Calcium Wave Propagation Research Articles

Acute downregulation of Cx43 alters P2Y receptor expression levels in mouse spinal cord astrocytes.

Propagation of intercellular calcium waves (ICW) between astrocytes depends on the diffusion of signaling molecules through gap junction channels and diffusion through the extracellular space of neuroactive substances acting on plasmalemmal receptors. The relative contributions of these two pathways vary in different brain regions and under certain pathological conditions. We have previously shown that in wild-type spinal cord astrocytes, ICW are primarily gap junction-dependent, but that deletion of the main gap junction protein (Cx43) by homologous recombination results in a switch in mode of ICW propagation to a purinoceptor-dependent mechanism. Such a compensatory mechanism for ICW propagation was related to changes in the pharmacological profile of P2Y receptors, from an adenine-sensitive P2Y(1), in wild-type, to a uridine-sensitive P2U receptor subtype, in Cx43 knockout (KO) astrocytes. Using oligonucleotide antisense to Cx43 mRNA for acute downregulation of connexin43 expression levels, we provide evidence for the molecular nature of such compensatory mechanism. Pharmacological studies and Western blot analysis indicate that there is a reciprocal regulation of P2Y(1) and P2Y(4) expression levels, such that downregulation of Cx43 leads to decreased expression of the adenine-sensitive P2Y(1) receptor and increased expression of the uridine-sensitive P2Y(4) receptor. This change in functional expression of the P2Y receptor subtype population in acutely downregulated Cx43 was paralleled by changes in the mode of ICW propagation, similar to that previously observed for Cx43 KO spinal cord astrocytes. On the basis of these results, we propose that Cx43 regulates both modes of ICW by altering P2Y receptor subtype expression in addition to providing intercellular coupling.

Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts.

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca(2+)](i) transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca(2+)](i) transients were recorded at depths </=100 microm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca(2+)](i) transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t(90-50%)) and from 50 to 10% (t(50-10%)) of the peak [Ca(2+)](i) were (means +/- SE) 73 +/- 4 and 126 +/- 10 ms, respectively, and at 2 Hz, 62 +/- 3 and 94 +/- 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca(2+)](i) decay was markedly slower in fura-2-loaded hearts (t(90-50%) at 1 Hz, 128 +/- 9 ms and at 2 Hz, 88 +/- 5 ms; t(50-10%) at 1 Hz, 214 +/- 18 ms and at 2 Hz, 163 +/- 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca(2+)](i) decline resulted from increased cytosolic Ca(2+) buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca(2+) chelator BAPTA. Propagating calcium waves and [Ca(2+)](i) amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.

Storage and Release of ATP from Astrocytes in Culture

ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A(1). ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently.

血管内皮細胞におけるCa2+伝播とギャップジャンクションとの関係に関する検討

血管内皮細胞におけるCa2+伝播とギャップジャンクションとの関係に関する検討

Calcium wave pacemakers in eggs.

During the past 25 years, the characterization of sperm-triggered calcium signals in eggs has progressed from the discovery of a single calcium increase at fertilization in the medaka fish to the observation of repetitive calcium waves initiated by multiple meiotic calcium wave pacemakers in the ascidian. In eggs of all animal species, sperm-triggered inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] production regulates the vast array of calcium wave patterns observed in the different species. The spatial organization of calcium waves is driven either by the intracellular distribution of the calcium release machinery or by the localized and dynamic production of calcium-releasing second messengers. In the highly polarized egg cell, cortical endoplasmic reticulum (ER)-rich clusters act as pacemaker sites dedicated to the initiation of global calcium waves. The extensive ER network made of interconnected ER-rich domains supports calcium wave propagation throughout the egg. Fertilization triggers two types of calcium wave pacemakers depending on the species: in mice, the pacemaker site in the vegetal cortex of the egg is probably a site that has enhanced sensitivity to Ins(1,4,5)P(3); in ascidians, the calcium wave pacemaker may rely on a local source of Ins(1,4,5)P(3) production apposed to a cluster of ER in the vegetal cortex.

Photolytic flash-induced intercellular calcium waves using caged calcium ionophore in cultured astrocytes from newborn rats.

