Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro

Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus 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.     

Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.     

Nitrate reductase activity of plasma membranes from cultured carrot cells

Summary Cultured carrot cells (Daucus carota L.) reduced nitrate to nitrite at a slow rate (0.4 moles/g dry wt · h) without any additions to the reaction medium. This rate was doubled or tripled in presence of 100 M NADH. Ethanol and other alcohols stimulated the basal rate 8–10-fold. Isolated carrot plasma membranes also reduced nitrate to nitrite at a rate of 80 nmoles/mg protein · h. This plasma membrane-bound nitrate reductase activity was estimated to be 1.7% of the total activity. Nitrate reduction by carrot cells was inhibited 56% by sodium tungstate, 57% by potassium cyanide, and 87% by gold chloride. It was stimulated by plasma membrane electron transport inhibitors (retinoic acid and chloroquine) and ATPase inhibitors (diethylstilbestrol). From differential effects of some stimulators or inhibitors in the presence or absence of NADH, it can be implied that the nitrate reductase activity of cultured carrot cells was due to a transmembrane enzyme exhibiting an exogenous nitrate reductase activity when NADH was added.Abbreviation DMSO dimethyl sulfoxide - SHAM salicyl hydroxamic acid     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

Retinoic acid modulates gap junctional permeability: a comparative study of dye spreading and ionic coupling in cultured cells

All-trans retinoic acid (RA), which was recently identified as a morphogen, affects gap junctional permeability in a dose- and time-dependent manner. In five different established mammalian cell lines (FL, BRL, BICR/M1Rk, HEL37, BT5C1) 100 mumol/liter RA reduced Lucifer yellow spreading within 30 min to 20-50% of the control. Ionic coupling, however, remained almost unaffected under the same conditions. Freeze-fractured membranes of untreated and RA-treated cells were similar with regard to frequency and sizes of gap junction plaques. With concentrations of less than 10 mumol/liter RA the dye spreading increased significantly in the human amniotic cell line FL, pointing to a possible modulatory effect of RA on junctional communication.     

Nontransformed cells can normalize gap junctional communication with transformed cells

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.     

Biochemical and biophysical analysis of cell-to-cell channels and regulation of gap junctional permeability

Gap junctional communication is a universal property of metazoan animals. Biochemical, immunological, molecular biological, ultrastructural, biophysical and physiological studies of gap junctions have permitted increasingly detailed modelling of gap junctional structure and function. In spite of this progress the questions to be addressed are whether the channel is a mixed oligomer and the stoichiometry for each tissue is fixed. Also the extent of homology among gap junction proteins in different tissues and their possible regulatory function have to be clarified. As long as the different channels are not cloned and expressed, the ultrastructural correlates of the physiological concepts such as channel gating, selectivity and regulation, as well as assembly and disassembly cannot be determined. The genetic approach is in full progress. The observed differences between gap junction proteins from different tissues and the multiplicity of subunits in even one channel implies a functional specialization for gap junctions. Correlative studies on the molecular and cellular level should help to clarify the physiological meaning of intercellular communication by gap junctions.     

The role of stem cells and gap junctional intercellular communication in carcinogenesis

Understanding the process of carcinogenesis will involve both the accumulation of many scientific facts derived from molecular, biochemical, cellular, physiological, whole animal experiments and epidemiological studies, as well as from conceptual understanding as to how to order and integrate those facts. From decades of cancer research, a number of the "hallmarks of cancer" have been identified, as well as their attendant concepts, including oncogenes, tumor suppressor genes, cell cycle biochemistry, hypotheses of metastasis, angiogenesis, etc. While all these "hallmarks" are well known, two important concepts, with their associated scientific observations, have been generally ignored by many in the cancer research field. The objective of the short review is to highlight the concept of the role of human adult pluri-potent stem cells as "target cells" for the carcinogenic process and the concept of the role of gap junctional intercellular communication in the multi-stage, multi-mechanism process of carcinogenesis. With these two concepts, an attempt has been made to integrate the other well-known concepts, such as the multi-stage, multimechanisn or the "initiation/promotion/progression" hypothesis; the stem cell theory of carcinogenesis; the oncogene/tumor suppression theory and the mutation/epigenetic theories of carcinogenesis. This new "integrative" theory tries to explain the well-known "hallmarks" of cancers, including the observation that cancer cells lack either heterologous or homologous gap junctional intercellular communication whereas normal human adult stem cells do not have expressed or functional gap junctional intercellular communication. On the other hand, their normal differentiated, non-stem cell derivatives do express connexins and express gap junctional intercellular communication during their differentiation. Examination of the roles of chemical tumor promoters, oncogenes, connexin knock-out mice and roles of genetically-engineered tumor and normal cells with connexin and anti-sense connexin genes, respectively, seems to provide evidence which is consistent with the roles of both stem cells and gap junctional communication playing a major role in carcinogenesis. The integrative hypothesis provides new strategies for chemoprevention and chemotherapy which focuses on modulating connexin gene expression or gap junctional intercellular communication in the premalignant and malignant cells, respectively.     

