The Role of Gap Junctions in the Generation of Smooth Muscle Cells from Bone Marrow Mesenchymal Stem Cells.

Background: Studies have shown that stem cell transplantation can improve smooth muscle cell (SMC) regeneration and remodelling. Gap junctions can enhance the cytoprotective effects of neighbouring cells. We investigated the effect of gap junctions on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into SMCs. Materials and Methods: Rat BMSCs and SMCs were obtained from the bone marrow and bladder of Sprague-Dawley rats, respectively. Flow cytometry and multilineage differentiation were performed to assess the characteristics of these cells. BMSCs and SMCs were incubated together in cocultures in the presence and absence of heptanol, an uncoupler of gap junctions. Cocultures were divided into three groups consisting of a contact coculture, noncontact coculture, and contact coculture plus heptanol groups. The expression of BMSC-specific markers and the effect of gap junctions on the differentiation of BMSCs were evaluated by performing real-time reverse transcription-polymerase chain reaction, immunofluorescence analysis, and western blotting after cocultures. Results: CD90 and CD44 were markedly expressed, and CD31 and CD45 were weakly or not expressed in BMSCs. The cells also showed good osteogenic and adipogenic differentiation ability. Compared with the noncontact coculture group, the SMC markers such as α-SMA, calponin, and connexin43 increased in the contact coculture group. The effect of contact in the coculture group was significantly weakened by heptanol. Conclusions: The results suggested that gap junctions play an important role in the generation of SMCs from BMSCs. The formation of SMCs can potentially be used to repair the sphincter muscle of patients with stress urinary incontinence.

Involvement of sphingosine-1-phosphate receptors 2/3 in IR-induced sudden cardiac death.

It has been demonstrated that S1P receptors affect heart ischaemia-reperfusion (IR) induced injury. However, whether S1P receptors affect IR-induced cardiac death has not been investigated. The aim of this paper is to demonstrate the role of S1P receptors in IR-induced cardiac death. Healthy adult male Sprague-Dawley rats were assigned to the following groups: non-operation control group, sham operation group, IR group, IR group pretreated with DMSO, IR group pretreated with S1P3 agonist, IR group pretreated with an antagonist of S1P3, IR group pretreated with S1P2 and S1P3 antagonists, IR group pretreated with heptanol and antagonists of S1P2/3, and IR group pretreated with Gap26 and antagonists of S1P2/3 (heptanol acts as a Cx43 uncoupler and the mimic peptide Gap26 as Cx43 blocker). The groups with S1P2 or S1P3 agonist application before reperfusion were used to assess whether these can be used for therapy of IR. The haemodynamics, electrocardiograms (ECG), infarction area, and mortality rates were recorded. Immunohistological connexin 43 (Cx43) expression in the heart was detected in each group. Blocking S1P2/3 receptors with specific antagonists resulted in an increment of IR-induced mortality, increased infarction size, redistribution of Cx43 expression, as well as affecting the heart function. The infarction size, heart function, and mortality were totally or partially restored in the S1P2, S1P3 agonist-pretreated IR group, and the heptanol/Gap26-treated S1P2/3-blocked IR group. The S1P receptor S1P2/3 and Cx43 are involved in the IR-induced cardiac death.

Hearts during ischemia with or without HTK-protection analysed by dielectric spectroscopy.

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.

Antinociceptive effect of Aristolochia trilobata stem essential oil and 6-methyl-5-hepten-2yl acetate, its main compound, in rodents.