Waves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. However, the mechanisms responsible for the initiation and propagation of astrocytic calcium waves remain unclear. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium ionophore (DMNPE-caged Br A23187) in cultured astrocytes from newborn rats. The focal photolysis of the caged compound resulted in the increase in intracellular calcium in a single astrocyte, and this increase then propagated to neighboring astrocytes. We also analyzed the spatiotemporal characteristics of the intercellular calcium waves, and estimated the propagation pathways for them. The method using a caged calcium ionophore described in this study provides a new in vitro model for the analysis of intercellular calcium waves.

GAP junctional channel inhibition alters actin organization and calcium propagation in rat cultured astrocytes

Astrocytes are connected by gap junctions, which provide intercellular pathways that allow a direct exchange of ions and small metabolites including second messengers and the propagation of electric currents. The roles of gap junctional communication on whole-cell morphology, cytoskeletal organization, and intercellular communication in astrocytes are not yet clear even in vitro, though there are many studies that have examined the active relation between gap junctions and actin filaments in astrocytes. Here we examined the effects of gap junction inhibitors, which do not interrupt the formation but rather the function of gap junctions, on whole-cell morphology, cytoskeletal organization, and intercellular communication in rat cultured astrocytes. Functional blockade of gap junctions during the formation of an astrocytic monolayer resulted in discordance of actin stress fibers between neighboring cells, even though whole-cell morphology of these cells did not change by such treatment. Mechanical stimulation-induced calcium wave propagation was significantly reduced in these actin-discordance cells even after thorough wash out. Differentiation of astrocytes in the presence of gap junction inhibitors was associated with morphological disarrangement among neighboring cells due to disordered alignment of actin stress fibers between cells. Our results indicate that gap junctional communication enables cell-to-cell coordination of actin stress fibers in astrocytes, thus enhancing intercellular communication through calcium spread.

Altered Calcium-Mediated Cell Signaling in Keratinocytes Cultured from Patients with Neurofibromatosis Type 1

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes cultured from control persons and patients with type 1 neurofibromatosis. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in Ca(2+) signaling between these cells. Keratinocytes cultured from patients with type 1 neurofibromatosis (NF1) had a tendency to form cultures with markedly altered calcium-related signaling characteristics. Specifically, the resting Ca(2+) levels, intracellular Ca(2+) stores, capacitative calcium influx, and gap-junctional signal transduction were defective in NF1 keratinocytes. Western transfer analysis revealed apparently equal connexin 43 protein levels in normal control and in NF1 keratinocytes. Indirect immunofluorescence, however, demonstrated that connexin 43 was relatively evenly distributed in NF1 cells and did not form typical gap-junctional plaques between keratinocytes. Furthermore, the speed of the calcium wave was reduced in NF1 cells compared to normal keratinocytes. The results demonstrate that keratinocytes cultured from patients with NF1 display altered calcium-mediated signaling between cells.

Gap junctions: structure and function (Review).

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.

Role of mitochondria in Ca 2+ oscillations and shape of Ca 2+ signals in pancreatic acinar cells

We studied the role of mitochondria in Ca 2+ signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide- p-tryfluoromethoxyphenyl hydrazone or rotenone (assessed by confocal microscopy using rhodamine-123) induced a partial but statistically significant reduction in the decay of Ca 2+ signals under different experimental conditions. Spreading of Ca 2+ waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca 2+ concentration ([Ca 2+] i) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide- p-tryfluoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca 2+] i oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca 2+ concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba 2+ for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca 2+ clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca 2+ waves and in the development of [Ca 2+] i oscillations.

