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Background Time-domain and non-linear methods can be used to quantify beat-to-beat repolarization variability but whether measures of repolarization variability can predict ventricular arrhythmogenesis in mice have never been explored. Methods Left ventricular monophasic action potentials (MAPs) were recorded during constant right ventricular 8 ​Hz pacing in Langendorff-perfused mouse hearts, in the presence or absence of the gap junction and sodium channel inhibitor heptanol (0.1, 0.5, 1 or 2 ​mM). Results Under control conditions, mean action potential duration (APD) was 39.4 ​± ​8.1 ​ms. Standard deviation (SD) of APDs was 0.3 ​± ​0.2 ​ms, coefficient of variation was 0.9 ​± ​0.8% and the root mean square (RMS) of successive differences in APDs was 0.15 ​± ​0.14 ​ms. Poincaré plots of APDn+1 against APDn revealed ellipsoid morphologies with a SD along the line-of-identity (SD2) to SD perpendicular to the line-of-identity (SD1) ratio of 4.6 ​± ​2.1. Approximate and sample entropy were 0.61 ​± ​0.12 and 0.76 ​± ​0.26, respectively. Detrended fluctuation analysis revealed short- and long-term fluctuation slopes of 1.49 ​± ​0.27 and 0.81 ​± ​0.36, respectively. Heptanol at 2 ​mM induced ventricular tachycardia in five out of six hearts. None of the above parameters were altered by heptanol during which reproducible electrical activity was observed (KW-ANOVA, P ​> ​0.05). Contrastingly, SD2/SD1 decreased to 2.03 ​± ​0.41, approximate and sample entropy increased to 0.82 ​± ​0.12 and 1.45 ​± ​0.34, and short-term fluctuation slope decreased to 0.82 ​± ​0.19 during the 20-s period preceding spontaneous ventricular tachy-arrhythmias (n ​= ​6, KW-ANOVA, P ​, Graphical abstract Image 1, Highlights • None of the parameters on variability determined using time-domain or non-linear analyses was altered by heptanol. • In the immediate periods preceding the onset of spontaneous ventricular tachycardia, the following changes were observed. • SD2/SD1 was decreased, approximate and sample entropy were increased, and short-term fluctuation slope was decreased. • These measures of repolarization variability could be used to predict impending ventricular arrhythmias in mouse hearts.

Melatonin (MT) increases oocyte maturation by reducing reactive oxygen species level and enhancing oocyte antioxidant capacity. However, the mechanisms via which MT works are still poorly understood. In the present study, the effects of MT on the maturation rate and development ability of bovine oocytes were investigated. Then, the transcriptome of oocytes treated by MT was sequenced. Finally, the expression of gap junction protein alpha 4 (GJA4) protein and cAMP level were detected in bovine oocytes, and isoprenaline (enhancer of gap junctional intercellular communication (GJIC)) and heptanol (inhibitor of GJIC) were used to investigate the effect of MT on GJIC activity in bovine oocytes. Our results showed that MT significantly improved the maturation, developmental ability and mRNA expression of GJA4 of bovine oocytes. Meanwhile, MT significantly increased GJA4 protein level and cAMP level in bovine oocytes. In contrast to heptanol, both isoproterenol and MT significantly increased GJIC activity, nuclear maturation and the development ability of bovine oocytes. However, MT significantly restored the nuclear maturation and developmental ability of oocytes treated by heptanol. In conclusion, our results showed that MT improves the maturation and developmental ability of bovine oocytes by enhancing GJIC activity via up-regulating GJA4 protein expression in IVM progress.

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.

Aims Treatment of arrhythmias evoked by hypothermia/rewarming remains challenging, and the underlying mechanisms are unclear. This in vitro experimental study assessed cardiac electrophysiology in isolated rabbit hearts at temperatures occurring in therapeutic and accidental hypothermia. Methods and results Detailed ECG, surface electrogram, and panoramic optical mapping were performed in isolated rabbit hearts cooled to moderate (31°C) and severe (17°C) hypothermia. Ventricular activation was unchanged at 31°C while action potential duration (APD) was significantly prolonged (176.9 ± 4.2 ms vs. 241.0 ± 2.9 ms, P, Graphical Abstract Graphical Abstract

In this work, the action of heptanol and ethanol was investigated in a two-dimensional (2D) model of cardiac tissue: the neonatal rat ventricular myocyte monolayer. Heptanol is known in electrophysiology as a gap junction uncoupler but may also inhibit voltage-gated ionic channels. Ethanol is often associated with the occurrence of arrhythmias. These substances influence sodium, calcium, and potassium channels, but the complete mechanism of action of heptanol and ethanol remains unknown. The optical mapping method was used to measure conduction velocities (CVs) in concentrations of 0.05–1.8 mM heptanol and 17–1342 mM ethanol. Heptanol was shown to slow the excitation wave significantly, and a mechanism that involves a simultaneous action on cell coupling and activation threshold was suggested. Whole-cell patch-clamp experiments showed inhibition of sodium and calcium currents at a concentration of 0.5 mM heptanol. Computer modeling was used to estimate the relative contribution of the cell uncoupling and activation threshold increase caused by heptanol. Unlike heptanol, ethanol slightly influenced the CV at clinically relevant concentrations. Additionally, the critical concentrations for re-entry formation in ethanol were determined.

Ventricular arrhythmic and electrophysiological properties were examined during normokalaemia (5.2 mM [K+]), hypokalaemia (3 mM [K+]) or hypokalaemia in the presence of 0.1 or 2 mM heptanol in Langendorff-perfused mouse hearts. Left ventricular epicardial or endocardial monophasic action potential recordings were obtained during right ventricular pacing. Hypokalaemia induced ventricular premature beats (VPBs) in 5 of 7 and ventricular tachycardia (VT) in 6 of 7 hearts (P0.05), reducing excitation wavelengths (λ, CV × VERP) from 7.9±1.1 to 5.1±0.3 mm (P0.001). Heptanol (0.1 mM) prevented VT, restored effective refractory period (ERP) to 45.2±2.9 msec without altering CV or APD, returning λ to control values (P>0.05) and CI to 8.4±3.8 msec (P0.05), returning λ and CI to control values (P>0.05). Anti-arrhythmic effects of heptanol during hypokalaemia were explicable by ERP changes, scaling λ and CI.