Aristolochia trilobata L. is an aromatic plant, popularly known as "mil-homens", and its essential oil (EO) is generally used to treat colic, diarrhea and dysentery disorders. We evaluated the antinociceptive effect of A. trilobata stem EO and of its major compound, the (R)-(-)-6-methyl-5-hepten-2-yl acetate (sulcatyl acetate: SA), using acetic acid (0.85%)-induced writhing response and formalin-induced (20 µL of 1%) nociceptive behavior in mice. We also evaluated the EO and SA effect on motor coordination, using the rota-rod apparatus. EO (25, 50 and 100 mg/kg) or SA (25 and 50 mg/kg) reduced nociceptive behavior in the writhing test (p<0.001). EO (100 mg/kg) and SA (25 and 50 mg/kg) decreased the nociception on the first phase of the formalin test (p<0.05). On the second phase, EO (25: p<0.01; 50: p<0.05 and 100 mg/kg: p<0.001) and SA (25 and 50 mg/kg; p<0.001) reduced the nociceptive response induced by formalin. EO and SA were not able to cause changes in the motor coordination of animals. Together, our results suggest that EO has an analgesic profile and SA seems to be one of the active compounds in this effect.

Serum lipidomics analysis of ovariectomized rats under Curcuma comosa treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma comosa Roxb. (C. comosa) or Wan Chak Motluk, Zingiberaceae family, has been used in Thai traditional medicine for the treatment of gynecological problems and inflammation. AIM OF THE STUDY: This study aimed to investigate the therapeutic potential of C. comosa by determining the changes in the lipid profiles in the ovariectomized rats, as a model of estrogen-deficiency-induced hyperlipidemia, after treatment with different components of C. comosa using an untargeted lipidomics approach. MATERIALS AND METHODS: Lipids were extracted from the serum of adult female rats subjected to a sham operation (SHAM; control), ovariectomy (OVX), or OVX with 12-week daily doses of estrogen (17ß-estradiol; E2), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD; a phytoestrogen from C. comosa), powdered C. comosa rhizomes or its crude ethanol extract. They were then analyzed by liquid chromatography-mass spectrometry, characterized, and subjected to the orthogonal projections to latent structures discriminant analysis statistical model to identify tentative biomarkers. RESULTS: Levels of five classes of lipids (ceramide, ceramide-1-phosphate, sphingomyelin, 1-O-alkenyl-lysophosphatidylethanolamine and lysophosphatidylethanolamine) were elevated in the OVX rats compared to those in the SHAM rats, while the monoacylglycerols and triacylglycerols were decreased. The E2 treatment only reversed the levels of ceramides, whereas treatments with DPHD, C. comosa extract or powder returned the levels of all upregulated lipids back to those in the SHAM control rats. CONCLUSIONS: The findings suggest the potential beneficial effects of C. comosa on preventing the increased ceramide levels in OVX rats, a possible cause of metabolic disturbance under estrogen deficiency. Overall, the results demonstrated the power of untargeted lipidomics in discovering disease-relevant biomarkers, as well as evaluating the effectiveness of treatment by C. comosa components (DPHD, extract or powder) as utilized in Thai traditional medicine, and also providing scientific support for its folklore use.

Heptanol application to the mouse round window: a model for studying cochlear lateral wall regeneration.

OBJECTIVE: Identify cells supporting cochlear lateral wall regeneration. STUDY DESIGN: Prospective controlled trial. SETTING: Laboratory. Human presbyacusis occurs, in part, secondary to age-related degeneration of cochlear lateral wall structures such as the stria vascularis and spiral ligament fibrocytes. This degeneration is likely linked to the diminished regenerative capacity of lateral wall cells with age. While lateral wall regeneration is known to occur after an acute insult, this process remains poorly understood and the cells capable of self-replication unidentified. We hypothesized that spiral ligament fibrocytes constitute these proliferative cells. SUBJECTS AND METHODS: To test the hypothesis, an acute ototoxic insult was created in 65 normal-hearing, young adult mice via cochlear exposure to heptanol. Sacrifice occurred at 1 to 60 days posttreatment. Auditory brainstem responses, 5-ethynyl-2'-deoxyuridine assay, and immunostaining were used to assess regeneration. RESULTS: Posttreatment hearing thresholds were elevated in nearly all treated mice. Selective fibrocyte apoptosis and strial injury were observed at the time of peak hearing loss around 1 to 7 days posttreatment. Cellular proliferation was detected in the region of type II fibrocytes during this time. Hearing thresholds plateaued at 7 days posttreatment followed by a significant recovery of both hearing and morphologic appearance. Permanent outer hair cell degeneration was observed. CONCLUSIONS: Heptanol application to the round window of young adult mice is a rapid, selective, and reliable technique for investigating proliferation in the cochlear lateral wall. The data indirectly showed that spiral ligament fibrocytes may be the proliferative cells of the cochlear lateral wall. Further studies of this process are needed.