Gap Junctional Channels Regulate Acid Secretion in the Mammalian Gastric Gland

Gap junction channels are regarded as a primary pathway for intercellular message transfer, including calcium wave propagation. Our study identified two gap junctional proteins, connexin26 and connexin32, in rat gastric glands by RT-PCR, Western blot analysis, and immunofluorescence. We demonstrated a potential physiological role of the gap junctional channels in the acid secretory process using the calcium indicator fluo-3, and microinjection of Lucifer Yellow. Application of gastrin (10-7 m) to the basolateral membrane resulted in the induction of uniphasic calcium signals in adjacent parietal cells. In addition, single parietal cell microinjections in intact glands with the cell-impermeant dye Lucifer Yellow resulted in a transfer of dye from the injected cell to the adjacent parietal cell following gastrin stimulation, demonstrating gastrin-induced cell-to-cell communication. Both calcium wave propagation and Lucifer Yellow transfer were blocked by the gap junction inhibitor 18alpha-glycyrrhetinic acid. Our studies demonstrate that functional gap junction channels in gastric glands provide an effective means for rapid cell-to-cell communication and allow for the rapid onset of acid secretion.

Calmodulin Kinase Pathway Mediates the K+-Induced Increase in Gap Junctional Communication between Mouse Spinal Cord Astrocytes

Astrocytes are coupled to one another by gap junction channels that allow the diffusion of ions and small molecules throughout the interconnected syncytium. In astrocytes, gap junctions are believed to participate in spatial buffering removing the focal excess of potassium resultant from intense neuronal activity by current loops through the syncytium and are also implicated in the propagation of astrocytic calcium waves, a form of extraneuronal signaling. Gap junctions can be modulated by several factors, including elevation of extracellular potassium concentration. Because K(+) elevation is a component of spinal cord injury, we evaluated the extent to which cultured spinal cord astrocytes is affected by K(+) levels and obtained evidence suggesting that a Ca(2+)-calmodulin (CaM) protein kinase is involved in the K(+)-induced increased coupling. Exposure of astrocytes to high K(+) solutions induced a dose-dependent increase in dye coupling; such increased coupling was greatly attenuated by reducing extracellular Ca(2+) concentration, prevented by nifedipine, and potentiated by Bay-K-8644. These results indicate that K(+)-induced increased coupling is mediated by a signaling pathway that is dependent on the influx of Ca(2+) through L-type Ca(2+) channels. Evidence supporting the participation of the CaM kinase pathway on K(+)-induced increased coupling was obtained in experiments showing that calmidazolium and KN-93 totally prevented the increase in dye and electrical coupling induced by high K(+) solutions. Because no changes in connexin43 expression levels or distribution were observed in astrocytes exposed to high K(+) solutions, we propose that the increased junctional communication is related to an increased number of active channels within gap junction plaques.

Stochastic Effects in Intercellular Calcium Spiking in Hepatocytes

We carry out a Monte Carlo simulation of stochastic effects for two models of intercellular calcium wave propagation in rat hepatocytes. Both models involve gap junction diffusion by a second messenger. We find that, in general, the stochastic effects improve agreement with experiment, for a reasonable choice of model parameters. Both stochastic models exhibit baseline fluctuations and variations in the peak heights of Ca2+. In addition, we find for one model that there is a distribution of latency times, rather than a single latency time, with a distribution width which is comparable to the experimental observation of spike widths. We also find for the other model with low gap junction diffusion that it is possible for cell multiplets to oscillate independently initially, but to subsequently become synchronized.

Calcium Waves in Agarose Gel with Cell Organelles: Implications of the Velocity Curvature Relationship

Calcium oscillations and waves have been observed not only in several types of living cells but also in less complex systems of isolated cell organelles. Here we report the determination of apparent Ca 2+ diffusion coefficients in a novel excitable medium of agarose gel with homogeneously distributed vesicles of skeletal sarcoplasmic reticulum. Spatiotemporal calcium patterns were visualized by confocal laser scanning fluorescence microscopy. To obtain characteristic parameters of the velocity curvature relationship, namely, apparent diffusion coefficient, velocity of plane calcium waves, and critical radius, positively and negatively curved wave fronts were analyzed. It is demonstrated that gel-immobilized cell organelles reveal features of an excitable medium. Apparent Ca 2+ diffusion coefficients of the in vitro system, both in the absence or in the presence of mitochondria, were found to be higher than in cardiac myocytes and lower than in unbuffered agarose gel. Plane calcium waves propagated markedly slower in the in vitro system than in rat cardiac myocytes. Whereas mitochondria significantly reduced the apparent Ca 2+ diffusion coefficient of the in vitro system, propagation velocity and critical size of calcium waves were found to be nearly unchanged. These results suggest that calcium wave propagation depends on the kinetics of calcium release rather than on diffusion.

