Propagation of fast and slow intercellular Ca(2+) waves in primary cultured arterial smooth muscle cells

Smooth muscle contraction is regulated by changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). In response to stimulation, Ca²⁺ increase in a single cell can propagate to neighbouring cells through gap junctions, as intercellular Ca²⁺ waves. To investigate the mechanisms underlying Ca²⁺ wave propagation between smooth muscle cells, we used primary cultured rat mesenteric smooth muscle cells (pSMCs). Cells were aligned with the microcontact printing technique and a single pSMC was locally stimulated by mechanical stimulation or by microejection of KCl. Mechanical stimulation evoked two distinct Ca²⁺ waves: (1) a fast wave (2 mm/s) that propagated to all neighbouring cells, and (2) a slow wave (20 μm/s) that was spatially limited in propagation. KCl induced only fast Ca²⁺ waves of the same velocity as the mechanically induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptors, shows that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca²⁺ influx through voltage-operated Ca²⁺ channels, whereas, the slow wave was due to Ca²⁺ release primarily through IP₃ receptors. Altogether, these results indicate that temporally and spatially distinct mechanisms allow intercellular communication between SMCs. In intact arteries this may allow fine tuning of vessel tone.     

The effect of heterogeneously-distributed RyR channels on calcium dynamics in cardiac myocytes

Calcium plays an essential role in excitation-contraction coupling in muscle, and derangements in calcium handling can produce a variety of potentially harmful conditions, especially in cardiac muscle. In cardiac tissue specialized invaginations of the sarcolemma, called T-tubules, penetrate deep into each sarcomere, and depolarization of the SL leads to an influx of calcium through voltage-sensitive channels in the T-tubules that in turn triggers further calcium release from the sarcoplasmic reticulum via ryanodine-sensitive calcium channels. Under certain conditions, such as elevated external Ca²⁺, cardiac cells can release calcium from the sarcoplasmic reticulum spontaneously, producing a calcium ’spark’ and propagating traveling waves of elevated Ca²⁺ concentration, without depolarization of the SL (Wier and Blatter, 1991a, Cell Calcium 12, 241–254; Williams, 1993, Cell Calcium 14, 724–735; Cheng et al., 1993a, Science 262, 740–744). However, under normal resting conditions these potentially harmful waves seldom occur. In this paper we investigate the role of the periodic distribution of ryanodine-sensitive channels in determining whether a spark can trigger a wave, using a modification of the kinetic model proposed by Tang and Othmer, 1994b, Biophys. J. 67, 2223–2235, for calcium-induced calcium release. We show that the spatial localization of these channels near the T-tubules has a significant effect on both wave propagation and the onset of oscillations in this system. Spatial localization provides a possible explanation for the differing effects of various experimental protocols on the system’s ability to propagate a traveling wave. Supported in part by NIH Grant GM29123.     

Fast calcium wave propagation mediated by electrically conducted excitation and boosted by CICR

We have investigated synchronization and propagation of calcium oscillations, mediated by gap junctional excitation transmission. For that purpose we used an experimentally based model of normal rat kidney (NRK) cells, electrically coupled in a one-dimensional configuration (linear strand). Fibroblasts such as NRK cells can form an excitable syncytium and generate spontaneous inositol 1,4,5-trisphosphate (IP(3))-mediated intracellular calcium waves, which may spread over a monolayer culture in a coordinated fashion. An intracellular calcium oscillation in a pacemaker cell causes a membrane depolarization from within that cell via calcium-activated chloride channels, leading to an L-type calcium channel-based action potential (AP) in that cell. This AP is then transmitted to the electrically connected neighbor cell, and the calcium inflow during that transmitted AP triggers a calcium wave in that neighbor cell by opening of IP(3) receptor channels, causing calcium-induced calcium release (CICR). In this way the calcium wave of the pacemaker cell is rapidly propagated by the electrically transmitted AP. Propagation of APs in a strand of cells depends on the number of terminal pacemaker cells, the L-type calcium conductance of the cells, and the electrical coupling between the cells. Our results show that the coupling between IP(3)-mediated calcium oscillations and AP firing provides a robust mechanism for fast propagation of activity across a network of cells, which is representative for many other cell types such as gastrointestinal cells, urethral cells, and pacemaker cells in the heart.     

