Gap Junction Permeability Research Articles

Local dissipation and coupling properties of cellular oscillators: A case study on calcium oscillations

Synchronised signal transduction between cells is crucial, since it assures fast and immutable information processing, which is vital for flawless functioning of living organisms. The question arises how to recognise the ability of a cell to be easily coupled with other cells. In the present paper, we investigate the system properties that determine best coupling abilities and assure the most efficient signal transduction between cells. A case study is done for intercellular calcium oscillations. For a particular diffusion-like coupled system of cellular oscillators, we determined the minimal gap-junctional permeability that is necessary for synchronisation of initially asynchronous oscillators. Our results show that dissipation is a crucial system property that determines the coupling ability of cellular oscillators. We found that low dissipation assures synchronisation of coupled cells already at very low gap-junctional permeability, whereas highly dissipative oscillators require much higher gap-junctional permeability in order to synchronise. The results are discussed in the sense of their biological importance for systems where the synchronous responses of cells were recognised to be indispensable for appropriate physiological functioning of the tissue.

Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes.

Mitogenic stimulation of cardiomyocytes is associated with decreased gap junction coupling and protein kinase C (PKC)-mediated phosphorylation of the gap junction protein connexin43 (Cx43). Identification of and interference with the amino acid(s) that becomes phosphorylated in response to stimulation are important steps towards defining the relationship between Cx43 phosphorylation and cell cycle. Using immunoblotting and phosphospecific antibodies we were able to show that serine-262 (S262) on Cx43 becomes phosphorylated in response to growth factor or PKC stimulation of cardiomyocytes. To examine the effect of Cx43, S262 phosphorylation and cell-cell contact (and/or coupling) on DNA synthesis, we overexpressed wild-type (wt) or mutant Cx43, carrying a S262-to-alanine (S262A, simulating the unphosphorylated state) or a S262-to-aspartate (S262D, simulating constitutive phosphorylation) substitutions in cultures of cell-cell contact forming or isolated cardiomyocytes. Overexpression of wt-Cx43 caused a significant decrease in DNA synthesis irrespective of the presence of cell-cell contact. In cell-cell contact forming cultures, the S262D mutation reversed while the S262A mutation increased the inhibitory effect of Cx43. In the absence of cell-cell contact, the S262-Cx43 mutations had no significant effect on Cx43 inhibition of DNA synthesis. Dye-coupling, evaluated by scrape-loading, indicated increased gap junction permeability in S262A (compared to wt or S262D) overexpressing myocytes. We conclude that Cx43 inhibits cardiomyocyte DNA synthesis irrespectively of cell-cell contact or coupling. Cell-cell contact, and possibly gap junction-mediated communication is required, however, in order to reverse Cx43 inhibition of DNA synthesis by S262 phosphorylation.

Protective role of gap junctions in preconditioning against myocardial infarction.

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue.

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells.

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+.

Wrong connexin signals cataracts

Besides allowing ions to flow between adjacent cells, gap junctions selectively conduct larger signaling molecules. What determines their selectivity is unknown. Martinez-Wittinghan et al. (page 969) explored this problem by extensively manipulating the genetic loci of the two connexin proteins that make up gap junctions in the lens of the eye. The results show that the developing lens is exquisitely sensitive to changes in the composition of gap junctions, and that alterations in gap junction permeability could underlie human diseases ranging from cataracts to deafness. Figure Dye coupling (left) is sharply reduced in cells with a mixture of connexins (right). In previous studies, mice lacking the connexin 46 gene, which is expressed primarily in fiber cells beneath the lens epithelium, grew lenses of normal size but with severe cataracts. Deletion of the connexin 50 gene, which is expressed in both the lens epithelium and the fiber cells, caused a severe growth defect in the lens but only mild cataracts. The authors have now generated new mouse strains in which the coding sequences of connexins 46 and 50 were deleted from and swapped between their normal genetic loci in a variety of combinations, producing a spectrum of expression patterns. When connexins 46 and 50 are both expressed in the epithelium as well as the fiber cells, the lens grows to normal size but has severe cataracts. Expressing only connexin 46 in the epithelium and fiber cells prevents cataracts but produces a growth defect. Ionic coupling between lens cells appears to be adequate in the mutants. The authors conclude that connexin 46 maintains lens clarity, and connexin 50 ensures proper lens growth, by determining the permeability of gap junctions not just to ions but to larger biochemical signals. In humans, mutations in connexin genes are associated with hearing loss, inherited cataracts, and several skin diseases. The new work suggests that these mutations might change the composition of gap junctions, altering their permeability and generating signaling errors during development. ▪

Effect of endothelin-1 on astrocytic protein content.