Nitric oxide signalling is involved in diarylheptanoid-induced increases in femoral arterial blood flow in ovariectomized rats.

The mechanisms by which the hexane extract of Curcuma comosa increases femoral blood flow (FBF) in ovariectomized rats are not known. Thus, the aim of the present study was to investigate the acute effects and modes of action of the diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3), a phyto-oestrogen isolated from C. comosa, on FBF in ovariectomized rats. On Day 7 after ovariectomy, rats were injected once intra-arterially with D3 (100, 200, 400 and 800 µg/kg), 17ß-oestradiol (E2; 1, 2, 4 and 8 µg/kg) or vehicle. In some experiments, rats were injected with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) 120 min after 800 µg/kg D3 or 4 µg/kg E2. In other experiments, rats were injected with 10 mg/kg L-NAME, 900 µg/kg 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 900 µg/kg ICI 182 780 30 min prior to the injection of 800 µg/kg D3 or 4 µg/kg E2. Mean arterial blood pressure (mABP) and FBF were recorded using a pressure transducer and a laser Doppler flow meter, respectively. Both D3 and E2 dose-dependently increased FBF without changing mABP or heart rate. The EC(50) at 120 min for D3 and E2 was 195.8 and 1.8 µg/kg, respectively. In addition, D3 and E2 dose-dependently decreased femoral vascular resistance (FVR). The EC(50) of D3 was about 100-fold greater than that of E2. The effects of D3 and E2 on FBF and FVR were diminished by intravenous injection of 10 mg/kg l-NAME. Furthermore, 30 min pretreatment with L-NAME (10 mg/kg), ODQ (900 µg/kg) or ICI 182 780 (900 µg/kg) blocked the effects of D3 and E2 on FBF and FVR. The results of the present study suggest that the phyto-oestrogen D3 increases FBF in ovariectomized rats via oestrogen receptor and nitric oxide-guanylyl cyclase signalling, which, in turn, relaxes femoral vascular resistance.

Mechanical preconditioning enables electrophysiologic coupling of skeletal myoblast cells to myocardium.

OBJECTIVE: The effect of mechanical preconditioning on skeletal myoblasts in engineered tissue constructs was investigated to resolve issues associated with conduction block between skeletal myoblast cells and cardiomyocytes. METHODS: Murine skeletal myoblasts were used to generate engineered tissue constructs with or without application of mechanical strain. After in vitro myotube formation, engineered tissue constructs were co-cultured for 6 days with viable embryonic heart slices. With the use of sharp electrodes, electrical coupling between engineered tissue constructs and embryonic heart slices was assessed in the presence or absence of pharmacologic agents. RESULTS: The isolation and expansion procedure for skeletal myoblasts resulted in high yields of homogeneously desmin-positive (97.1% ± 0.1%) cells. Mechanical strain was exerted on myotubes within engineered tissue constructs during gelation of the matrix, generating preconditioned engineered tissue constructs. Electrical coupling between preconditioned engineered tissue constructs and embryonic heart slices was observed; however, no coupling was apparent when engineered tissue constructs were not subjected to mechanical strain. Coupling of cells from engineered tissue constructs to cells in embryonic heart slices showed slower conduction velocities than myocardial cells with the embryonic heart slices (preconditioned engineered tissue constructs vs embryonic heart slices: 0.04 ± 0.02 ms vs 0.10 ± 0.05 ms, P = .011), lower maximum stimulation frequencies (preconditioned engineered tissue constructs vs embryonic heart slices: 4.82 ± 1.42 Hz vs 10.58 ± 1.56 Hz; P = .0009), and higher sensitivities to the gap junction inhibitor (preconditioned engineered tissue constructs vs embryonic heart slices: 0.22 ± 0.07 mmol/L vs 0.93 ± 0.15 mmol/L; P = .0004). CONCLUSIONS: We have generated skeletal myoblast-based transplantable grafts that electrically couple to myocardium.