Aims The α2-adrenoceptor inducer dexmedetomidine (Dex) provides renoprotection against ischemia/reperfusion (I/R) injury, but the mechanism of this effect is largely unknown. The present study investigated the effect of Dex on apoptosis induced by hypoxia/reoxygenation (H/R) and the relationship between this effect and gap junction intercellular communication (GJIC). Main methods In vitro, two cell lines of normal rat kidney proximal tubular cells (NRK-52E) and HeLa cells that were transfected with a connexin 32 (Cx32) plasmid were exposed to H/R. The role of Dex in the modulation of H/R-induced apoptosis was explored by the manipulation of connexin expression, and hence gap junction (GJ) function, using a GJIC inhibitor, heptanol, and a GJIC inducer, retinoic acid. GJ function and the Cx32 protein level were determined by the parachute dye-coupling assay and Western blotting, respectively. Key findings Dex and heptanol significantly reduced H/R-induced apoptosis in NRK-52E cells. The anti-apoptosis effect of Dex was exhibited only in Cx32-expressing HeLa cells. One hour Dex exposure inhibited GJ function mainly via a decrease in Cx32 protein levels in NRK-52E cells. Significance Our data suggest that Dex reduced H/R-induced apoptosis through the inhibition of GJ activity by reducing Cx32 protein levels.

We examined junctional conductance (gj) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic β-cells. Only a very small fraction (∼0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased gj, while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and gj increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and β-cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase gj by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.

A novel scaffold-free self-assembled cartilage construct has been generated and used to repair particular chondral defects effectively. However, the mechanisms related to the construction of these self-assembled cartilages have not yet been fully elucidated. We hypothesize that gap junction intercellular communication (GJIC) plays a critical role in the development of self-assembled constructs upon GDF-5 induction. In this study, we investigated the effect of connexin 43 (C×43) mediated GJIC on GDF-5 modulation of chondrogenesis from two aspects, cell monolayer culture and 3-D self-assembly culture. We induced cells or self-assembled constructs with chondrogenic media (CM), growth differentiation factor 5 (GDF-5) or 1-heptanol for 3 weeks. At the end of that time, the results of quantitative fluorescence redistribution after photobleaching (FRAP) assay and immunofluorescence demonstrated that GDF-5 improved both GJIC and chondrogenic differentiation to a significant degree while 1-heptanol nearly offset the expected improvements in chondrogenesis. Biochemical assay and histology showed that GDF-5 can obviously enhance GAG, C×43 and type II collagen expressions. Conversely, we also showed that while 1-heptanol weakened GAG and type II collagen expression in self-assembled constructs, it had no effect on C×43 expression. Furthermore, real-time polymerase chain reaction showed that GDF-5 enhanced GAG and type II collagen transcription while 1-heptanol reduced them, but was affectless on C×43 transcription. This suggests that the generation of scaffold-free self-assembled cartilage from human mesenchymal stem cells upon GDF-5 induction may be mediated, at least in part, via the modulation of GJIC.

This is the final version of the article. It first appeared from Spandidos via https://doi.org/ 10.3892/mmr.2016.5738, In the current study, arrhythmogenic effects of the gap junction inhibitor heptanol (0.05 mM) were examined in Langendorff-perfused mouse hearts. Monophasic action potential recordings were obtained from the left ventricular epicardium during right ventricular pacing. Regular activity was observed both prior and subsequent to application of heptanol in all of the 12 hearts studied during 8 Hz pacing. By contrast, induced ventricular tachycardia (VT) was observed after heptanol treatment in 6/12 hearts using a S1S2 protocol (Fisher's exact test; P0.05). Consequently, excitation wavelengths (λ; CV x ERP) were reduced from 9.1±0.6 to 6.5±0.6 mm (P0.05). Together, these observations demonstrate for the first time, to the best of our knowledge, that inhibition of gap junctions alone using a low heptanol concentration (0.05 mM) was able to reduce CV, which alone was sufficient to permit the induction of VT using premature stimulation by reducing λ, which therefore appears central in the determination of arrhythmic tendency.

Background Gap junction (GJ) dysfunctions predispose cardiac tissues to various arrhythmias. Sinoatrial node (SAN) and pulmonary veins (PVs) are closely related atrial dysrhythmia. This study evaluated whether GJ modifications modulate SAN and PVs electrical activities. Methods Conventional microelectrodes were used to record action potentials in isolated rabbit SAN, PVs, and connected PV-SAN tissue preparations before and after heptanol (GJ inhibitor) and PQ1 (GJ enhancer) administration with and without isoproterenol. A whole-cell patch clamp was used to record the electrical activities before and after heptanol in single SAN and PV cardiomyocytes. Results Heptanol (1, 3, and 10μM) reduced the spontaneous beating rates of isolated SAN preparations but not PVs. Heptanol (10μM) decelerated the SAN leading rhythm in the PV-SAN preparations and induced PV burst firings without (3 of 6, 50%) and with (6 of 6, 100%) isoproterenol (1μM). Heptanol (10μM) also reduced the spontaneous beating rates in single SAN cardiomyocyte, but not PV cardiomyocyte, with a decreased pacemaker current. PQ1 (50 and 500nM) treatment did not change the spontaneous beating rates in isolated SAN and PV preparations. In the connected PV-SAN preparations, PQ1 (500nM) did not induce any PV firing even having additional isoproterenol treatment (1μM). Moreover, PQ1 (500nM) prevented heptanol-induced electrical changes in SAN and PVs preparations. Conclusion GJ dysfunction modulates SAN and PV electrical activity, which may contribute to atrial arrhythmogenesis. GJ enhancer has a therapeutic potential in SAN dysfunction and atrial arrhythmogenesis.

OBJECTIVE We investigated canine hearts during ischemia after aortic cross clamping (UI, n = 20) and after HTK-cardioplegia (HTK, n = 24) at 35 °C, 25 °C, 15 °C, and 5 °C with the aim to compare tissue changes caused by the activity of anaerobic metabolism(AAM), cell membrane destruction(CD), and gap junction uncoupling(GJU). APPROACH We measured continuously the complex dielectric spectrum(DS), ATP- and lactate content, extracellular pH, and rigor contracture. To identify changes in DS caused by AAM, CD, and GJU we performed additional experiments on the gap junction-free skeletal muscle. We used heart model simulations to calculate the effect of temperature. MAIN RESULTS AAM affected the DS at 10 MHz and we found a strong correlation between DS and the proton concentration with a maximum of DS at 10 mmol g-1 dry weight in ATP-concentration. The time of GJU was detected by a characteristic increase in DS and CD by a characteristic decrease at 13 kHz. In comparison to UI, GJU, AAM and CD were delayed by HTK and by hypothermia, indicating a minimization of energy consumption and an improved preservation of tissue structure. SIGNIFICANCE The novel findings were that in UI at 5 °C GJU occurred earlier and AAM remained constant, indicating a less effective preservation in UI by deep hypothermia in contrast to HTK.