The effects of cryptorchidism on the regulation of steroidogenesis and gap junctional communication in equine testes

INTRODUCTION: Evidence collected over the years has demonstrated that cryptorchidism is associated with a defect in spermatogenesis and, as a consequence, with either reduced fertility or infertility. However, the effect of cryptorchidism on Leydig cell function is less clear. The aim of our study therefore was to investigate the regulation of steroid hormone biosynthesis and, additionally, intercellular communication in the cryptorchid equine testes. MATERIAL AND METHODS: Testes of mature bilaterally cryptorchid horse and healthy stallions were used for this study. The expression of luteinising hormone receptor (LHR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), aromatase and connexin43 (Cx43) was detected by means of immunohistochemistry. Testosterone and oestradiol levels were measured in testicular homogenates using appropriate radioimmunoassays. RESULTS: In the testes of both normal and cryptorchid stallions, immunostaining for LHR, 3beta-HSD and aromatase was confined to the Leydig cells. In the cryptorchid horse, the intensity of the staining for LHR and 3beta-HSD was weaker, whereas the staining for aromatase was clearly stronger than that of the normal stallion. Radioimmunological analysis revealed disturbance of the androgen-oestrogen balance in the cryptorchid testes. Additionally, in both the seminiferous tubules and interstitial tissue of the cryptorchid a clear reduction of the Cx43 signal was observed. CONCLUSIONS: Decreased expression of LHR and 3beta-HSD and increased expression of aromatase in the cryptorchid testes suggest that hormonal imbalance was caused both by reduced testosterone synthesis and by increased androgen aromatisation. Impaired expression of Cx43 in the seminiferous tubules as well as in the interstitial tissue of the cryptorchid horse indicates that cryptorchidism affects intercellular communication in the testes.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

High glucose promotes gap junctional communication in cultured neonatal cardiac fibroblasts via AMPK activation

Cardiac fibroblasts are known to be essential for adaptive responses in the pathogenesis of cardiovascular diseases, and increased intercellular communication of myocardial cells and cardiac fibroblasts acts as a crucial factor in maintaining the functional integrity of the heart. AMP-activated kinase (AMPK) is a key stress signaling kinase, which plays an important role in promoting cell survival and improving cell function. However, the underlying link between AMPK and gap junctional communication (GJIC) is still poorly understood. In this study, a connection between AMPK and GJIC in high glucose-mediated neonatal cardiac fibroblasts was assessed using fibroblast migration, measurement of dye transfer and connexin43 (Cx43) expression. 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and Compound C (CC) were used to regulate AMPK activity. The levels of cell migration and Cx43 protein expression in neonatal cardiac fibroblasts increased during high glucose treatment, accompanied by developed dye transfer. In addition, high glucose induced abundant phosphorylation of AMPK. Suppression of AMPK phosphorylation using CC reduced dye transfer, cell migration and Cx43 protein expression in neonatal cardiac fibroblasts, whereas the activation of AMPK using AICAR mimicked the high glucose-mediated cell migration, Cx43 protein expression and dye transfer enhancement. AMPK appears to participate in regulating GJIC in high-glucose-treated neonatal cardiac fibroblasts, including cell migration, dye transfer, Cx43 expression and distribution.     

Gap junctional regulation of signal transduction in bone cells

The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions.     

Aluminum impairs gap junctional intercellular communication between astroglial cells in vitro

The purpose of the present study was to investigate the effect of aluminum on gap junctional intercellular communication (GJIC) in cultured astrocytes. In the CNS the extracellular environment and metabolic status of neurons is dependent upon astrocytes, which are known to exhibit GJIC. This cell-to-cell communication provides a cytoplasmic continuity between adjacent cells, allowing exchange of diverse ions, second messengers, and metabolites. To study the effects of aluminum intoxication on GJIC in cultured glial cells, astroglial cell cultures obtained from fetal rat brains were exposed to aluminum lactate for 2-6 weeks. To demonstrate the metabolic coupling of neighboring cells, the technique of microinjection of the gap junction permeable substance neurobiotin was performed. Whereas in controls intensive GJIC was observed by dye transfer of neurobiotin from the microinjected cell into the adjacent astrocytes, aluminum treatment significantly impaired this cellular communication. As aluminum is known to affect cytoskeletal elements, additional investigations into the organization of intermediate filaments (glial fibrillary acid protein, GFAP) and microfilaments in control astrocytes and subsequent aluminum exposure were performed with the aid of fluorescence microscopy and rapid-freeze, deep-etch electron microscopy. Aluminum exposure led to an aggregation of GFAP-positive filaments near to the cell nucleus, accompanied by a destruction of the actin cytoskeleton, especially close to the cell membrane. Ultrastructurally these data could be verified as prominent areas without actin filaments contacting the cell membrane detectable in aluminum-treated astrocytes. Immunohistochemical staining of Cx43 revealed an impaired trafficking of this connexin into the cell prolongations following aluminum treatment, although electron-microscopic data revealed that gap junctions between adjacent astrocytes were still present after aluminum incubation for 24 days. In conclusion, in cultured astrocytes the morphological integrity of microfilaments and the intermediate filament network seem to be fundamental for the translocation of connexins from Golgi complex into the cellular prolongation to exhibit proper and extensive cellular communication through gap junctions.     

