A model of smooth muscle cell synchronization in the arterial wall

Vasomotion is a rhythmic variation in microvascular diameter. Although known for more than 150 years, the cellular processes underlying the initiation of vasomotion are not fully understood. In the present study a model of a single cell is extended by coupling a number of cells into a tube. The simulated results point to a permissive role of cGMP in establishing intercellular synchronization. In sufficient concentration, cGMP may activate a cGMP-sensitive calcium-dependent chloride channel, causing a tight spatiotemporal coupling between release of sarcoplasmic reticulum calcium, membrane depolarization, and influx of extracellular calcium. Low [cGMP] is associated only with unsynchronized waves. At intermediate concentrations, cells display either waves or whole cell oscillations, but these remain unsynchronized between cells. Whole cell oscillations are associated with rhythmic variation in membrane potential and flow of current through gap junctions. The amplitude of these oscillations in potential grows with increasing [cGMP], and, past a certain threshold, they become strong enough to entrain all cells in the vascular wall, thereby initiating sustained vasomotion. In this state there is a rhythmic flow of calcium through voltage-sensitive calcium channels into the cytoplasm, making the frequency of established vasomotion sensitive to membrane potential. It is concluded that electrical coupling through gap junctions is likely to be responsible for the rapid synchronization across a large number of cells. Gap-junctional current between cells is due to the appearance of oscillations in the membrane potential that again depends on the entrainment of sarcoplasmic reticulum and plasma membrane within the individual cell.     

Injection of inositol trisphosphorothioate into Limulus ventral photoreceptors causes oscillations of free cytosolic calcium

Limulus ventral photoreceptors contain calcium stores sensitive to release by D-myo-inositol 1,4,5 trisphosphate (InsP3) and a calcium-activated conductance that depolarizes the cell. Mechanisms that terminate the response to InsP3 were investigated using nonmetabolizable DL-myo-inositol 1,4,5 trisphosphorothioate (InsPS3). An injection of 1 mM InsPS3 into a photoreceptor's light-sensitive lobe caused an initial elevation of cytosolic free calcium ion concentration (Cai) and a depolarization lasting only 1-2 s. A period of densensitization followed, during which injections of InsPS3 were ineffective. As sensitivity recovered, oscillations of membrane potential began, continuing for many minutes with a frequency of 0.07-0.3 Hz. The activity of InsPS3 probably results from the D-stereoisomer, since L-InsP3 was much less effective than InsP3. Injections of 1 mM InsP3 caused an initial depolarization and a period of densensitization similar to that caused by 1 mM InsPS3, but no sustained oscillations of membrane potential. The initial response to InsPS3 or InsP3 may therefore be terminated by densensitization, rather than by metabolism. Metabolism of InsP3 may prevent oscillations of membrane potential after sensitivity has recovered. The InsPS3-induced oscillations of membrane potential accompanied oscillations of Cai and were abolished by injection of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. Removal of extracellular calcium reduced the frequency of oscillation but not its amplitude. Under voltage clamp, oscillations of inward current were observed. These results indicate that periodic bursts of calcium release underly the oscillations of membrane potential. After each burst, the sensitivity of the cell to injected InsP3 was greatly reduced, recovering during the interburst interval. The oscillations may, therefore, result in part from a periodic variation in sensitivity to a constant concentration of InsPS3. Prior injection of calcium inhibited depolarization by InsPS3, suggesting that feedback inhibition of InsPS3-induced calcium release by elevated Cai may mediate desensitization between bursts and after injections of InsPS3.     

Effects of cytochalasin B and local anesthetics on electrical and morphological properties in L cells

Spontaneous oscillations of membrane potential observed in L cells were inhibited rapidly and reversibly in the presence of cytochalasin B (CB). Sustained hyperpolarization induced by high external Ca²⁺ was also depressed by the drug. However, Ca²⁺ injection into the cytoplasm elicited a sustained hyperpolarization, even in the presence of CB. These observations strongly suggest that CB inhibits calcium transport system in cell membrane. Morphological alterations associated with the CB treatment were decreased adhesiveness and rounding of the cells, with concomitant changes in surface architecture. Similar changes in electrophysiological and morphological properties were observed in cells treated with local anesthetics. Since such morphological changes induced by CB and local anesthetics were always preceded by electrical changes, it was suggested that the morphological changes are secondary phenomena resulting from inhibition of the Ca²⁺ transport.     

Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.

There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture.     

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.     

Oscillations in calcium-cyclic AMP control loops form the basis of pacemaker activity and other high frequency biological rhythms.