Objective To study the effect of cumulus denudation on in vitro maturation of rabbit oocytes. Design Experimental animal study. Setting Academic institution. Animal(s) Rabbits and mice. Intervention(s) Rabbit oocytes were observed compared with mouse oocytes. Main Outcome Measure(s) Developmental competence, membrane integrity, and apoptotic status of oocytes after cumulus denudation. Result(s) Although in vitro maturation of mouse cumulus-denuded oocytes was unaffected, rabbit cumulus-denuded oocytes could not mature. However, 50% of rabbit cumulus-intact oocytes matured normally when their gap junctions were sealed with 1-heptanol. Coculture with cumulus cells did not improve maturation of rabbit cumulus-denuded oocytes unless with an intact corona radiata. Staining with Hoechst 33258, Bcl-2 antibodies, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling showed membrane breaches or apoptosis of rabbit cumulus-denuded oocytes, contrary to the mouse cumulus-denuded oocytes. Ultrastructurally, rabbit oocytes showed no perivitelline space but numerous long cell junctions projecting into the egg cortex, contrary to the mouse oocytes. However, the damaging effect of cumulus denudation was much relieved after preincubation of rabbit cumulus-intact oocytes with phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and some cumulus-denuded oocytes prepared after preincubation matured and developed into blastocysts. Conclusion(s) [1] Cumulus denudation severely damaged rabbit oocytes leading to their apoptosis or degeneration, possibly because of the deep-set junctional complexes anchoring the oocyte and corona cells; and [2] preincubation with phosphodiesterase inhibitor may provide a method to avoid the damaging effect of cumulus denudation on rabbit oocytes.

Heptanol and 18 beta-glycyrrhetinic acid (18 beta GA) block gap junctions, but have other actions on transmitter release that have not been characterised. This study investigates the prejunctional and postjunctional effects of these compounds in guinea pig and mouse vas deferens using intracellular electrophysiological recording and confocal Ca(2+) imaging of sympathetic nerve terminals. In mice, heptanol (2 mM) reversibly decreased the amplitude of purinergic excitatory junction potentials (EJPs; 52+/-5%, P0.05) while having little effect on spontaneous excitatory junction potentials (sEJPs). Heptanol (2 mM) reversibly abolished the nerve terminal Ca(2+) transient in 52% of terminals. 18 beta GA (10 microM) decreased the mean EJP amplitude, and increased input resistance in both mouse (137+/-17%, P0.05) and guinea pig (354+/-50%, P0.001) vas deferens indicating gap junction blockade. Further, 18 beta GA increased the sEJP frequency significantly in guinea pigs (by 71+/-25%, P0.05) and in 5 out of 6 tissues in mice (19+/-3%, P0.05). Moreover, 18 beta GA depolarised cells from both mice (11+/-1%, P0.01) and guinea pigs (8+/-1%, P0.005). Therefore, we conclude that heptanol (2 mM) decreases neurotransmitter release (given the decrease in EJP amplitude) by abolishing the nerve terminal action potential in a proportion of nerve terminals. 18 betaGA (10 microM) effectively blocks the gap junctions, but the increase in sEJP frequency suggests an additional prejunctional effect, which might involve the induction of spontaneous nerve terminal action potentials.

The purpose of this study is to provide direct evidence for the role of intercellular communications in electrical synchronization and mechanical function of myocardium. We used heptanol, a reversible inhibitor of gap junctions, at low (0.16 mM) and high (0.5 mM) concentration as perfusate for 18 Langendorff-perfused rabbit hearts to study its effects on myocardial electrical and mechanical functions. Optical mapping was performed to measure conduction velocity (CV) and action potential duration (APD). Ultrasonic integrated backscatter and Doppler tissue imaging (DTI) were used to evaluate the intrinsic and global myocardial contractile performance. The CV decreased during low-dose heptanol infusion and became much slower at high dose (high dose vs. baseline, 50.8 +/- 10.2 cm/s vs. 69.3 +/- 8.8 cm/s, p0.001). After washout of heptanol, CV completely recovered. The alterations of APD by heptanol infusion were similar to CV. The APD dispersion, standard deviation of APD(80), was increased after heptanol infusion (low dose vs. baseline, 5.9 +/- 1.1 ms vs. 4.3 +/- 1.1 ms, p = 0.004; high dose, 6.0 +/- 1.3 ms, vs. baseline, p = 0.035). However, washout did not restore the APD dispersion which became even larger after washout (13.6 +/- 1.9 ms vs. high dose and baseline, both p0.001). Regarding contractile function, heptanol treatment resulted in a progressive decrease of cardiac cycle-dependent variations of integrated backscatter (CVIBS; low dose vs. baseline, 6.1 +/- 1.7 dB vs. 7.2 +/- 1.8 dB, p = 0.007; high dose 1.7 +/- 0.3 dB vs. baseline, p0.001) and peak systolic strain rate (low dose vs. baseline, -1.5 +/- 0.6 1/s vs. -1.9 +/- 0.6 1/s, p = 0.014; high dose -0.4 +/- 0.2 1/s; vs. baseline, p0.001). That both CVIBS and strain rate incompletely recovered after heptanol washout may be attributed to the increased APD dispersion. In conclusion, uncoupling of gap junctions resulted in slowing CV, increased repolarization heterogeneity, reduced CVIBS and impaired myocardial contractility. There was a reversible dose-response relationship between the myocardial electromechanical functions and gap junction coupling.

To investigate how intercellular coupling can be changed during Ca2+ overloading of ventricular muscle, we studied Ca2+ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca2+ images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous Ca2+ transients in response to field stimulation over a field of view of 0.3 × 0.3 mm2. In severely Ca2+-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of Ca2+ transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca2+-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely Ca2+-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in Ca2+-overloaded heart. Inactivation of Na+ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay.

