Electrotonic Coupling between Human Atrial Myocytes and Fibroblasts Alters Myocyte Excitability and Repolarization

Atrial fibrosis has been implicated in the development and maintenance of atrial arrhythmias, and is characterized by expansion of the extracellular matrix and an increased number of fibroblasts (Fbs). Electrotonic coupling between atrial myocytes and Fbs may contribute to the formation of an arrhythmogenic substrate. However, the role of these cell-cell interactions in the function of both normal and diseased atria remains poorly understood. The goal of this study was to gain mechanistic insight into the role of electrotonic Fb-myocyte coupling on myocyte excitability and repolarization. To represent the system, a human atrial myocyte (hAM) coupled to a variable number of Fbs, we employed a new ionic model of the hAM, and a variety of membrane representations for atrial Fbs. Simulations elucidated the effects of altering the intercellular coupling conductance, electrophysiological Fb properties, and stimulation rate on the myocyte action potential. The results demonstrate that the myocyte resting potential and action potential waveform are modulated strongly by the properties and number of coupled Fbs, the degree of coupling, and the pacing frequency. Our model provides mechanistic insight into the consequences of heterologous cell coupling on hAM electrophysiology, and can be extended to evaluate these implications at both tissue and organ levels.     

Gap junction coupling and calcium waves in the pancreatic islet

The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of β-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet.     

Mean-field coupling of calcium oscillations in a multicellular system of rat hepatocytes

In a multicellular system of rat hepatocytes and even in an intact liver, cytoplasmic calcium oscillations are synchronized and highly coordinated. In this paper, the mean-field coupling term has been introduced to describe the coupling flux, which is more efficient than gap junctional coupling terms. An optimal coupling strength and an optimal stimulation level for the synchronization of the coupled system have been observed in this paper. Moreover, it has been proved that these results are independent of the cells number. Interestingly, it has been observed that the intracellular noise and the extracellular noise have different effects on the synchronization of the coupled system.     

Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 μM GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca²⁺ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.     

Bursting synchronization dynamics of pancreatic β-cells with electrical and chemical coupling

Based on bifurcation analysis, the synchronization behaviors of two identical pancreatic β-cells connected by electrical and chemical coupling are investigated, respectively. Various firing patterns are produced in coupled cells when a single cell exhibits tonic spiking or square-wave bursting individually, irrespectively of what the cells are connected by electrical or chemical coupling. On the one hand, cells can burst synchronously for both weak electrical and chemical coupling when an isolated cell exhibits tonic spiking itself. In particular, for electrically coupled cells, under the variation of the coupling strength there exist complex transition processes of synchronous firing patterns such as “fold/limit cycle” type of bursting, then anti-phase continuous spiking, followed by the “fold/torus” type of bursting, and finally in-phase tonic spiking. On the other hand, it is shown that when the individual cell exhibits square-wave bursting, suitable coupling strength can make the electrically coupled system generate “fold/Hopf” bursting via “fold/fold” hysteresis loop; whereas, the chemically coupled cells generate “fold/subHopf” bursting. Especially, chemically coupled bursters can exhibit inverse period-adding bursting sequence. Fast–slow dynamics analysis is applied to explore the generation mechanism of these bursting oscillations. The above analysis of bursting types and the transition may provide us with better insight into understanding the role of coupling in the dynamic behaviors of pancreatic β-cells.     

The Role of Cellular Coupling in the Spontaneous Generation of Electrical Activity in Uterine Tissue

The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition.     

Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.

The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.     

Characterization of the glutamine synthetase amplifiable eukaryotic expression system applied to an integral membrane protein--the human thyrotropin receptor.