A PKC wave follows the calcium wave after activation of Xenopus eggs

Calcium waves are well-known hallmarks of egg activation that trigger resumption of the cell cycle and development of the embryo. These waves rapidly and efficiently assure that activation signals are transmitted to all regions of the egg. Although the mechanism by which the calcium wave propagates across an egg as large as that of Xenopus is not known, two models prevail. One model is a wave of calcium-induced calcium release (CICR) and the other is propagation by inositol-induced calcium release (IICR). IICR requires a wave of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, generating two second messengers, IP3, which then releases calcium and DAG, which activates protein kinase C (PKC). We show here that a wave of PKC-green fluorescent protein travels across the egg immediately following, and at the same velocity as, the calcium wave. This is the first example of a PKC wave in a vertebrate egg and supports the IICR model of wave propagation.     

Intercellular Ca2+ wave propagation through gap-junctional Ca2+ diffusion: a theoretical study

Intercellular regenerative calcium waves in systems such as the liver and the blowfly salivary gland have been hypothesized to spread through calcium-induced calcium release (CICR) and gap-junctional calcium diffusion. A simple mathematical model of this mechanism is developed. It includes CICR and calcium removal from the cytoplasm, cytoplasmic and gap-junctional calcium diffusion, and calcium buffering. For a piecewise linear approximation of the calcium kinetics, expressions in terms of the cellular parameters are derived for 1) the condition for the propagation of intercellular waves, and 2) the characteristic time of the delay of a wave encountered at the gap junctions. Intercellular propagation relies on the local excitation of CICR in the perijunctional space by gap-junctional calcium influx. This mechanism is compatible with low effective calcium diffusivity, and necessitates that CICR can be excited in every cell along the path of a wave. The gap-junctional calcium permeability required for intercellular waves in the model falls in the range of reported gap-junctional permeability values. The concentration of diffusive cytoplasmic calcium buffers and the maximal rate of CICR, in the case of inositol 1,4,5-trisphosphate (IP3) receptor calcium release channels set by the IP(3) concentration, are shown to be further determinants of wave behavior.     

Calcium Wave Propagation in Networks of Endothelial Cells: Model-based Theoretical and Experimental Study

In this paper, we present a combined theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. We consider multicellular structures composed of a single chain of cells as well as a chain of cells with a side branch, namely a “T” structure. In the experiments, we investigate the result of applying mechano-stimulation to induce signaling in the form of calcium waves along the chain and the effect of single and dual stimulation of the multicellular structure. The experimental results provide evidence of an effect of architecture on the propagation of calcium waves. Simulations based on a model of calcium-induced calcium release and cell-to-cell diffusion through gap junctions shows that the propagation of calcium waves is dependent upon the competition between intracellular calcium regulation and architecture-dependent intercellular diffusion.     

The role of calcium and potassium conductance in electrogenesis of smooth-muscle cells of the basilar artery

The role of calcium and potassium conductances in electrogenesis of smooth muscle cells of the bovine basilar artery has been investigated using blocking agents of calcium and potassium channels both in the normal Krebs solution and in hyperpotassium solution under anelectrotonic repolarization of the cell membrane. It is shown that both voltage-operated calcium and potassium conductances participate in generation of gradual action potentials evoked by electrical stimulation. A higher contribution of potassium conductance into the total membrane conductance during depolarization is found to be the main factor interfered with development of full-size action potential.     

Agonist-induced calcium transients in cultured smooth muscle cells: measurements with fura-2 loaded monolayers

Elevation of cytosolic Ca2+ in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDT1, A10, rabbit aorta) loaded with the either quin-2 or fura-2, and assayed either in suspension or in monolayer cultures attached to plastic cover slips. Agonists (norepinephrine, vasopressin) induced both the release of intracellular Ca2+ and the influx of extracellular Ca2+. Agonist-induced Ca2+ influx was not blocked by dihydropyridines, and depolarization did not induce Ca2+ influx. However, in fura-2 loaded monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive Ca2+ influx. Thus cultured smooth muscle cells appear to express receptor-operated Ca2+ channels, but not functional voltage-operated Ca2+ channels.     

Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole cell oscillations in smooth muscle cells

In vitro, alpha-adrenoreceptor stimulation of rat mesenteric small arteries often leads to a rhythmic change in wall tension, i.e., vasomotion. Within the individual smooth muscle cells of the vascular wall, vasomotion is often preceded by a period of asynchronous calcium waves. Abruptly, these low-frequency waves may transform into high-frequency whole cell calcium oscillations. Simultaneously, multiple cells synchronize, leading to rhythmic generation of tension. We present a mathematical model of vascular smooth muscle cells that aims at characterizing this sudden transition. Simulations show calcium waves sweeping through the cytoplasm when the sarcoplasmic reticulum (SR) is stimulated to release calcium. A rise in cGMP leads to the experimentally observed transition from waves to whole cell calcium oscillations. At the same time, membrane potential starts to oscillate and the frequency approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes a uniform opening of L-type calcium channels on the cell surface, stimulating a synchronized release of SR calcium and inducing the shift from waves to whole cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion.     

