplification of W by Ca” activation of rise times of these waves require that they be gener- PLC. The distinguishing featme of these waves is that II?J is the ated by a fast amplitier and suggest that they are diffusing messenger and Ca2’ release is triggered by II$gated driven by Ca2+ diffusion. Many cellular waves, release of Ca2+ from intracellular stores.
LOCALIZED CALCIUM SPIKES AND PROPAGATING CALCIUM WAVES
It is conceivable, however, that the waves. measured slow rise times do not accurately reflect the true local rise time since many studies were performed without high spatial resolution. Nevertheless the wide range of velocities and rise times suggests that a multiplicity of mechanisms exists for propagation of Ca2+ waves. Variations in Ca2+ buffer speed and capacity , and in the degradation kinetics of IP3 may further enrich the diversity of Ca2+signaIling. The hypothesis of amplification units
We propose that Ca2+ wave propagation by IF’3 often occurs by diffusion of IP3 between amplification units. An amplification unit is he= defined as a cytoplasmic region where IPs-gated Ca2’ channels are sufficiently close to PLC such that released Ca2’ can enhance the production of IP3. In an arnplifica-
tion unit, an increase in Ca2’ generated by a primary stimulus, such as receptor txiggered IP3 production, would be amplified locally by an autocatalytic release of Ca2’ frm the Ca2’ channel (Fii. 4). The xkased Ca2+ would diffuse to nearby PLC molecules and accelerate the production of IP3, thus producing lP3 that then diffuses to the next amplification unit and nz&rts the cycle. In this situation, both IP3 and Ca2’ act as diffusive messengers. The distance between the amplification units would be limited by the length over which IP3 could travel before it is degraded A small cell (< 20 pm). for example, could function as an amplitication unit in a chain of gap-junction connected cells. For elongated cells. IP3 amplification units could be distributed along the long axis of the cell, with each unit being triggered sequentially. The localization of PLC would have no importance as long as it is near some of the calcium release sites in each amplification unit (Fig. 4). Even if PLC is localized
Fig. 4 Diagmm of a postulated amplifiition
unit that participates in the generation of Ca2’ waves.
consists of two mechanisms of Ca2’ amplification
Ca2’-activated Ip3 cecep~,
In this model, au amplification unit
and Ca2’-activated PLC. Tke
close proximity and mutually support each other: Ca2’ released by E% further opens Ca” chanaeb~ and also generates more &.
am in Each
amplification unit (AU) is coupled to others by the dif%sion of IPs. Hmce, the dimensions of the amplifkation unit are detetmked by the range of action of Ca2’ and the distance between amplification units is dekrmined by the range of action of ll%.
solely to the plasma membrane, IP3 could release calcium from stores located deep inside a cell. Depending on the lifetime of IPs, this Ca2+ release could be more than 100 pm away from the plasma membrane.
The cellular roles of local calcium signals and calcium waves A potential role of Ca2’ waves is to synchronize the activity of individual cells and of groups of gapjunction connected cells [8, 37, 44, 461. How is synchronization between cells achieved? Ca2’ buffering dramatically increases the time it takes for the Ca2+ concentration to rise after passing through a gap junction. Only a small fraction of the Ca2+will be free and about a lOO-fold(the buffering ratio f) mom Ca2’ ions must pass through the pore to give a concentration increase in free Ca2+ equivalent to that of lP3. Because it is much less buffered than Ca2+, the long ranged messenger, lP3, is better suited for cell-to-cell signaling. Indeed, it has recently been shown that Ca2’ is not the messenger that propagates Ca2’ waves across gap junctions  and blockage of these waves by heparin implicates IP3 as the long-ranged messenger that synchronizes cell assemblies. In cells smaller than 20 pm, IP3 mediated Ca2’ responses are expected to be nearly synchronous in all regions of the cell due to its rapid diffusion. Ca2’ feedback onto PLC is therefore a poor mechanism to generate rapid and localized Ca2’ spikes. In contrast, the direct feedback from Ca2’ onto Ca2’ channels is ideally suited to simultaneously activate clusters of Ca2+channels and to generate localized Ca2’ spikes that can reach concentrations of tens of p.M for short time periods. In fact, a key physiologic significance of repetitive calcium spikes could be to permit such spikes to be localized to subcellular domains. In myocytes and in other specialized cells with high densities of Ca2’ activated Ca2’ channels, localized spikes trigger propagating Ca2’ waves independent of Ca2’ activated IPs production 147-491. In cells that contain both types of Ca2’ amplification processes, an initial global rise in Ca2’ could be triggered by IPs diffusion and Ca2’ feedback onto phospholipase C, which is then amplified
locally by Ca2+ activated Ca2’ channels to activate selected target enzymes . Punctionally, these two types of feedback are likely to activate effector systems with different Ca2’ afftity and may serve as an additional means for selective target activation. High afllnity Ca2+ signaling pathways, such as many cahnodulin activated enzymes, could be maximahy activated by long-ran!? submicromolar increases in the cytosolic Ca concentration. On the other hand, low affinity Ca2+signaling pathways, used for example in secretion, may only be activated by localized Ca2’ spikes. lWme studies will have to determine whether the co-localization of low affinity Ca2+sensors to clusters of Ca2’ channels can indeed be demonstrated for a large class of calcium-dependent processes.
Acknowledgements We thank L. Stryer and A. Kindman for stimulating discussions and Marc Langan for graphic arts work. This work was supported by a National Institute of Health Research Fellowship Award 5F32AIO814203 (NLA), a National Institute of Mental Health grant (MH45324). a David and Lucile Paclmd Fellowship (92-5199). and a Shannon Award (I-R55-GM-48113-01).
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Please send reprint requests to : Dr Tobias Meyer, Departments of Cell Biology and Pharmacology, Duke University Medical Center, Box 3709, Durham, NC 27710, USA. Received : 28 July 1993 Accepted : 28 July 1993