An amplifiable eukaryotic expression system, based upon glutamine synthetase, has been applied to the production of a complex integral membrane glycoprotein, the human receptor for the polypeptide hormone thyrotropin (TSH). Production of recombinant protein was achieved in chinese hamster ovary (CHO) cells at levels at least 10-fold higher than has been achieved in any other system. After amplification of the inserted gene, the gene copy number was found to be increased in most (but not all) subclones in the range of 3- to 50-fold; mRNA levels of the individual cell lines broadly followed their gene copy number. The level of protein production (measured both functionally and structurally, by radioligand binding and cytofluorimetry, respectively) also reflected these increases in DNA and RNA, but appeared to be limited to a maximum value which we conclude is the maximum that the cells can tolerate without impairing their viability. The receptor is efficiently coupled to adenylate cyclase (22-45 pM TSH producing a 50% response), although the coupling mechanism appeared to be saturated at higher receptor numbers. The high level of expression has allowed, for the first time, the detection of recombinant TSH receptor by immunochemical means. This expression system should prove very useful, not only in facilitating characterization of the TSH receptor, but also for the production of many other integral membrane proteins in their native form.     

Steady states and dynamics of urokinase-mediated plasmin activation in silico and in vitro

Plasmin (PLS) and urokinase-type plasminogen activator (UPA) are ubiquitous proteases that regulate the extracellular environment. Although they are secreted in inactive forms, they can activate each other through proteolytic cleavage. This mutual interplay creates the potential for complex dynamics, which we investigated using mathematical modeling and in vitro experiments. We constructed ordinary differential equations to model the conversion of precursor plasminogen into active PLS, and precursor urokinase (scUPA) into active urokinase (tcUPA). Although neither PLS nor UPA exhibits allosteric cooperativity, modeling showed that cooperativity occurred at the system level because of substrate competition. Computational simulations and bifurcation analysis predicted that the system would be bistable over a range of parameters for cooperativity and positive feedback. Cell-free experiments with recombinant proteins tested key predictions of the model. PLS activation in response to scUPA stimulus was found to be cooperative in vitro. Finally, bistability was demonstrated in vitro by the presence of two significantly different steady-state levels of PLS activation for the same levels of stimulus. We conclude that ultrasensitive, bistable activation of UPA-PLS is possible in the presence of substrate competition. An ultrasensitive threshold for activation of PLS and UPA would have ramifications for normal and disease processes, including angiogenesis, metastasis, wound healing, and fibrosis.     

Altered Kir and gap junction channels in temporal lobe epilepsy

Since astrocytes may sense and respond to neuronal activity these cells are now considered important players in brain signaling. Astrocytes form large gap junction coupled syncytia allowing them to clear the extracellular space from K⁺ and neurotransmitters accumulating during neuronal activity, and redistribute it to sites of lower extracellular concentrations. Increasing evidence suggests a crucial role for dysfunctional astrocytes in the etiology of epilepsy. Notably, alterations in expression, localization and function of astroglial K⁺ channels as well as impaired K⁺ buffering was observed in specimens from patients with pharmacoresistant temporal lobe epilepsy and in chronic epilepsy models. Altered astroglial gap junction coupling has also been reported in epileptic tissue which, however, seems to play a dual role: (i) junctional coupling counteracts hyperactivity by facilitating clearance of elevated extracellular K⁺ and glutamate while (ii) it also provides a pathway for energetic substrates and fuels neuronal activity. Dysfunctional astrocytes should be considered promising targets for new therapeutic strategies.     

Modulation of human fibroblast gap junction intercellular communication by hyaluronan

The composition of the extracellular matrix changes during dermal repair. Initially, hyaluronan (HA) concentration is high, however, by day 3, HA is eliminated. HA optimizes collagen organization within granulation tissue. One possible mechanism of HA modulation of collagen packing is through the promotion of gap junction intercellular communication (GJIC). Gap junctions are gated channels that allow rapid intercellular communication and synchronization of coupled cell activities. The gap junction channel is composed of connexin (Cx) proteins that form a gated channel between coupled cells. HA is reported to enhance Cx43 expression in transformed fibroblasts. GJIC was quantified by the scrape loading technique and reported as a coupling index. The coupling index for human dermal fibroblasts was 4.6 +/- 0.2, while the coupling index for fibroblasts treated with HA more than doubled to 10.6 +/- 0.7. By Western blot analysis no differences were appreciated in the protein levels of Cx43 or beta-catenin, a protein involved in the translocation of Cx to the cell surface. By immuno-histology Cx43 and beta-catenin were evenly distributed throughout the cell in controls, but in cells treated with HA these proteins were co-localized to the cell surface. Coupled fibroblasts are reported to enhance the organization of collagen fibrils. It is proposed that HA increases the accumulation of Cx43 and beta-catenin on the cell surface, leading to greater GJIC and enhanced collagen organization.     