Object The gap junction is important in the propagation of dilation/constriction signals along vessels for coordinated behavior in control of vascular tone. The authors hypothesized that gap junctions might play a role in cerebral vasospasm following subarachnoid hemorrhage (SAH). The aims of the present study were to investigate the role of gap junctions and to observe the potential therapeutic efficacy of gap junction blockers in cerebral vasospasm in vitro and in vivo. Methods For the in vitro investigation, the effect of heptanol on the oxyhemoglobin (HbO2)-induced contraction of isolated rabbit basilar arteries (BAs) was observed by using an isometric tension-recording method. For the in vivo experiments, the potential therapeutic efficacy of heptanol and carbenoxolone was surveyed after it was given intravenously in the rabbit double-hemorrhage model. Light microscopy was performed to assess the morphological changes in the arteries examined. Results For the in vitro method, heptanol significantly inhibited the sustained contraction induced both by HbO2 and K+ in the BA rings. The magnitude of the heptanol-induced relaxation was dose dependent. The inhibitory effect of heptanol on the K+-induced vasoconstriction was weaker than that on the HbO2-induced constriction. After arterial rings were pretreated for 10 minutes, heptanol significantly decreased their responses to the HbO2-induced contraction. For the in vivo method, heptanol and carbenoxolone significantly decreased the narrowing of BAs when given intravenously in the rabbit double-hemorrhage model. In both treated groups, the diameters of the arteries had not changed significantly on Day 7 compared with those of the arteries in the SAH + vehicle and the SAH-only group. Conclusions Heptanol and carbenoxolone significantly inhibited the experimental cerebral vasospasm both in vitro and in vivo. Blockage of gap junctions is a probable candidate for a new approach in the treatment of cerebral vasospasm. Gap junctions may play a pathophysiological role in cerebral vasospasm.

We examined the effects of two putative gap junction blockers, heptanol and carbenoxolone, on noradrenaline-induced contractions in guinea pig vas deferens. The force generated due to the exogenously added noradrenaline (20 μM) consisted of two components: the tonic and the oscillatory. 2 mM heptanol abolished the oscillatory contractions and drastically suppressed both the maximum force (by 85.4 ±18.2%) as well as the tonic component (by 28.8 ± 5.1%) (P < 0.01, n = 7). However, the effects of carbenoxolone (50 μM) were strikingly different, with the spikes of the oscillatory component being merged into a steady, “fused” contraction, without affecting the maximum force developed. The L-type Ca2+ channel blocker nifedipine (2 μM) abolished the oscillatory component of the contractions and significantly reduced the maximum force and tonic component (by 82.4 ± 6.8% and 19.7 ± 6.4% respectively; P < 0.01, n = 4), in a manner similar to that elicited by heptanol. Our results indicate that (i) while carbenoxolone specifically blocks gap junctions, heptanol appears to exert its actions through non-gap junctional mechanisms, possibly by blocking VGCCs in smooth muscle; (ii) gap junctions play a significant modulatory role in the generation of noradrenaline-induced contractions in guinea pig vas deferens, particularly in the emergence of oscillatory contractions, while the maximum force developed may be independent of gap junctional contribution., © Elsevier

Intercellular coupling between ventricular myocytes and myofibroblasts was studied by co-culturing adult rabbit ventricular myocytes with previously prepared layers of cardiac myofibroblasts. Intercellular coupling was examined by: (i) tracking the movement of the fluorescent dye calcein; (ii) immunostaining for connexin 43 (Cx43); and (iii) measurement of intracellular [Ca2+] ([Ca2+]i). The effects of stimulating ventricular myocytes on the underlying myofibroblasts was examined by confocal measurements of [Ca2+]i using fluo-3. When ventricular myocytes were preloaded with calcein and co-cultured with myofibroblasts for 24 h, calcein fluorescence was detected in 52+/-4% (n=8 co-cultures) of surrounding myofibroblasts. Treatment with the gap junction uncoupler heptanol significantly reduced the movement of calcein (12+/-3%, n=6 co-cultures). Immunostaining showed expression of Cx43 in co-cultured myofibroblasts and myocytes. Field stimulation of ventricular myocytes co-cultured with myofibroblasts increased myofibroblast [Ca2+]i, no response was observed after treatment with heptanol or stimulation of fibroblasts in the absence of ventricular myocytes. Action potential parameters of ventricular myocytes in co-culture were similar to control values. However, application of the hormone sphingosine-1-phosphate (S-1-P) to the co-culture caused a depolarization of ventricular myocytes to approximately -20 mV. Sphingosine-1-phosphate had no effect on ventricular myocytes alone. Voltage-clamp measurements of isolated myofibroblasts indicated that S-1-P activated a significant quasi-linear current with a reversal potential of approximately -40 mV. In conclusion, this study shows that stimulation of the ventricular myocyte influences the intracellular Ca2+ of the linked myofibroblast via connexons. These intercellular links also allow the myofibroblasts to influence the electrical activity of the myocyte. This work indicates the nature of the gap junction-mediated bi-directional interactions that occur between ventricular myocyte and myofibroblast.

We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis).ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining.In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM.GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

It has been shown that cell-to-cell chemical coupling may persist during severe myocardial hypoxia or ischemia. We aimed to analyze the effects of different, chemically unrelated gap junction uncouplers on the progression of ischemic injury in hypoxic myocardium. First, we analyzed the effects of heptanol, 18α-glycyrrhetinic acid, and palmitoleic acid on intracellular Ca2+ concentration during simulated hypoxia (2 mM NaCN) in isolated cardiomyocytes. Next, we analyzed their effects on developed and diastolic tension and electrical impedance in 47 isolated rat hearts submitted to 40 min of hypoxia and reoxygenation. All treatments were applied only during the hypoxic period. Cell injury was determined by lactate dehydrogenase (LDH) release. Heptanol, but not 18α-glycyrrhetinic acid nor palmitoleic acid, attenuated the increase in cytosolic Ca2+ concentration induced by simulated ischemia in cardiomyocytes and delayed rigor development (rigor onset at 7.31 ± 0.71 min in controls vs. 14.76 ± 1.44 in heptanol-treated hearts, P < 0.001) and the onset of the marked changes in electrical impedance (tissue resistivity: 4.02 ± 0.29 vs. 7.75 ± 1.84 min, P = 0.016) in hypoxic rat hearts. LDH release from hypoxic hearts was minimal and was not significantly modified by drugs. However, all gap junction uncouplers, given during hypoxia, attenuated LDH release during subsequent reoxygenation. Dose-response analysis showed that increasing heptanol concentration beyond the level associated with maximal effects on cell coupling resulted in further protection against hypoxic injury. In conclusion, gap junction uncoupling during hypoxia has a protective effect on cell death occurring upon subsequent reoxygenation, and heptanol has, in addition, a marked protective effect independent of its uncoupling actions.