The astrocytic endothelin (ET) receptors, ET(A) and ET(B), modulate calcium signaling and the astrocytic gap junctional network. The nonselective ET receptor ligand ET-1 inhibits gap junction permeability, an effect that can be blocked by tolbutamide. This mechanism may play a role in pathophysiological conditions such as ischemic stroke, characterized by elevated tissue ET-1 levels and hypertrophic-appearing reactive astrocytes. Therefore, the effect of ET-1 on cellular protein content was investigated in confluent once-passaged rat astrocyte cultures under serum-free conditions, by the Lowry method. Gap junction permeability was determined by the dye transfer technique. ET-1 prevented the decrease in astrocytic protein content observed in controls. The effect of ET-1 on cellular protein content was most pronounced in cultures seeded at high density, but it was attenuated in ET(B)-deficient (sl/sl) astrocytes. This effect could be blocked by the nonselective ET antagonist LU 302872 (10 micro M), as well as by the protein synthesis inhibitor cycloheximide (10 micro M). This increase in astrocytic protein content was inhibited by the ATP-sensitive K(+) channel blocker tolbutamide, which also antagonized the ET-1-induced reduction of gap junction permeability and reversed the morphological changes observed in astrocytes upon ET-1 treatment. Cytosine arabinoside (10 micro M), a DNA synthesis blocker, inhibited the ET-1-induced BrdU uptake without affecting the ET-1-induced increase in astrocytic protein content. To conclude, ET-1 induces an increase in astrocytic protein content as well as changes in astrocyte morphology in vitro. This hypertrophic response involves uncoupling of the astrocytic gap junctional network and is not dependent on DNA synthesis.

Opposing effects of nitric oxide on different connexins expressed in the vascular system.

Gap junctions--clusters of intercellular channels built by connexins (Cx)--are thought to be important for vascular cell functions such as differentiation, control of tone, or growth. In the vascular system, gap junctions can be formed by four different connexins (Cx37, Cx40, Cx43 and Cx45). The permeability of these connexin-formed gap junctions determines the amount of intercellular coupling and can be modulated by several vasoactive substances such as prostacyclin or nitric oxide (NO). We demonstrate here that NO has specific effects on certain connexins. Using two different techniques--injection of a fluorescent dye in single cells as well as detection of the de novo formation of gap junctions by a flow cytometry based technique--we found that NO decreases the functional coupling in Cx37 containing gap junctions whereas it increases the de novo formation of gap junctions containing Cx40. We conclude that NO, in addition to its known vasomotor effects, has a novel role in controlling intercellular coupling resulting in opposing effects depending on the specific connexin expressed in the cells.

Microinjected Anti-actin Antibodies Decrease Gap Junctional Intercellular Commmunication in Cultured Astrocytes

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.

Cultured periodontal ligament fibroblasts express diverse connexins

Recent studies have suggested multiple functions of periodontal ligament fibroblasts (PDLFs) which may relate to the permeability of gap junctions composed of various types of connexins (Cxs). At present, 15 types of Cxs are known to exist, and six of their antibodies, anti-Cx26, Cx32, Cx37, Cx40, Cx43, and Cx45 are commercially available. This study aims to examine which types of Cxs are expressed in cultured PDLFs by an immunohistochemical method, western blotting, and RT-PCR. The study confirmed the expressions of Cx32, Cx40, Cx43, and Cx45 in PDLFs, while Cx26 and Cx37 were not detected. Considering previous reports, Cx32 may relate to the secretory function, and Cx40 and Cx45 to the contractile function of PDLFs, however, a function for Cx43 has not been specified. In the immunohistochemical examination, different localizations of Cx40/43 and Cx32/45 were established. The former were observed punctately, suggesting that a large part of Cx40/43 may exist in the cell membrane and construct gap junctions. In contrast, the latter were observed uniformly in all the cells, indicating that they are present both in the cell membrane and in the cytoplasm of the cells.