Theory of excitation-contraction coupling in cardiac muscle.

The consequences of cardiac excitation-contraction coupling by calcium-induced calcium release were studied theoretically, using a series of idealized models solved by analytic and numerical methods. "Common-pool" models, those in which the trigger calcium and released calcium pass through a common cytosolic pool, gave nearly all-or-none regenerative calcium releases (in disagreement with experiment), unless their loop gain was made sufficiently low that it provided little amplification of the calcium entering through the sarcolemma. In the linear (small trigger) limit, it was proven rigorously that no common-pool model can give graded high amplification unless it is operated on the verge of spontaneous oscillation. To circumvent this problem, we considered two types of "local-control" models. In the first type, the local calcium from a sarcolemmal L-type calcium channel directly stimulates a single, immediately opposed SR calcium release channel. This permits high amplification without regeneration, but requires high conductance of the SR channel. This problem is avoided in the second type of local control model, in which one L-type channel triggers a regenerative cluster of several SR channels. Statistical recruitment of clusters results in graded response with high amplification. In either type of local-control model, the voltage dependence of SR calcium release is not exactly the same as that of the macroscopic sarcolemmal calcium current, even though calcium is the only trigger for SR release. This results from the existence of correlations between the stochastic openings of individual sarcolemmal and SR channels. Propagation of regenerative calcium-release waves (under conditions of calcium overload) was analyzed using analytically soluble models in which SR calcium release was treated phenomenalogically. The range of wave velocities observed experimentally is easily explained; however, the observed degree of refractoriness to wave propagation requires either a strong dependence of SR calcium release on the rate of rise of cytosolic calcium or localization of SR release sites to one point in the sarcomere. We conclude that the macroscopic behavior of calcium-induced calcium release depends critically on the spatial relationships among sarcolemmal and SR calcium channels, as well as on their kinetics.     

Calcium wave propagation by calcium-induced calcium release: An unusual excitable system

We discuss in detail the behaviour of a model, proposed by Goldbeteret al. (1990.Proc. natn. Acad. Sci. 87, 1461–1465), for intracellular calcium wave propagation by calcium-induced calcium release, focusing our attention on excitability and the propagation of waves in one spatial dimension. The model with no diffusion behaves like a generic excitable system, and threshold behaviour, excitability and oscillations can be understood within this general framework. However, when diffusion is included, the model no longer behaves like a generic excitable system; the fast and slow variables are not distinct and previous results on excitable systems do not necessarily apply. We consider a piecewise linear simplification of the model, and construct travelling pulse and periodic plane wave solutions to the simplified model. The analogous behaviour in the full model is studied numerically. Goldbeter's model for calciuminduced calcium release is an excitable system of a type not previously studied in detail.     

The initiation and propagation of the fertilization wave in sea urchin eggs

Calcium waves sweep across most eggs of the deuterostome lineage at fertilization. The precise timing of the initiation and propagation of a fertilization calcium wave has been best studied in sea urchin embryos, since the rapid depolarization caused by sperm egg fusion can be detected as a calcium influx using confocal imaging of calcium indicator dyes. The time between sperm egg fusion and the first sign of the calcium increase that constitutes the calcium wave is comparable to the time it takes for the wave to sweep across the egg, once initiated. The latency and rise time of the calcium response is sensitive to inhibitors of the InsP3 signalling pathway, as reported previously. Using calcium green dextran and confocal microscopy, we confirm that the propagation time of the calcium wave is lengthened and that initiation of the calcium wave involves activation of calcium release at hot spots that may represent clusters of calcium release channels, as has been seen in other cell types.     

Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells.

Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.     