Improvements of insulin resistance in ovariectomized rats by a novel phytoestrogen from Curcuma comosa Roxb.

BACKGROUND: Curcuma comosa Roxb. (C. comosa) is an indigenous medicinal herb that has been used in Thailand as a dietary supplement to relieve postmenopausal symptoms. Recently, a novel phytoestrogen, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol or compound 049, has been isolated and no study thus far has investigated the role of C. comosa in preventing metabolic alterations occurring in estrogen-deprived state. The present study investigated the long-term effects (12 weeks) of C. comosa hexane extract and compound 049 on insulin resistance in prolonged estrogen-deprived rats. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) and treated with C. comosa hexane extract (125 mg, 250 mg, or 500 mg/kg body weight (BW)) and compound 049 (50 mg/kg BW) intraperitoneally three times per week for 12 weeks. Body weight, food intake, visceral fat weight, uterine weight, serum lipid profile, glucose tolerance, insulin action on skeletal muscle glucose transport activity, and GLUT-4 protein expression were determined. RESULTS: Prolonged ovariectomy resulted in dyslipidemia, impaired glucose tolerance and insulin-stimulated skeletal muscle glucose transport, as compared to SHAM. Treatment with C. comosa hexane extract and compound 049, three times per week for 12 weeks, markedly reduced serum total cholesterol and low-density lipoprotein levels, improved insulin sensitivity and partially restored uterine weights in ovariectomized rats. In addition, compound 049 or high doses of C. comosa hexane extract enhanced insulin-mediated glucose uptake in skeletal muscle and increased muscle GLUT-4 protein levels. CONCLUSIONS: Treatment with C. comosa and its diarylheptanoid derivative improved glucose and lipid metabolism in estrogen-deprived rats, supporting the traditional use of this natural phytoestrogen as a strategy for relieving insulin resistance and its related metabolic defects in postmenopausal women.

QRS widening and QT prolongation under bupropion: a unique cardiac electrophysiological profile.

QRS widening and QT prolongation are associated with bupropion. The objectives were to elucidate its cardiac electrophysiological properties. Patch-clamp technique was used to assess the I(Kr) -, I(Ks) -, and I(Na) -blocking effects of bupropion. Langendorff retroperfusion technique on isolated guinea-pig hearts was used to evaluate the MAPD(90) -, MAP amplitude-, phase 0 dV/dt-, and ECG-modulating effects of bupropion and of two gap junction intercellular communication inhibitors: glycyrrhetinic acid and heptanol. To evaluate their effects on cardiac intercellular communication, fluorescence recovery after photobleaching (FRAP) technique was used. Bupropion is an I(Kr) blocker. IC(50) was estimated at 34 µm. In contrast, bupropion had hardly any effect on I(Ks) and I(Na) . Bupropion had no significant MAPD(90) -modulating effect. However, as glycyrrhetinic acid and heptanol, bupropion caused important reductions in MAP amplitude and phase 0 dV/dt. A modest but significant QRS-widening effect of bupropion was also observed. FRAP experiments confirmed that bupropion inhibits gap junctional intercellular communication. QT prolongation during bupropion overdosage is due to its I(Kr) -blocking effect. QRS widening with bupropion is not related to cardiac sodium channel block. Bupropion rather mimics the QRS-widening, MAP amplitude- and phase 0 dV/dt -reducing effect of glycyrrhetinic acid and heptanol. Unlike class I anti-arrhythmics, bupropion causes cardiac conduction disturbances by reducing cardiac intercellular coupling.