Gap junctions do not underlie changes in whole-cell conductance in anoxic turtle brain

An acute reduction in cell membrane permeability could provide an effective strategy to prolong anoxic survival. A previous study has shown that in the western painted turtle whole-cell neuronal conductance ( G w) decreases during anoxia, which may be mediated by the activation of adenosine A 1 receptors and calcium. Reduction in G w is thought to be the result of ion channel closure, but closure of gap junctions could also be responsible for this phenomenon. In our study, antibody staining of connexin 32 and 43 (Cx32 and Cx43) suggested the presence of gap junctional components in the turtle cortex. To examine if gap junctions were involved in the previously measured anoxic decrease in G w, neuronal connectivity was assessed through the measurement of whole-cell capacitance ( C w). Turtle cortical sheets were perfused with normoxic (95%O 2/5%CO 2), anoxic (95%N 2/5%CO 2), high calcium (4 mM) and adenosine (200 μm) artificial cerebral spinal fluid (aCSF). No significant change in C w was observed under any of the above conditions. However, during hypo-osmotic aCSF perfusion C w decreased significantly, with the lowest value of 50±10.4 pF ( P<0.05) occurring at 30 min. To visualize changes in gap junction permeability lucifer yellow was loaded into turtle neurons during normoxic, anoxic, 0 calcium, hypo-osmotic, cold shock, (+)-isoproterenol, nitric oxide donor S-nitoso-acetyl penicillamine, and 8-bromo-guanosine 3′,5′-cyclic monophosphate aCSF perfusion. Dye propagation was only observed in 3 of 20 cold shock experiments (4 °C). We conclude that gap junctions are not involved in the acute reduction in G w previously observed during anoxia and that our results support the hypothesis that ion channel arrest is involved.

Expression and regulation of gap junctions in rat cholangiocytes

Hepatocytes and other digestive epithelia exchange second messengers and coordinate their functions by communicating through gap junctions. However, little is known about intercellular communication in cholangiocytes. The aim of this study was to examine expression and regulation of gap junctions in cholangiocytes. Connexin expression was determined by confocal immunofluorescence in rat bile ducts and in normal rat cholangiocyte (NRC) cells, a polarized cholangiocyte cell line. Intercellular Ca 2+ signaling was monitored by fluorescent microscopy. Microinjection studies assessed regulation of gap junction permeability in NRC cells and in SKHep1 cells, a liver-derived cell line engineered to express connexin 43. Immunochemistry showed that cholangiocytes from normal rat liver as well as the NRC cells express connexin 43. Localization of apical, basolateral, and tight junction proteins confirmed that NRC cells are well polarized. Apical exposure to ATP induced Ca 2+ oscillations that were coordinated among neighboring NRC cells, and inhibition of gap junction conductance desynchronized the Ca 2+ oscillations. NRC cells transfected with a connexin 43 antisense were significantly less coupled. Transcellular dye spreading was inhibited by activation of protein kinase A or protein kinase C. The same was observed in transfected SKHep1 cells, which expressed only connexin 43. Rat cholangiocytes and NRC cells express connexin 43, which permits synchronization of Ca 2+ signals among cells. Permeability of connexin 43-gap junctions is negatively regulated by protein kinases A and C. In conclusion, cholangiocytes have the capacity for intercellular communication of second messenger signals via gap junctions in a fashion that is under hormonal control. (H EPATOLOGY 2002;36:631-640.)