Synchronizing genetic oscillators by signaling molecules

The authors examine collective rhythms in a general multicell system with both linearly diffusive and nondiffusive couplings. The effect of coupling on synchronization through intercellular signaling in a population of Escherichia coli cells is studied. In particular, a synchronization solution is given through the auxiliary individual system for 2 types of couplings. The sufficient conditions for the global synchronization of such a coupled system are derived based on the Lyapunov function method. The authors show that an appropriate design of the coupling and the inner-linking matrix can ensure global synchronization of the coupled synthetic biological system. Moreover, they demonstrate that the dynamics of an individual cell with coupling and without coupling may be qualitatively different; one is oscillatory, and the other is steady state. The change from a nonoscillatory state to an oscillatory one is induced by appropriate coupling, which also entrains all cells to synchronization. These results establish not only a theoretical foundation but also a quantitative basis for understanding the essential cooperative dynamics, such as collective rhythms or synchronization, in a population of cells.     

Estimation of the junctional resistance between electrically coupled receptor cells in Necturus taste buds

Junctional resistance between coupled receptor cells in Necturus taste buds was estimated by modeling the results from single patch pipette voltage clamp studies on lingual slices. The membrane capacitance and input resistance of coupled taste receptor cells were measured to monitor electrical coupling and the results compared with those calculated by a simple model of electrically coupled taste cells. Coupled receptor cells were modeled by two identical receptor cells connected via a junctional resistance. On average, the junctional resistance was approximately 200-300 M omega. This was consistent with the electrophysiological recordings. A junctional resistance of 200-300 M omega is close to the threshold for Lucifer yellow dye-coupling detection (approximately 500 M omega). Therefore, the true extent of coupling in taste buds might be somewhat greater than that predicted from Lucifer yellow dye coupling. Due to the high input resistance of single taste receptor cells (> 1 G omega), a junctional resistance of 200-300 M omega assures a substantial electrical communication between coupled taste cells, suggesting that the electrical activity of coupled cells might be synchronized.     

Active transport of thallous ions by Streptococcus lactis.

Tl+ ions have been shown to mimic or compete with K+ in a number of membrane systems. We confirmed that in starved, valinomycin-treated cells of Streptococcus lactis 7962, Tl+ ions distributed themselves across the bacterial membrane in response to the potassium diffusion potential. In glucose-energized cells, however, Tl+ was taken up by a system specifically stimulated by sodium salts. The intracellular levels of Tl+ exceeded those attained by [3H]triphenylmethylphosphonium ion, a lipophilic cation which accumulates in response to the membrane potential. The uptake of Tl+ by (Na+ and glucose)-stimulated cells was strongly inhibited by potassium salts. These experiments suggest that metabolic energy is coupled to Tl+ transport by means of a high energy phosphate compound and that Tl+ ions are actively transported by a membrane carrier whose normal substrate is K+. The uptake of Tl+ is not a valid method for determining the streptococcal membrane potential.     

Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling.

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.     

Simultaneous measurement of CYP1A2 activity, regioselectivity, and coupling: Implications for environmental sensitivity of enzyme-substrate binding

The cytochrome P450 (CYP) reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature. A high-throughput, optical method was developed to simultaneously measure the rate of substrate depletion, NADPH depletion, and generation of the O-dealkylated product. Increasing the phosphate buffer concentration and temperature increased both the NADPH and EOMCC depletion rates by 6-fold, whereas coupling was constant at 7.9% and the regioselectivity of O-dealkylation to other coupled pathways was constant at 21.7%. Varying the buffer type and pH increased NADPH depletion by 2.5-fold and EOMCC depletion by 3.5-fold; however, neither coupling nor regioselectivity was constant, with variations of 14.4% and 21.6%, respectively. Because the enzyme–substrate binding interaction is a primary determinant of both coupling and regioselectivity, it is reasonable to conclude that ionic strength, as varied by the buffer concentration, and temperature alter the rate without affecting binding while buffer type and pH alter both.     