Connexon-mediated cell adhesion drives microtissue self-assembly.

Microtissue self-assembly is thought to be driven primarily by cadherins, while connexons have been examined mainly in intercellular coupling. We investigated whether connexon 43 (Cx43)-mediated cell adhesion modulates self-assembly of human KGN granulosa cells, normal human fibroblasts (NHFs), and MCF-7 breast cancer cells seeded into nonadhesive agarose gels. We found that treatment with anti-Cx43 E2 (112 µg/ml), which suppresses Cx43 docking, significantly inhibited the kinetics of KGN and NHF self-assembly compared to the preimmune sera control (41.1 ± 4.5 and 24.5 ± 10.4% at 8 h, respectively). Likewise, gap junction inhibitor carbenoxolone also inhibited self-assembly of KGN, NHF, and MCF-7 cells in a dose-dependent manner that was specific to cell type. In contrast, Gap26 connexin mimetic peptide, which inhibits channel permeability but not docking, accelerated self-assembly of KGN and NHF microtissues. Experiments using selective enzymatic digestion of cell adhesion molecules and neutralizing N-cadherin antibodies further showed that self-assembly was comparably disrupted by inhibiting connexin- and cadherin-mediated adhesion. These findings demonstrate that connexon-mediated cell adhesion and intercellular communication differentially influence microtissue self-assembly, and that their contributions are comparable to those of cadherins.

Pathways for ATP release by bovine ciliary epithelial cells, the initial step in purinergic regulation of aqueous humor inflow.

ATP release by nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells is the enabling step in purinergic regulation of aqueous humor formation, but the release pathways are unknown. We measured ATP release from primary cultures of bovine mixed NPE and PE (bCE) cells and transformed bovine NPE and PE cells, using the luciferin-luciferase reaction. Hypotonicity-triggered bCE ATP release was inhibited by the relatively selective blocker of pannexin-1 (PX1) hemichannels (probenecid, 1 mM, 47 ± 2%), by a connexin inhibitor (heptanol, 1 mM, 49 ± 4%), and by an inhibitor of vesicular release (bafilomycin A1, 25 ± 2%), but not by the P2X(7) receptor (P2RX(7)) antagonist KN-62. Bafilomycin A1 acts by reducing the driving force for uptake of ATP from the cytosol into vesicles. The reducing agent dithiothreitol reduced probenecid-blockable ATP release. Similar results were obtained with NPE and PE cell lines. Pannexins PX1-3, connexins Cx43 and Cx40, and P2RX(7) were identified in native cells and cell lines by RT-PCR. PX1 mRNA expression was confirmed by Northern blots; its quantitative expression was comparable to that of Cx43 by real-time PCR. Heterologous expression of bovine PX1 in HEK293T cells enhanced swelling-activated ATP release, inhibitable by probenecid. We conclude that P2RX(7)-independent PX1 hemichannels, Cx hemichannels, and vesicular release contribute comparably to swelling-triggered ATP release. The relatively large response to dithiothreitol raises the possibility that the oxidation-reduction state is a substantial regulator of PX1-mediated ATP release from bovine ciliary epithelial cells.

Gap junction channel modulates pulmonary vascular permeability through calcium in acute lung injury: an experimental study.