The mechanism of propagation of intracellular calcium waves in cultured human uterine myocytes

Objective: The primary goal of this work was to determine the relative importance of sarcoplasmic reticulum inositol 1,4,5-triphosphate receptors and ryanodine receptors in the mechanism of intracellular calcium wave propagation in human uterine myocytes. A secondary goal was to identify the rate-determining step of calcium wave propagation. Study Design: Pregnant human myometrium was obtained at the time of cesarean delivery, enzymatically dispersed, and cultured through several passages. Intracellular calcium wave velocities were measured with video fluorescence microscopy and the calcium-dependent fluorescent dye calcium green 1. Experimental conditions were modified by exposure of the cells to ruthenium red (blocked ryanodine receptor), ryanodine (locked open ryanodine receptor), oxytocin (increased inositol-1,4,5-triphosphate), sodium butyrate (intracellular acidification), ammonium chloride (intracellular alkalinization), and elevation of temperature (from 19°C to 30°C). Results: Wave velocities were found to be the same for spontaneously occurring (9.6 ± 2.6 μm/s) and oxytocin-stimulated (10.3 ± 3.4 μm/s) waves. Advance treatment of the cells with ryanodine or ruthenium red failed to change oxytocin-stimulated wave velocities from control values. The temperature dependence of calcium wave velocities was studied across the range 19°C to 30°C. Plots of wave velocities versus the inverse of the temperature yielded apparent activation energies that were the same for spontaneous (13.2 ± 0.3 kcal/mol) and oxytocin-induced (14.3 ± 1.6 kcal/mol) waves. After intracellular acidification by treatment with butyrate (20 mmol/L) wave velocities increased by 44%. Wave velocities decreased by 35% after treatment with ammonium chloride (20 mmol/L). Conclusion: Propagation of intracellular calcium waves in cultured human uterine myocytes exhibited mechanisms of sarcoplasmic reticulum calcium release that could use either inositol 1,4,5-triphosphate receptors alone or ryanodine receptors alone, or both together. The rate-determining step for calcium wave propagation was diffusion of calcium though a highly buffered cytoplasm. (Am J Obstet Gynecol 2001;184:1228-34.)

Vasopressin receptor distribution in the liver controls calcium wave propagation and bile flow.

SPECIFIC AIMSA gradient in hormone receptor density in the liver lobule is responsible for the spatial organization of hormone-induced intercellular calcium waves. In this study, we show that the l...

Propagation of Intercellular Calcium Waves in Retinal Astrocytes and Müller Cells

Intercellular Ca(2+) waves are believed to propagate through networks of glial cells in culture in one of two ways: by diffusion of IP(3) between cells through gap junctions or by release of ATP, which functions as an extracellular messenger. Experiments were conducted to determine the mechanism of Ca(2+) wave propagation between glial cells in an intact CNS tissue. Calcium waves were imaged in the acutely isolated rat retina with the Ca(2+) indicator dye fluo-4. Mechanical stimulation of astrocyte somata evoked Ca(2+) waves that propagated through both astrocytes and Müller cells. Octanol (0.5 mm), which blocks coupling between astrocytes and Müller cells, did not reduce propagation into Müller cells. Purinergic receptor antagonists suramin (100 microm), PPADS (20-50 microm), and apyrase (80 U/ml), in contrast, substantially reduced wave propagation into Müller cells (wave radii reduced to 16-61% of control). Suramin also reduced wave propagation from Müller cell to Müller cell (51% of control). Purinergic antagonists reduced wave propagation through astrocytes to a lesser extent (64-81% of control). Mechanical stimulation evoked the release of ATP, imaged with the luciferin-luciferase bioluminescence assay. Peak ATP concentration at the surface of the retina averaged 78 microm at the stimulation site and 6.8 microm at a distance of 100 microm. ATP release propagated outward from the stimulation site with a velocity of 41 microm/sec, somewhat faster than the 28 microm/sec velocity of Ca(2+) waves. Ejection of 3 microm ATP onto the retinal surface evoked propagated glial Ca(2+) waves. Together, these results indicate that Ca(2+) waves are propagated through retinal glial cells by two mechanisms. Waves are propagated through astrocytes principally by diffusion of an internal messenger, whereas waves are propagated from astrocytes to Müller cells and from Müller cells to other Müller cells primarily by the release of ATP.

Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity.

Information storage in the nervous system requires transcription triggered by synaptically evoked calcium signals. It has been suggested that translocation of calmodulin into the nucleus, initiated by submembranous calcium transients, relays synaptic signals to CREB. Here we show that in hippocampal neurons, signaling to CREB can be activated by nuclear calcium alone and does not require import of cytoplasmic proteins into the nucleus. The nucleus is particularly suited to integrate neuronal firing patterns, and specifies the transcriptional outputs through a burst frequency-to-nuclear calcium amplitude conversion. Calcium release from intracellular stores promotes calcium wave propagation into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors. Pharmacological or genetic modulation of nuclear calcium may directly affect transcription-dependent memory and cognitive functions.

Propagation of intercellular calcium waves in C6 glioma cells transfected with connexins 43 or 32.

In this study, gap junction-deficient C6 glioma cells, transfected with either connexin 43 (Cx43) or 32 (Cx32), have been used to evaluate the ability of these connexins to pass intercellular Ca2+ waves. Ca2+ waves, observed with fluorescence imaging using fura-2 or fluo-3, were initiated by mechanical stimulation in the presence of a supra-perfusion of the extracellular fluid or by the non-contact technique of flash photolysis of intracellular caged-IP3. Following manual mechanical stimulation, the parental C6 glioma cells and cells expressing Cx43 and Cx32 gap junctions all propagated intercellular Ca2+ waves. Ca2+ waves in cells expressing Cx43 traveled approximately twice the distance as compared to waves in cells expressing Cx32 or parental cells. The cells expressing Cx43 were also about twice as sensitive to ATP as cells expressing Cx32. In the presence of a supra-perfusion of extracellular fluid, the Ca2+ waves in parental cells were almost abolished while the mechanically induced Ca2+ waves in the cells expressing Cx43 and Cx32 propagate similar but limited distances of several cells in a direction opposite to the fluid flow. The photolytic release of IP3, but not Ca2+, in cells expressing Cx43 or Cx32 resulted in the propagation of Ca2+ waves that traveled distances similar to those observed in the presence of supra-perfusion. Parental C6 glioma cells did not initiate intercellular Ca2+ waves when stimulated by photolysis. From these studies we conclude that (1) both Cx43 and Cx32 based gap junctions are permeable to IP3 and can serve to communicate Ca2+ waves, (2) that Ca2+ wave propagation via gap junctions was dependent on the diffusion of IP3 but not Ca2+, (3) that an extracellular messenger capable of communicating waves is released from only the stimulated cell, and (4) that simultaneous intracellular and extracellular signaling can occur to enhance the propagation of intercellular Ca2+ waves.

Underlying Mechanisms of Symmetric Calcium Wave Propagation in Rat Ventricular Myocytes

Calcium waves in heart cells are mediated by diffusion-coupled calcium-induced calcium release. The waves propagate in circular fashion. This is counterintuitive in view of the accepted ultrastructure of the cardiac myocyte. The density of calcium release sites in the transverse direction is four times higher than in the longitudinal direction. Simulations with release sites localized along Z-lines and isotropic diffusion yielded highly elliptical, nonphysiological waves. We hypothesized that subcellular organelles counteracted the higher release site density along the Z-lines by acting as transverse diffusion barriers and sites of active calcium uptake. We quantified the reduction of transverse diffusion by microinjecting cells with the nonreactive dye fluorescein. The ratio of the radial diffusion coefficient to the longitudinal coefficient was 0.39. Inhibition of mitochondrial uptake by rotenone accelerated the wave in the transverse direction. Simulations with release sites clustered at the Z-lines and a transverse diffusion coefficient 50% of the longitudinal coefficient generated waves of ellipticity 2/1 (major axis along the Z-line). Introducing additional release sites between the Z-lines at a density 20% of that on the Z-lines produced circular waves. The experiments and simulations support the presence of transverse diffusion barriers, additional uptake sites, and possibly intermediate release sites as well.