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN’s apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.     

Mechanosensitivity and intercellular communication in HOBIT osteoblastic cells: a possible role for gap junction hemichannels

Mechanically induced intercellular Ca2+ signalling was investigated in differentiated HOBIT osteoblastic cells. HOBIT cells express connexin43 clustered at the cell-to-cell boundary and display functional intercellular coupling assessed by intercellular transfer of Lucifer yellow. Mechanical stimulation of single cells, besides leading to an intracellular Ca2+ rise, induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. Suramin and apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signalling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18alpha-glycyrrhetinic acid.     

Opening hemichannels in nonjunctional membrane stimulates gap junction formation

We studied gap junction formation in pairs of Xenopus laevis oocytes expressing connexins that form functional hemichannels and found no correlation between junctional conductance (G(j)) and whole-cell hemichannel conductances (G(hemi)) within the first few hours of pairing. However, opening hemichannels to a threshold current stimulated a rapid G(j) increase. Moreover, cx46 hemichannel current stimulated cx40 G(j) even though cx40 and cx46 do not form heteromeric or heterotypic gap junctions. Initial growth rate and final steady-state level of stimulated G(j) were proportional to the product of hemichannel conductances. External calcium affected the growth rate of stimulated G(j) but not the final steady-state value. Time constants of formation were short in low [Ca(2+)](out) (3 min in 200 micro M Ca(2+)) and long in high [Ca(2+)](out) (15 min in 1 mM Ca(2+)), but in oocyte pairs pretreated with lectins to reduce steric hindrance imposed by large membrane glycoproteins the time constant was short and Ca(2+)-independent. We suggest that hemichannel activity stimulates G(j) by collapsing the extracellular volume between membranes to allow the end-to-end binding between hemichannels. These studies suggest the possibility that functional hemichannels could trigger or enhance junctional formation in vivo in response to appropriate stimuli.     

Chondroitin sulfate at the plasma membranes of cultured fibroblasts

We have previously shown that in confluent human fibroblast cultures chondroitin sulfate proteoglycan is a component of the fibronectin-containing pericellular matrix fibers. In the present work the distribution of chondroitin sulfate was studied in subconfluent cell cultures using antibodies that bind to a chemically defined carbohydrate fragment of chondroitinase ABC-modified chondroitin sulfate proteoglycan. Using immunofluorescence microscopy, we observed, in addition to the fibrillar matrix staining, chondroitin sulfate diffusely distributed at the cell surface. In indirect immunoferritin electron microscopy this staining corresponded to patchy binding of ferritin close (24 nm) to the outer aspect of the plasma membrane. The patchy organization appeared uniform in all cell surfaces. The cell surface chondroitin sulfate could not be removed from the plasma membrane by agents that dissociate electrostatic interactions. These data show that in fibroblasts chondroitin sulfate is a component of the outer aspect of the plasma membrane, and raise the possibility of an integral plasma membrane chondroitin sulfate proteoglycan.     

Perturbing plasma membrane hemichannels attenuates calcium signalling in cardiac cells and HeLa cells expressing connexins

Many cell signalling pathways are driven by changes in cytosolic calcium. We studied the effects of a range of inhibitors of connexin channels on calcium signalling in cardiac cells and HeLa cells expressing connexins. Gap 26 and 27, peptides that mimic short sequences in each of the extracellular loops of connexin 43, and anti-peptide antibodies generated to extracellular loop sequences of connexins, inhibited calcium oscillations in neonatal cardiac myocytes, as well as calcium transients induced by ATP in HL-1 cells originating from cardiac atrium and HeLa cells expressing connexin 43 or 26. Comparison of single with confluent cells showed that intracellular calcium responses were suppressed by interaction of connexin mimetic peptides and antibodies with hemichannels present on unapposed regions of the plasma membrane. To investigate how inhibition of hemichannels in the plasma membrane by the applied reagents was communicated to calcium store operation in the endoplasmic reticulum, we studied the effect of Gap 26 on calcium entry into cells and on intracellular IP3 release; both were inhibited by Gap 26. Calcium transients in both connexin 43- and connexin 26-expressing HeLa cells were inhibited by the peptides suggesting that the extended cytoplasmic carboxyl tail domain of larger connexins and their interactions with intracellular scaffolding/auxiliary proteins were unlikely to feature in transmitting peptide-induced perturbations at hemichannels in the plasma membrane to IP3 receptor channel central to calcium signalling. The results suggest that calcium levels in a microenvironment functionally connecting plasma membrane connexin hemichannels to downstream IP3-dependent calcium release channels in the endoplasmic reticulum were disrupted by the connexin mimetic peptide, although implication of other candidate hemichannels cannot be entirely discounted. Since calcium signalling is fundamental to the maintenance of cellular homeostasis, connexin hemichannels emerge as therapeutic targets open to manipulation by reagents interacting with external regions of these channels.