Calcium-induced calcium release in smooth muscle: the case for loose coupling

This article reviews the key experiments demonstrating calcium-induced calcium release (CICR) in smooth muscle and contrasts the biophysical and molecular features of coupling between the sarcolemmal (L-type Ca²⁺ channel) and sarcoplasmic reticulum (ryanodine receptor) Ca²⁺ channels in smooth and cardiac muscle. Loose coupling refers to the coupling process in smooth muscle in which gating of ryanodine receptors is non-obligate and may occur with a variable delay following opening of the sarcolemmal Ca²⁺ channels. These features have been observed in the earliest studies of CICR in smooth muscle and are in marked contrast to cardiac CICR, where a close coupling between T-tubular and SR membranes results in tight coupling between the gating events. The relationship between this “loose coupling” and distinct subcellular release sites within smooth muscle cells, termed frequent discharge sites, is discussed.     

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration-response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd(3+), N(W)-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd(3+) had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100?mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide - cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.     

Ultrafast Ca wave in cultured vascular smooth muscle cells aligned on a micropatterned surface

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which propagates as Ca²⁺ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca²⁺ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca²⁺ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca²⁺]_i dynamics and membrane potential events along the network of cells. The rate of [Ca²⁺]_i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca²⁺ channels (VOCCs) inhibition suppressed the ultrafast Ca²⁺ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca²⁺ entry from the external medium through VOCCs, modeled as an ultrafast Ca²⁺ wave. This wave may trigger and drive slower Ca²⁺ waves observed ex vivo and in vivo.     

Stretch-induced calcium release in smooth muscle

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.     

Velocity-curvature relationship of colliding spherical calcium waves in rat cardiac myocytes.

Colliding spherical calcium waves in enzymatically isolated rat cardiac myocytes develop new wavefronts propagating perpendicular to the original direction. When investigated by confocal laser scanning microscopy (CLSM), using the fluorescent Ca2+ indicator fluo-3 AM, "cusp"-like structures become visible that are favorably approximated by double parabolae. The time-dependent position of the vertices is used to determine propagation velocity and negative curvature of the wavefront in the region of collision. It is evident that negatively curved waves propagate faster than positively curved, single waves. Considering two perfectly equal expanding circular waves, we demonstrated that the collision of calcium waves is due to an autocatalytic process (calcium-induced calcium release), and not to a simple phenomenon of interference. Following the spatiotemporal organization in simpler chemical systems maintained under conditions far from the thermodynamic equilibrium (Belousov-Zhabotinskii reaction), the dependence of the normal velocity on the curvature of the spreading wavefront is given by a linear relation. The so-called velocity-curvature relationship makes clear that the velocity is enhanced by curvature toward the direction of forward propagation and decreased by curvature away from the direction of forward propagation (with an influence of the diffusion coefficient). Experimentally obtained velocity data of both negatively and positively curved calcium waves were approximated by orthogonal weighted regression. The negative slope of the straight line resulted in an effective diffusion coefficient of 1.2 x 10(-4) mm2/s. From the so-called critical radius, which must be exceeded to initiate a traveling calcium wave, a critical volume (with enhanced [Ca2+]i) of approximately 12 microm3 was calculated. This is almost identical to the volume that is occupied by a single calcium spark.     

Evidence that Ca2+-waves in Xenopus melanotropes depend on calcium-induced calcium release: a fluorescence correlation microscopy and linescanning study

The neuroendocrine melanotrope cell displays Ca2+ oscillations that are build up by several discrete Ca2+ rises ('steps'). Each step is linked to Ca2+-entry across the plasma membrane via voltage-operated calcium channels and associated with a fast Ca2+-wave travelling from the plasma membrane to the central parts of the cell. Previously, linescanning with confocal laser scanning microscopy (CLSM) supported that these waves have high speeds (between 30 and 80 microm/s), which is considered indicative of the involvement of a calcium-induced calcium release (CICR) mechanism in fast-wave propagation. However, to firmly establish the presence of a CICR mechanism one must rule out the possibility that the Ca2+ signal is artifactually accelerated by the presence of a highly mobile Ca2+ probe and also eliminate imaging artifacts inherent to single wavelength imaging. In the present study both problems are addressed. Mobility and intracellular distribution of a generally used Ca2+ probe, Oregon-green 488 BAPTA-1 (O-green-1), were established using fluorescence correlation microscopy. We then used the ratio signal of co-loaded O-green-1 and Fura-Red to quantify the relative [Ca2+]i during linescanning. It was found that O-green-1 displays different diffusion times when regions near the plasma membrane and in the center of the cell are compared. However, the calculated diffusion constant of the probe was too low to account for the observed high speed of the Ca2+ wave. In conclusion, we established the authenticity of the high speed of Ca2+-waves in Xenopus melanotropes, providing evidence for the involvement of a CICR mechanism in wave propagation.