Exogenous corticosterone and nest abandonment: A study in a long-lived bird, the Adélie penguin

Breeding individuals enter an emergency life-history stage when their body reserves reach a minimum threshold. Consequently, they redirect current activity toward survival, leading to egg abandonment in birds. Corticosterone (CORT) is known to promote this stage. How and to what extent CORT triggers egg abandonment when breeding is associated with prolonged fasting, however, requires further investigation.We manipulated free-living male Adélie penguins with CORT-pellets before their laying period. We then examined their behavioral response with respect to nest abandonment in parallel with their prolactin levels (regulating parental care), and the subsequent effects of treatment on breeding success in relieved birds.Exogenous CORT triggered nest abandonment in 60% of the treated penguins ~ 14 days after treatment and induced a concomitant decline in prolactin levels. Interestingly, prolactin levels in treated penguins that did not abandon their nest were higher at the point of implantation and also after being relieved by females, when compared with abandoning penguins. Among successful birds, the treatment did not affect the number of chicks, nor the brood mass.Our results show the involvement of CORT in the decision-making process regarding egg abandonment in Adélie penguins when incubation is associated with a natural long fast. However, we suggest that CORT alone is not sufficient to trigger nest abandonment but that 1) prolactin levels need to reach a low threshold value, and 2) a rise in proteolysis (i.e. utilization of protein as main energy substrate) seems also to be required.     

Uncoupled Active Transport Mechanisms Accounting for Low Selectivity in Multidrug Carriers: P-Glycoprotein and SMR Antiporters

The extraordinarily low substrate specificity of P-glycoprotein conflicts with the notion that specific substrate interactions are required in the control of the reaction path in an active transport system. The difficulty is shown to be overcome by a half-coupled mechanism in which the ATP reaction is linked to carrier transformations, as in a fully coupled system, but in which the transported substrate plays a passive role. The mechanism, which requires no specific interaction with the substrate, brings about uphill transport. A half-coupled mechanism is directly supported by two observations: (i) almost completely uncoupled ATPase activity in purified P-glycoprotein, and (ii) a pattern of substrate specificity like that of passive systems, where maximum rates for different substrates vary little (unlike active systems, where maximum rates vary greatly). The mechanism accommodates other findings: partial inhibition of ATPase activity by an actively transported substrate; simultaneous binding and translocation of more than one substrate molecule; and stimulation or inhibition of the transport of one substrate molecule by another. A half-coupled system associated with an internal competitive inhibitor should behave as if tightly coupled, in agreement with the effects of the synthetic peptide, polytryptophan. The degree of coupling in the intact system is yet to be determined, however. A half-coupled ATPase mechanism could originally have evolved in a flippase, where immersion of the carrier in its substrate, the membrane lipid, precludes uncoupled ATP hydrolysis. These concepts may have wider application. An uncoupled antiport mechanism, driven by a proton gradient rather than ATP, can explain low selectivity in the SMR multidrug carriers of bacteria, and a half-coupled mechanism for the ion-driven cotransport of water (the substrate in which the carrier site is immersed) can explain a recently proposed uphill flow of water. Received: 23 April 1999/Revised: 29 July 1999     

Type 2 metabotropic glutamate receptor (mGluR2) fails to negatively couple to cGMP in stably transfected cells

The group II metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3) share sequence homology, common pharmacology and negative coupling to cAMP. We recently discovered that mGluR3 also is negatively coupled through a G-protein to the cGMP transduction pathway in rat cerebellar granule cells and astrocytes. To test the hypothesis that mGluR2 also has access to the cGMP pathway, C6 glioma cells were stably transfected with mGluR2 and mGluR3 cDNA and their coupling to cGMP levels was characterized. In contrast to many other cell lines, C6 has a robust cGMP response that makes it attractive in the study of receptor coupling to this second messenger pathway. Consistent with prior studies, the mGluR3 receptor was negatively coupled to cGMP and this coupling was blocked by PTX. In contrast, mGluR2 agonists failed to reduce sodium nitroprusside stimulated cGMP levels in transfected cell lines where the receptor was negatively coupled to cAMP. These data provide further support for the functional divergence between these two closely related receptors.