In this paper theoretical and experimental evidence is presented which indicates that oscillations in internal calcium and cyclic AMP concentrations due to an instability in their common control loops are possible and indeed may be widespread. Further, it is demonstrated that fluctuations in various cellular properties, in particular membrane potential, are a direct consequence of these second messenger oscillations. Given the central importance of calcium and cyclic AMP to the regulation of metabolism, these oscillations would influence most metabolic processes especially rhythmic behaviour. We propose that these oscillations form the basis of several biological rhythms including, potential oscillations in cardiac pacemaker cells, neurones and insulin secreting β-cells, the minute rhythm in smooth muscle, cyclic AMP pulses in Dictyostelium, rhythmical cytoplasmic streaming in Physarum and transepitheliel potential oscillations in Calliphora salivary gland. This model makes possible an explanation of the frequency and amplitude effects of hormones.     

Calcium Oscillations and Waves Generated by Multiple Release Mechanisms in Pancreatic Acinar Cells

We explore the dynamic behavior of a model of calcium oscillations and wave propagation in the basal region of pancreatic acinar cells [Sneyd, J., et al., Biophys. J. 85: 1392–1405, 2003]. Since it is known that two principal calcium release pathways are involved, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR), we study how the model behavior depends on the density of each receptor type. Calcium oscillations can be mediated either by IPR or RyR. Continuous increases in either RyR or IPR density can lead to the appearance and disappearance of oscillations multiple times, and the two receptor types interact via their common effect on cytoplasmic calcium concentration and the subsequent effect on the total amount of calcium inside the cell. Increases in agonist concentration can stimulate oscillations via the RyR by increasing calcium influx. Using a two time-scale approach, we explain these complex behaviors by treating the total amount of cellular calcium as a slow parameter. Oscillations are controlled by the shape of the slow manifold and where it intersects the nullcline of the slow variable. When calcium diffusion is included, the existence of traveling waves in the model equation is strongly dependent on the interplay between the total amount of calcium in the cell and membrane transport, a feature that can be experimentally tested. Our results help us understand the behavior of a model that includes both receptors in comparison to the properties of each receptor type in isolation.     

Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body

Neurogenesis is known to occur in the specific niches of the adult mammalian brain, but whether germinal centers exist in the neural-crest-derived peripheral nervous system is unknown. We have discovered stem cells in the adult carotid body (CB), an oxygen-sensing organ of the sympathoadrenal lineage that grows in chronic hypoxemia. Production of new neuron-like CB glomus cells depends on a population of stem cells, which form multipotent and self-renewing colonies in vitro. Cell fate mapping experiments indicate that, unexpectedly, CB stem cells are the glia-like sustentacular cells and can be identified using glial markers. Remarkably, stem cell-derived glomus cells have the same complex chemosensory properties as mature in situ glomus cells. They are highly dopaminergic and produce glial cell line-derived neurotrophic factor. Thus, the mammalian CB is a neurogenic center with a recognizable physiological function in adult life. CB stem cells could be potentially useful for antiparkinsonian cell therapy.     

IgE receptor-mediated depolarization of rat basophilic leukemia cells measured with the fluorescent probe bis-oxonol

Receptor-mediated changes in plasma membrane potential were recorded in rat basophilic leukemia (RBL) cells with the potential-sensitive fluorescent indicator bis-oxonol. Depolarization of the mitochondria with metabolic inhibitors was not detected by bis-oxonol, suggesting that only potential changes across the plasma membrane were being measured. The resting membrane potential of RBL cells was largely generated by the equilibrium distribution of K+ and not through electrogenic activity of the sodium pump. Depolarization was maintained as long as IgE receptors remained aggregated. We believe that at physiologic calcium concentrations a large portion of the measured potential change may be due to calcium influx across the plasma membrane. Prevention of calcium influx by lanthanum, disruption of aggregated receptors, or prior depolarization in a high K+ saline solution completely inhibited the antigen-induced depolarization. The time course of the antigen-stimulated increase in bis-oxonol fluorescence was similar, but not identical, to the antigen-stimulated rise in cytoplasmic free ionized calcium measured with fura-2. Antigen-stimulated depolarization was inhibited by removing both calcium and sodium and could be restored by the addition of either ion. Reduction of total cellular adenosine triphosphate inhibited depolarization in response to antigen stimulation.     

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering by cytoplasmic calcium binding proteins. Numerical integration of the model allows us to study the fluctuations in the cytosolic calcium concentration, the ER membrane potential, and the concentration of free calcium binding sites on a calcium binding protein. The model demonstrates the physiological features necessary for calcium oscillations and suggests that the level of calcium flux into the cytosol controls the frequency and amplitude of oscillations. The model also suggests that the level of buffering affects the frequency and amplitude of the oscillations. The model is supported by experiments indirectly measuring cytosolic calcium by calcium-induced chloride currents in Xenopus oocytes as well as cytosolic calcium oscillations observed in other preparations.     

Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.     

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx .     

The dynamics of calcium oscillations that activate mammalian eggs

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP(3)) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP(3). This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP(3). The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.     

Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein

Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratiometric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, K(d)s ranging from 470 nm to 29 microm, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery.     

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration [( Ca]i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged -66.9 +/- 0.7 mV (mean +/- SEM, n = 125). Endothelin (10(-10)-10(-6)M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca]i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca]i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10(-4)M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.