We have studied the effects of 1-heptanol and nifedipine on noradrenaline (NA)-induced contractions in order to explore the role of gap junctions and their interactions with L-type Ca2+ channel mediated [Ca2+]o entry in the generation of NA-induced contractions in the rat vas deferens. Application of 20 microM NA to rat vas deferens resulted in contractions with three different components, an initial phasic component followed by a tonic component overlapped with an oscillatory component. Heptanol (0.01-2 mM) induced a concentration dependent reduction of the contractions. 2 mM heptanol reduced the phasic component by 32.9 +/- 4.4% and the tonic component by 93.8 +/- 1.9% of control, while the oscillatory component was completely abolished (n=7). Nifedipine (2 microM) reduced the phasic component by 34.5 +/- 4.1% and the tonic component by 89.5 +/- 3.8% of control and abolished the oscillatory component (n=6). In the presence of heptanol and nifedipine together, the phasic component was reduced by 61.3 +/- 8.3% and the tonic component by 94.5 +/- 1.0% of control. The oscillatory component was completely abolished (n=6). These results allow the conclusion that phasic contraction is mainly due to the direct action of NA, independent of gap junctions, while the tonic and oscillatory contractions may depend significantly on cell-to-cell communication. These in turn may depend critically on the availability of extracellularly derived Ca2+.

This study examines the role of gap junctional communication in smooth muscle in relation to the frequency of stimulation and the innervation density of the tissue in the generation of neurogenic contractions. Toward this end the effects of heptanol, a gap junctional blocker, on the neurogenic contractions of guinea pig and rat vas deferens at different frequencies of stimulation (single pulse, 5, 10, 20, 40, 60, and 80 Hz) were studied. In both the prostatic and epididymal halves of these tissues, heptanol abolished the neurogenic contractions at the lower frequencies of stimulation. At higher frequencies, contractions were resistant to heptanol action. The effect of heptanol on the neurogenic contractions was found to decrease with increasing stimulation frequency. The neurogenic contractions of rat vas deferens were more resistant to heptanol than those of guinea pig vas deferens. Our data indicate that gap junctional communication is significant in the generation of neurogenic contractions in both guinea pig and rat vas deferens in a frequency-dependent manner, and we discuss the mechanisms underlying these findings.

Waves of contraction in the small intestine correlate with slow waves generated by the myenteric network of interstitial cells of Cajal. Coupled oscillator theory has been used to explain steplike gradients in the frequency (frequency plateaux) of contraction waves along the length of the small intestine. Inhibition of gap junction coupling between oscillators should lead to predictable effects on these plateaux and the wave dislocation (wave drop) phenomena associated with their boundaries. It is these predictions that we wished to test. We used a novel multicamera diameter-mapping system to measure contraction along 25- to 30-cm lengths of murine small intestine. There were typically two to three plateaux per length of intestine. Dislocations could be limited to the wavefronts immediately about the terminated wave, giving the appearance of a three-pronged fork, i.e., a fork dislocation; additionally, localized decreases in velocity developed across a number of wavefronts, ending with the terminated wave, which could appear as a fork, i.e., slip dislocations. The gap junction inhibitor carbenoxolone increased the number of plateaux and dislocations and decreased contraction wave velocity. In some cases, the usual frequency gradient was reversed, with a plateau at a higher frequency than its proximal neighbor; thus fork dislocations were inverted, and the direction of propagation was reversed. Heptanol had no effect on the frequency or velocity of contractions but did reduce their amplitude. To understand intestinal motor patterns, the pacemaker network of the interstitial cells of Cajal is best evaluated as a system of coupled oscillators.

The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18alpha-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.

To examine the effects of gap junction blockers on the endocochlear potential (EP) and endolymphatic potassium concentration ([K(+)](e)).The EP and [K(+)](e) were monitored using double-barreled ion-selective microelectrodes in the second turn of the guinea pig cochlea during perilymphatic perfusion.When the perilymphatic scalae of the cochlea were perfused with artificial perilymph containing 10 mM n-heptanol the EP was decreased by -8.8+/-1.4 mV (n=10), and this was accompanied by a decline in the [K(+)](e) of -6.7+/-2.1 mM (n=6). Perilymphatic application of 10 mM hexanol also produced declines in both the EP and [K(+)](e). In control studies, perilymphatic perfusion with 10 mM ethanol showed no remarkable changes in either the EP or [K(+)](e). Anoxia during perfusion with heptanol resulted in the generation of a negative EP, similar to the situation in controls.A decline in the EP together with a lowering of [K(+)](e) induced by long-chain n-alkanols, which act as gap junction blockers, may be explained by an interruption in potassium ion transport related to a gap junction dysfunction.

Transient inhibition of gap junction (GJ)-mediated communication with heptanol during myocardial reperfusion limits infarct size. However, inhibition of cell coupling in normal myocardium may be arrhythmogenic. The purpose of this study was to test the hypothesis that the consequences of GJ inhibition may be magnified in reperfused myocardium compared with normal tissue, thus allowing the inhibition of GJs in reperfused tissue while only minimally modifying overall macroscopic cell coupling in normal myocardium. Concentration-response curves were defined for the effects of heptanol, 18alpha-glycyrrhetinic acid, halothane, and palmitoleic acid on conduction velocity, tissue electrical impedance, developed tension and lactate dehydrogenase (LDH) release in normoxically perfused rat hearts (n= 17). Concentrations lacking significant effects on tissue impedance were added during the initial 15 min of reperfusion in hearts submitted to 60 min (n= 43) or 30 min (n= 35) of ischaemia. These concentrations markedly increased myocardial electrical impedance (resistivity and phase angle) in myocardium reperfused after either 30 or 60 min of ischaemia, and reduced reperfusion-induced LDH release after 1 h of ischaemia by 83.6, 57.9, 51.7 and 52.5% for heptanol, 18alpha-glycyrrhetinic acid, halothane and palmitoleic acid, respectively. LDH release was minimal in hearts submitted to 30 min of ischaemia, independently of group allocation. In conclusion, the present results strongly support the hypothesis that intercellular communication in postischaemic myocardium may be effectively reduced by concentrations of GJ inhibitors affecting only minimally overall electrical impedance in normal myocardium. Reduction of cell coupling during initial reperfusion was consistently associated with attenuated lethal reperfusion injury.