BACKGROUND: Increased pulmonary vascular permeability is a hallmark of acute lung injury (ALI). Gap junction channels (GJCs) connect adjacent cells and facilitate ion exchange. It remained unclear whether GJCs modulate pulmonary permeability in ALI through intracellular calcium. OBJECTIVES: This study aimed to verify if GJCs in pulmonary microvessel endothelial cells (PMVECs) modulate pulmonary vascular permeability in ALI via intracellular calcium. METHODS: Firstly, an animal model of ALI was studied using connexin 40 (Cx40) immunohistochemistry in the lung with Evans' blue (EB) leakage. Then cultured PMVECs were divided into three groups: G(control), G(serum) and G(blocker). Serum was obtained from animals with ALI following gunshot injury (injured serum). Initially, G(blocker) was treated with the blocker of GJCs, and then G(serum) and G(blocker) were stimulated with the injured serum, respectively. GJCs, the permeability of cell monolayers and intracellular Ca(2+) were assessed. RESULTS: Cx40 time-dependently decreased, whereas EB leakage increased. Cx40 and EB leakage exhibited a strong inverse correlation (rho = -0.934, p < 0.05). Injured serum decreased GJCs and expression of Cx40, whereas the blocker aggravated this effect. Similarly, when PMVEC monolayer was treated with injured serum, both permeability and intracellular Ca(2+) increased. These effects were also aggravated with the blocker. CONCLUSIONS: Depression of GJCs of PMVECs increased pulmonary vascular permeability in ALI; this effect may be mediated by the overload of intracellular calcium.

Damaging effect of cumulus denudation on rabbit oocytes.

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.

Gap junctions relay FGF8-mediated right-sided repression of Nodal in rabbit.

In vertebrate gastrula/neurula embryos, a cilia-driven leftward flow asymmetrically activates the Nodal cascade in the left lateral plate mesoderm (LPM). In frog embryos left-right axis formation was postulated to depend on gap junctions (GJs) during cleavage. Here, we show that GJs cooperate with fibroblast growth factor-8 (FGF8) to specify asymmetric Nodal in the rabbit embryo at gastrula/neurula. GJs and FGF signaling were manipulated in whole embryo and explant cultures of rabbit blastodiscs. These experiments demonstrate that right-sided inhibition of Nodal by FGF8 depended on intercellular communication by means of GJs, and that left-sided induction of Nodal required attenuation of gap junctional communication (GJC). Before flow, the left and right side were equally competent but actively prevented from Nodal induction through FGF8/GJ. Our data suggest that flow unilaterally attenuates FGF8/GJ-mediated repression of Nodal on the left side, integrating GJC and FGF8 into the flow-based mechanism of symmetry breakage in the vertebrate embryo.

Electron microprobe analysis of rabbit ciliary epithelium indicates enhanced secretion posteriorly and enhanced absorption anteriorly.

The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue's small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned, and freeze-dried for electron probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na(+),K(+)-ATPase increases Na(+) and decreases K(+) contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produced smaller changes in anterior PE cells, possibly reflecting higher Na(+) or K(+) permeability of anterior NPE cells. Inhibiting Na(+) entry selectively with amiloride, benzamil, or dimethylamiloride reduced anterior effects of ouabain by approximately 50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl(-) secretion. In contrast to ouabain's actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that 1) enhanced Na(+) reabsorption anteriorly, likely through Na(+) channels and Na(+)/H(+) exchange, mediates the regional dependence of ouabain's actions; and 2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.

Heterogeneous response of microvascular endothelial cells to shear stress.

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.

Isolated primary osteocytes express functional gap junctions in vitro.

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.

Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs.

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.

[The effect of gap blocker 1-heptanol on function and morphology of guinea pig cochlea].

OBJECTIVE: To investigate the role of gap junctions in hearing function of guinea pig in vivo. METHOD: Seven guinea pigs were inoculated with 1-heptanol into perilymphatic space after a small hole was drilled in the bony wall near tympanic scala of the base turn. Auditory brainstem response (ABR) was recorded before, 30 min, 60 min, 90 min 120 min after operation. Two hours after surgery, all the animals were killed and the ultrastructure of guinea cochlea was observed by making electron microscopy specimens. RESULT: The application of 1-heptanol into guinea pig cochlea resulted in large increase in ABR threshold, which showed 30 to 40 dB elevation in guinea pig. And 1-heptanol could destroy the gap conjunction structure between hair cells and Deiter's cells in guinea cochlea. CONCLUSION: The gap blocker 1-heptanol might significantly influence the guinea pig's hearing function, and the gap conjunction might play an important role in normal cochlear function and K+ recycle in inner ear of guinea pig.