Fluctuations of Contraction Rhythm During Simulated Ischemia/Reperfusion in Cultured Cardiac Myocytes from Neonatal Rats

Cardiac ischemia results in a rapid decrease of intracellular pH and in the rise of intracellular Ca 2+, changes that have been shown to reduce intercellular communication via gap junctions (GJ) between cardiac myocytes. Ischemia also results in electrical instability probably caused by the reduced GJ permeability contributing to an increased vulnerability to arrhythmias. This study aims at elucidating whether the fluctuations of contraction rhythm of spontaneously beating cardiac myocytes in culture changes during simulated ischemia/reperfusion. The coefficient of variation (CV) of contraction intervals, reflecting the fluctuation of contraction rhythm, increased significantly during simulated ischemia/reperfusion. However, the contraction rhythm of the cardiac myocytes in an aggregate remained synchronized during simulated ischemia/reperfusion. In contrast, pharmacological blockade of GJ with 12-doxyl stearic acid, a blocker of GJ permeability, resulted in the de-synchronization of contraction rhythm and in an increase in the CV of contraction intervals in normoxic conditions. The present findings lead to the suggestion that GJ remained open during simulated ischemia/reperfusion, and that a mechanism other than electrical uncoupling between myocytes contributed to the observed increase in the fluctuation of beating rhythm during ischemia.

Control and Plasticity of Intercellular Calcium Waves in Astrocytes: A Modeling Approach

Intercellular Ca2+ waves in astrocytes are thought to serve as a pathway of long-range signaling. The waves can propagate by the diffusion of molecules through gap junctions and across the extracellular space. In rat striatal astrocytes, the gap-junctional route was shown to be dominant. To analyze the interplay of the processes involved in wave propagation, a mathematical model of this system has been developed. The kinetic description of Ca2+ signaling within a single cell accounts for inositol 1,4,5-trisphosphate (IP3) generation, including its activation by cytoplasmic Ca2+, IP3-induced Ca2+ liberation from intracellular stores and various other Ca2+ transports, and cytoplasmic diffusion of IP3 and Ca2+. When cells are coupled by gap junction channels in a two-dimensional array, IP3 generation in one cell triggers Ca2+ waves propagating across some tens of cells. The spatial range of wave propagation is limited, yet depends sensitively on the Ca2+-mediated regeneration of the IP3 signal. Accordingly, the term "limited regenerative signaling" is proposed. The gap-junctional permeability for IP3 is the crucial permissive factor for wave propagation, and heterogeneity of gap-junctional coupling yields preferential pathways of wave propagation. Processes involved in both signal initiation (activation of IP3 production caused by receptor agonist) and regeneration (activation of IP3 production by Ca2+, loading of the Ca2+ stores) are found to exert the main control on the wave range. The refractory period of signaling strongly depends on the refilling kinetics of the Ca2+ stores. Thus the model identifies multiple steps that may be involved in the regulation of this intercellular signaling pathway.

Gap junction intercellular communication propagates cell death in cancerous cells

Gap junction intercellular communication (GJIC) or cell coupling has an important function in maintaining tissue homeostasis and is thus a critical factor in the life and death balance of cells. While the role of GJIC in cell growth regulation has been much studied, its involvement in apoptosis remains unclear. In this study we elucidated the possibility that cell death is propagated via gap junctions, employing the rat bladder carcinoma cell line BC31. BC31 cells proliferate quickly, are tumorigenic, and are well-coupled via gap junctions that contain the gap junction protein Connexin43 (Cx43). In addition, these cells are predisposed to spontaneous death by apoptosis, particularly upon achieving confluency. We found that many dying BC31 cells express Cx43 just as their non-apoptotic counterparts do. Furthermore, Cx43 in apoptotic cells could be functionally competent, supporting coupling of these cells with their non-apoptotic neighbors, and as a result, clusters of coordinately dying cells were observed. The role of Cx43 and GJIC in propagating cell death was shown by analysing clones of BC31 cells expressing a mutant of Cx43 that is a dominant negative inhibitor of GJIC, and by using beta-glycyrrhetinic acid to inhibit intrinsic cell coupling in BC31 cells: in both cases the formation of clusters of dying cells was abrogated, and the intensity of cell death was considerably decreased. These results suggest that GJIC spreads cell-killing signals initially generated by a single cell that spontaneously initiates apoptosis, into healthy surrounding cells, thus increasing the level of cell death. Treatment of BC31 cells with the sleep-inducing lipid Oleamide, which selectively restricts gap junction permeability to Ca(2+) ions, did not abrogate coordinated cell death by clusters, indicating that Ca(2+) ions are the most probable cell-killing signals spread through gap junctions.