To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43 −/− mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, g j , was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43 +/+ ) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43 +/− strands and was absent in Cx43 −/− strands. Cx45 signal was reduced by 46% in Cx43 +/− strands and to the limit of detection in Cx43 −/− strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in ≈ 2% of myocytes. Intercellular conductance, g j , was reduced by 32% in Cx43 +/− cell pairs and by 96% in Cx43 −/− cell pairs. The symmetrical dependence of g j on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43 −/− cells. Propagation in Cx43 −/− strands was very slow (2.1 cm/s versus 52 cm/s in Cx43 +/+ ) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands.

1 Heptanol, 18alpha-glycyrrhetinic acid (18alphaGA) and 18beta-glycyrrhetinic acid (18betaGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. 2 Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca(2+)-sensitive dyes), membrane potential and--as a measure of intercellular coupling--input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. 3 Heptanol (150 microm) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 microm did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 4 18betaGA (30 microm) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18alphaGA (100 microm) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. 5 We conclude that in mesenteric small arteries, heptanol and 18betaGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18betaGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18alphaGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

Aqueous humor is secreted by the bilayered ciliary epithelium. Solutes and water enter the pigmented ciliary epithelial (PE) cell layer, cross gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, and are released into the aqueous humor. Electrical measurements suggest that heptanol reduces transepithelial ion movement by interrupting PE-NPE communication and that gap junctions may be a regulatory site of aqueous humor formation. Several lines of evidence also suggest that net ciliary epithelial transport is strongly region dependent. Divided rabbit iris-ciliary bodies were incubated in chambers under control and experimental conditions, quick-frozen, cryosectioned, and freeze-dried. Elemental intracellular contents of NPE and PE cells were determined by electron probe X-ray microanalysis. With or without heptanol, ouabain produced concentration- and time-dependent changes more markedly in anterior than in posterior epithelium. Without heptanol, there were considerable cell-to-cell variations in Na gain and K loss. However, contiguous NPE and PE cells displayed similar changes, even when nearby cell pairs were little changed by ouabain in aqueous, stromal, or both reservoirs. In contrast, with heptanol present, ouabain added to aqueous or both reservoirs produced much larger changes in NPE than in PE cells. The results indicate that 1) heptanol indeed interrupts PE-NPE junctions, providing an opportunity for electron microprobe analysis of the sidedness of modification of ciliary epithelial secretion; 2) Na and K undergo faster turnover in anterior than in posterior epithelium; and 3) PE-NPE gap junctions differ from PE-PE and NPE-NPE junctions in permitting ionic equilibration between adjoining ouabain-stressed cells.

Little is known about the expression and possible functions of unopposed gap junction hemichannels in the brain. Emerging evidence suggests that gap junction hemichannels can act as stand-alone functional channels in astrocytes. With immunocytochemistry, dye uptake, and HPLC measurements, we show that astrocytes in vitro express functional hemichannels that can mediate robust efflux of glutamate and aspartate. Functional hemichannels were confirmed by passage of extracellular lucifer yellow (LY) into astrocytes in nominal divalent cation-free solution (DCFS) and the ability to block this passage with gap junction blocking agents. Glutamate/aspartate release (or LY loading) in DCFS was blocked by multivalent cations (Ca(2+), Ba(2+), Sr(2+), Mg(2+), and La(3+)) and by gap junction blocking agents (carbenoxolone, octanol, heptanol, flufenamic acid, and 18α-glycyrrhetinic acid) with affinities close to those reported for blockade of gap junction intercellular communication. Glutamate efflux via hemichannels was also accompanied by greatly reduced glutamate uptake. Glutamate release in DCFS, however, was not significantly mediated by reversal of the glutamate transporter: release did not saturate and was not blocked by glutamate transporter blockers. Control experiments in DCFS precluded glutamate release by volume-sensitive anion channels, P2X(7) purinergic receptor pores, or general purinergic receptor activation. Blocking intracellular Ca(2+) mobilization by BAPTA-AM or thapsigargin did not inhibit glutamate release in DCFS. Divalent cation removal also induced glutamate release from intact CNS white matter (acutely isolated optic nerve) that was blocked by carbenoxolone, suggesting the existence of functional hemichannels in situ. Our results indicated that astrocyte hemichannels could influence CNS levels of extracellular glutamate with implications for normal and pathological brain function.

Because of the variety of tissue structures, the interpretation of the passive complex dielectric permittivity spectrum epsilon (omega) of the heart is still a problem. The aim of this work was to correlate epsilon (omega) of heart tissue with physical processes on cellular level.epsilon (omega) of canine hearts was continuously measured in the range from 10 Hz to 400 MHz during cardioplegic perfusion and during following ischemia. Cardioplegic perfusion was performed with HTK (Custodiol) without or with heptanol, in order to produce electrical cell uncoupling via the closure of gap junctions. To analyse epsilon (omega), we present two heart models which consider cell shape, electrical cell coupling, and dielectric polarisation of cell membranes and membranes of intracellular structures.epsilon (omega) of heart tissue shows an alpha-, beta-, and gamma-dispersion. epsilon (omega) remains unchanged during cardioplegic perfusion with HTK, but if heptanol is added, there is an immediate decrease in the region of alpha-dispersion and an increase in the low frequency part of beta-dispersion. Similar changes are observed during ischemia following HTK perfusion without heptanol; additionally, the beta-dispersion shifts to higher frequencies. Using our models, we obtain analogue changes of epsilon (omega) by fitting model parameters which describe water content, water distribution, extra- and intracellular conductivity, and gap junction resistance.Changes of these tissue properties as calculated by our models based on the measurement of epsilon (omega) are consistent with intraischemic changes of heart tissue known from immunohistochemical, biochemical, and histological investigations. The next step will be to use our models for the prognosis of irreversible tissue damage.