Effective Reduction of Neuronal Death by Inhibiting Gap Junctional Intercellular Communication in a Rodent Model of Global Transient Cerebral Ischemia

Gap junctions assemble astrocytes into syncytia, allowing exchange of metabolites, catabolites, and second-messenger molecules. Connexin43 is the predominant connexin of astrocytic gap junctions. The distribution of gap junction protein connexin43 was analyzed in different subfields of the hippocampal formation as a function of time after transient forebrain ischemia. One decisive key step in understanding why an ischemic insult gradually expands may be to establish how gap junction channels permit dying cells in the ischemic focus to communicate, in particular, with viable cells. The role of gap junctional intercellular communication in the hippocampus under ischemic conditions could be decisive for cell death propagation. We found that the vulnerable CA1/CA2 subfields have a higher density of gap junctions than the resistant CA3/CA4 areas, that changes in the distribution of connexin43 immunoreactivity may correlate with the phenomenon of selective vulnerability, and that inhibition of astrocytic gap junction permeability by octanol restricts the flow of undesirable neurotoxins that could potentially exacerbate neuronal damage. This provides a novel perspective for analysis of the pathophysiology of cerebral ischemia.

Connexin45 Interacts with Zonula Occludens-1 and Connexin43 in Osteoblastic Cells

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.

Persistence of gap junction communication during myocardial ischemia.

During myocardial ischemia, severe ATP depletion induces rigor contracture followed by intracellular Ca2+ concentration ([Ca2+]i) rise and progressive impairment of gap junction (GJ)-mediated electrical coupling. Our objective was to investigate whether chemical coupling through GJ allows propagation of rigor in cardiomyocytes and whether it persists after rigor development. In end-to-end connected adult rat cardiomyocytes submitted to simulated ischemia the interval between rigor onset was 3.7 +/- 0.7 s, and subsequent [Ca2+]i rise was virtually identical in both cells, whereas in nonconnected cell pairs the interval was 71 +/- 12 s and the rate of [Ca2+]i rise was highly variable. The GJ blocker 18alpha-glycyrrhetinic acid increased the interval between rigor onset and the differences in [Ca(2+)]i between connected cells. Transfer of Lucifer yellow demonstrated GJ permeability 10 min after rigor onset in connected cell pairs, and 30 min after rigor onset in isolated rat hearts submitted to nonflow ischemia but was abolished after 2 h of ischemia. GJ-mediated communication allows propagation of rigor in ischemic myocytes and persists after rigor development despite acidosis and increased [Ca2+]i.

Intercellular Ca 2+ Wave Propagation through Gap-Junctional Ca 2+ 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 (IP 3) receptor calcium release channels set by the IP 3 concentration, are shown to be further determinants of wave behavior.

A Series of Biotinylated Tracers Distinguishes Three Types of Gap Junction in Retina

Gap junctions serve many important roles in various tissues, but their abundance and diversity in neurons is only beginning to be understood. The tracer Neurobiotin has revealed many different networks interconnected by gap junctions in retina. We compared the relative permeabilities of five different retinal gap junctions by measuring their permeabilities to a series of structurally related tracers. When large tracers were injected, the staining of coupled cells fell off more rapidly in some networks than others relative to Neurobiotin controls. Three distinctly different permeability profiles were found, suggesting that multiple neuronal connexin types were present. The most permeant to large molecules were gap junctions from A-type horizontal cells. The permeability of gap junctions of two types of amacrine cell were not distinguishable from those from B-type horizontal cells. The lowest permeability was found for gap junctions between cone bipolar cells and the AII amacrine cells to which they are coupled. Because only a single neural connexin type has been identified in retina, our results suggest more types remain to be found. To determine whether the unitary permeability of channels is altered by channel modulators, we reduced permeability with octanol and a cAMP analog. Although net permeability was substantially diminished, the proportion by which it declined was constant across tracer size. This suggests that these agents act only to close channels rather than alter individual channel permeabilities. This tracer series can therefore be used to contrast permeability properties of gap junctions in intact circuits, even at the level of individual channels.