The effects of two structurally distinct inhibitors of gap junction communication were studied by using three different forms of vasoconstriction in pressurized rat middle cerebral arteries. The sensitivity of myogenic tone (at 60 mmHg), vasopressin-induced tone (10 nM, at 20 mmHg), and depolarizing solution-induced tone (80 mM K(+), at 20 mmHg) to inhibition by heptanol (1.0 microM to 3.0 mM) or 18alpha-glycyrrhetinic acid (18alpha-GA, 1.0 to 50 microM) were determined. Pressure-induced myogenic tone was inhibited by heptanol (IC(50) = 0.75 +/- 0.09 mM) and 18alpha-GA ( approximately 30 microM). Vasopressin-induced vasoconstriction was also inhibited by heptanol (IC(50) = 0.4 +/- 0.3 mM) and 18alpha-GA (1 microM). Depolarizing solution-induced vasoconstriction was less sensitive to inhibition by heptanol compared to vasopressin (P0.01) or pressure-induced constriction (P0.05). However, 18alpha-GA did not inhibit depolarization-induced constriction. Sharp microelectrode experiments on isolated arteries revealed stable membrane potentials, with no detectable effect of heptanol (1 mM) or 18alpha-GA (20-30 microM) on the average membrane potential at 20 mmHg. However, approximately 20% of impaled cells (5 of 28) exhibited uncharacteristic oscillations in membrane potential after pharmacological uncoupling. At 60 mmHg a approximately 7- to 9-mV hyperpolarization and corresponding vasodilation (approximately 50%) was observed, and the frequency of membrane potential oscillations doubled (9 of 23 cells). These data indicate that gap junctions play an important role in the maintenance and modulation of membrane potential and tone in cerebral resistance arteries.

We report the induction of embryonic gene expression in epithelial HC-11 cells upon communication with blastomeres in compacting mouse embryos. In contrast to NIH3T3 fibroblasts, HC-11 epithelial cells form gap junctions with blastomeres after injection into cleavage-stage embryos, as shown by targeting of phosphorylated connexin43 (pCx43) to areas of cell-to-blastomere contact and dye coupling. This was accompanied by expression of the embrvo-specific transcription factor, Oct-3/4, in the HC-11 cells. Dye coupling and Oct-3/4 expression were abolished with heptanol and 18beta- glycyrrhetinic acid, two gap junction blockers. Oleamide, which blocks gap junction-mediated communication but not electrical conductance, also inhibited Oct-3/4 expression in HC-11 cells, suggesting that Oct-3/4 induction results from transfer of molecules of < 1 kDa through gap junctions. Inhibition of cAMP signaling in blastomeres abolishes Oct-3/4 expression in somatic cells despite gap junction formation. In addition, reprogramming of NIH3T3 fibroblasts in an extract of HC-11 cells enabled assembly of pCx43 and Oct-3/4 expression after contact of the reprogrammed cells with blastomeres. We propose that gap junction-mediated cAMP signaling between blastomeres and somatic cells results in changes in somatic cell gene expression.

The objective of this study was to determine whether connexin 32-type gap junctions contribute to the "contact effect" in follicular thyrocytes and whether the response is influenced by radiation quality. Our previous studies demonstrated that early-passage follicular cultures of Fischer rat thyroid cells express functional connexin 32 gap junctions, with later-passage cultures expressing a truncated nonfunctional form of the protein. This model allowed us to assess the role of connexin 32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions. The survival curves generated after gamma irradiation revealed that early-passage follicular cultures had significantly lower values of alpha (0.04 Gy(-1)) than later-passage cultures (0.11 Gy(-1)) (P0.0001, n = 12). As an additional way to determine whether connexin 32 was contributing to the difference in survival, cultures were treated with heptanol, resulting in higher alpha values, with early-passage cultures (0.10 Gy(-1)) nearly equivalent to untreated late-passage cultures (0.11 Gy(-1)) (P0.1, n = 9). This strongly suggests that the presence of functional connexin 32-type gap junctions was contributing to radiation resistance in gamma-irradiated thyroid follicles. Survival curves from proton-irradiated cultures had alpha values that were not significantly different whether cells expressed functional connexin 32 (0.10 Gy(-1)), did not express connexin 32 (0.09 Gy(-1)), or were down-regulated (early-passage plus heptanol, 0.09 Gy(-1); late-passage plus heptanol, 0.12 Gy(-1)) (P0.1, n = 19). Thus, for proton irradiation, the presence of connexin 32-type gap junctional channels did not influence their radiosensitivity. Collectively, the data support the following conclusions. (1) The lower alpha values from the gamma-ray survival curves of the early-passage cultures suggest greater repair efficiency and/or enhanced resistance to radiation-induced damage, coincident with the expression of connexin 32-type gap junctions. (2) The increased sensitivity of FRTL-5 cells to proton irradiation was independent of their ability to communicate through connexin 32 gap junctions. (3) The fact that the beta components of the survival curves from both gamma rays and proton beams were similar (average 0.022 +/- 0.008 Gy(-2), P0.1, n = 39) suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of radiation quality and the presence of functional gap junctions.

Role of mesangial cells and gap junctions in tubuloglomerular feedback. Background Tubuloglomerular feedback (TGF) is a process whereby the resistance of the afferent arterioles delivering blood to the glomeruli is regulated by the NaCl concentration of the forming urine in the lumen of the macula densa. Intraglomerular mesangial cells are located between capillaries within the glomerulus, while extraglomerular mesangial cells are located between the macula densa and the afferent arteriole. They are electrically and chemically coupled via gap junctions. The purpose of this study was to investigate the role of mesangial cells and gap junctions in TGF using the isolated, perfused juxtaglomerular apparatus. Method Juxtaglomerular apparatuses were dissected from male New Zealand white rabbits and perfused in vitro. The NaCl concentration at the macula densa was changed from 17/2 to 65/50 Na/Cl to initiate a TGF response. Afferent arterioles were perfused at 60 mm Hg throughout the experiment. Changes in luminal diameter caused by increasing the NaCl concentration at the macula densa were taken as the TGF response. TGF was measured before and after disrupting the gap junctions or damaging the mesangial cells in paired experiments. Results During the control period, TGF decreased afferent arteriole diameter by 2.9 ± 0.2 μm. After mesangial cells were damaged by perfusing Thy 1-1 antibody and complement into the afferent arteriole, the TGF response was completely eliminated. Separate experiments showed no statistically significant change in TGF response with time, or when antibody and complement were perfused into the macula densa lumen. The presence of Thy 1-1 antibody and complement in the afferent arteriole perfusate did not alter the ability of norepinephrine to constrict or acetylcholine to dilate the afferent arteriole. To investigate the role of gap junctions in TGF, we used heptanol to disrupt them. During the control period, TGF decreased afferent arteriole diameter by 2.9 ± 0.4 μm. After perfusing heptanol into the lumen of the afferent arteriole, the TGF response was completely eliminated. When heptanol was added to the bath, it had no significant effect on TGF response. Discussion The data show that after mesangial cells were selectively damaged, the constriction of the afferent arteriole induced by increasing the NaCl concentration at the macula densa was eliminated. However, such treatment had no effect when Thy 1-1 was perfused into the macula densa lumen, and did not alter the response of the afferent arteriole to norepinephrine or acetylcholine. Disruption of the gap junctions also eliminated the TGF response. These data indicate that the mesangial cells play a key role in mediating the TGF response, and that gap junctions among mesangial cells and between mesangial cells and vascular smooth muscle cells communicate the TGF signal to the afferent arteriole.

Background: Many stimuli can successfully protect the heart against ischemia. We investigated whether gap junction uncoupling before ischemia was myoprotective. We also studied the function of the adenosine triphosphate-dependent potassium channel, which has been implicated in the mechanism of pharmacologic preconditioning, with respect to gap junction physiology. Methods: Twenty-eight rabbit hearts were placed on a Langendorff perfusion apparatus. Five were given a 5-minute infusion of 1 mmol/L heptanol (a gap junction uncoupler), 5 were given 10 μmol/L 2,3-butanedione monoxime (an electromechanical uncoupler), and 6 were given no drug. The left anterior descending coronary artery was then occluded for 1 hour and reperfused for 2 hours. Six hearts received 10 μmol/L glybenclamide before heptanol to evaluate the role of the adenosine triphosphate-dependent potassium channel. Six hearts underwent ischemic preconditioning with 2 cycles of 5 minutes of global ischemia and reperfusion. Action-potential duration of the ischemic zone, left ventricular developed pressure, and coronary flow were measured continuously. Infarct size was determined at the end of reperfusion. Results: Heptanol significantly reduced infarct size (from 46% ± 2% to 22% ± 5%, P

Emerging evidence suggests that gap junction-mediated intercellular transmission of ions, metabolites and/or second messengers may serve as important determinants of myocyte viability. Our aim was to determine, using isolated buffer-perfused mouse hearts, whether the cardioprotection achieved with ischemic preconditioning (PC) is due in part to: (i) disruption of cell-cell coupling (manifest as a loss in the primary gap junction protein, connexin 43 [Cx43]) and resultant impaired transmission of a 'death' messenger, or conversely, (ii) transfer of a humoral 'survival' factor via existing gap junctions.To explore the first possibility, we employed immunostaining to visualize and quantify Cx43 in hearts subjected to 212 min of PC ischemia or a matched control period. To test the converse corollary, we assessed the effect of heptanol-an agent well recognized to rapidly and reversibly uncouple gap junctions--on the reduction of infarct size (delineated by tetrazolium staining) achieved with PC.We found no evidence of a deficit in Cx43 immunoreactive signal in response to the PC stimulus. Area of necrosis (AN) was, as expected, reduced in hearts that received PC ischemia vs. controls (31+/-3% vs. 40+/-3% of the left ventricle [LV]; P.01). However, treatment with heptanol rendered PC ineffective in eliciting protection (AN/LV: 42+/-1%).Our results suggest that gap junction-mediated transfer of an as-yet unknown 'survival' factor-rather than disrupted transfer of a 'death messenger'-may play a role in the increased resistance to infarction conferred by antecedent PC ischemia in mouse heart.

Stable expression of pannexin 1 (Panx1) and pannexin 3 (Panx3) resulted in functional gap junctions (GJs) in HeLa cells, but not in Neuro-2a (N2a) or PC-12 cells. The glycosylation pattern of expressed Panx1 varied greatly among different cell lines. In contrast to connexin (Cx) containing GJs (Cx-GJs), junctional conductance (G j) of pannexin GJs (Panx-GJs) is very less sensitive to junctional voltage. Both Panx1 and Panx3 junctions favoured anionic dyes over cations to permeate. Though, carbenoxolone (CBX) and probenecid blocked Panx1 hemichannel activity, they had no effect on Panx1-GJs or Panx3-GJs. Extracellular loop 1 (E1) of Panx1 possibly bears the binding pocket. The Cx-GJ blocker heptanol blocked neither Panx1 hemichannel nor Panx-GJs. Unlike the GJs formed by most Cxs, CO 2 did not uncouple Panx-GJs completely. Oxygen and glucose deprivation (OGD) caused lesser uncoupling of Panx-GJs compared to Cx43-GJs. These findings demonstrate properties of Panx-GJs that are distinctly different from Cx-GJs.

Connexins, the family of proteins that form vertebrate gap junctions, have key roles during development and in the adult. Previously, the physiological actions of connexins have been ascribed solely to formation of gap junction channels and thought to be mediated by the transfer of small molecules between neighboring cells. In conflict with this hypothesis here we demonstrate that Cx43 can affect cell growth independently of gap junction formation. This conclusion is based on four findings: (1) There is a lack of correlation between the action of Cx43 mutants Cx43-S255A, Cx43-S279A, and Cx43-S282A on growth and cell coupling in 3T3 A31 fibroblasts. (2) Blockade of gap junction formation, by either heptan-1-ol treatment or culturing cells at low density, had no effect on the ability of the Cx43 mutants to control growth. (3) Wildtype Cx43 inhibited growth of Neuro2a cells under conditions where gap junctions were unable to form. (4) The CT domain of Cx43, which lacks intrinsic gap junction activity, is as effective as the wildtype molecule in suppressing the growth of Neuro2a cells. These observations demonstrate that Cx43 has dual functions: first, its well-accepted role in forming a gap junction channel and, second, a direct action of the connexin protein on growth that is mediated via the cytoplasmic carboxyl domain.

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na(+) and Ca(2+) accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca(2+)]i and [Na(+)]i in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 microM fluo-3 AM for 30 min, or with 5 microM sodium green AM for 60 min at 37 degrees C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI+H (0.95 mM, 4.7 mM) for 60 min. Propidium iodide (PI, 25 microM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca(2+)]i and [Na(+)]i were then calculated by calibration. In some experiments, the effect of 10 microM tetrodotoxin (TTX) and 20 microM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca(2+)]i and [Na(+)]i in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca(2+)]i and [Na(+)]i in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca(2+)]i and [Na(+)]i overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na(+) and Ca(2+) channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.