Soluble factors from multipotent mesenchymal stromal cells have antinecrotic effect on cardiomyocytes in vitro and improve cardiac function in infarcted rat hearts.

The mechanisms underlying the functional improvement after injection of multipotent mesenchymal stromal cells (MSCs) in infarcted hearts remain incompletely understood. The aim of this study was to investigate if soluble factors secreted by MSCs promote cardioprotection. For this purpose, conditioned medium (CM) was obtained after three passages from MSC cultures submitted to 72 h of conditioning in serum-free DMEM under normoxia (NCM) or hypoxia (HCM) conditions. CM was concentrated 25-fold before use (NCM-25X, concentrated normoxia conditioned medium; HCM-25X, concentrated hypoxia conditioned medium). The in vitro cardioprotection was evaluated in neonatal ventricular cardiomyocytes by quantifying apoptosis after 24 h of serum deprivation associated with hypoxia (1% O(2)) in the absence or presence of NCM and HCM (nonconcentrated and 25-fold concentrated). The in vivo cardioprotection of HCM was tested in a model of myocardial infarction (MI) induced in Wistar male rats by permanent left coronary occlusion. Intramyocardial injection of HCM-25X (n = 14) or nonconditioned DMEM (n = 16) was performed 3 h after coronary occlusion and cardiac function was evaluated 19-21 days after medium injection. Cardiac function was evaluated by electro- and echocardiogram, left ventricular catheterization, and treadmill test. The in vitro results showed that HCM was able to decrease cardiomyocyte necrosis. The in vivo results showed that HCM-25X administered 3 h after AMI was able to promote a significant reduction (35%) in left ventricular end-diastolic pressure and improvement of cardiac contractility (15%) and relaxation (12%). These results suggest that soluble factors released in vitro by MSCs are able to promote cardioprotection in vitro and improve cardiac function in vivo.

Cell therapy in dilated cardiomyopathy: from animal models to clinical trials

Dilated cardiomyopathy can be the end-stage form and common denominator of several cardiac disorders of known cause, such as hypertensive, ischemic, diabetic and Chagasic diseases. However, some individuals have clinical findings, such as an increase in ventricular chamber size and impaired contractility (classical manifestations of dilated cardiomyopathy) even in the absence of a diagnosed primary disease. In these patients, dilated cardiomyopathy is classified as idiopathic since its etiology is obscure. Nevertheless, regardless of all of the advances in medical, pharmacological and surgical procedures, the fate of patients with dilated cardiomyopathy (of idiopathic or of any other known cause) is linked to arrhythmic episodes, severe congestive heart failure and an increased risk of sudden cardiac death. In this review, we will summarize present data on the use of cell therapies in animal models of dilated cardiomyopathies and will discuss the few clinical trials that have been published so far involving patients affected by this disease. The animal models discussed here include those in which the cardiomyopathy is produced by genetic manipulation and those in which disease is induced by chemical or infectious agents. The specific model used clearly creates restrictions to translation of the proposed cell therapy to clinical practice, insofar as most of the clinical trials performed to date with cell therapy have used autologous cells. Thus, translation of genetic models of dilated cardiomyopathy may have to wait until the use of allogeneic cells becomes more widespread in clinical trials of cell therapies for cardiac diseases.

Cell therapy in dilated cardiomyopathy: from animal models to clinical trials.

Dilated cardiomyopathy can be the end-stage form and common denominator of several cardiac disorders of known cause, such as hypertensive, ischemic, diabetic and Chagasic diseases. However, some individuals have clinical findings, such as an increase in ventricular chamber size and impaired contractility (classical manifestations of dilated cardiomyopathy) even in the absence of a diagnosed primary disease. In these patients, dilated cardiomyopathy is classified as idiopathic since its etiology is obscure. Nevertheless, regardless of all of the advances in medical, pharmacological and surgical procedures, the fate of patients with dilated cardiomyopathy (of idiopathic or of any other known cause) is linked to arrhythmic episodes, severe congestive heart failure and an increased risk of sudden cardiac death. In this review, we will summarize present data on the use of cell therapies in animal models of dilated cardiomyopathies and will discuss the few clinical trials that have been published so far involving patients affected by this disease. The animal models discussed here include those in which the cardiomyopathy is produced by genetic manipulation and those in which disease is induced by chemical or infectious agents. The specific model used clearly creates restrictions to translation of the proposed cell therapy to clinical practice, insofar as most of the clinical trials performed to date with cell therapy have used autologous cells. Thus, translation of genetic models of dilated cardiomyopathy may have to wait until the use of allogeneic cells becomes more widespread in clinical trials of cell therapies for cardiac diseases.

A safety and feasibility study of cell therapy in dilated cardiomyopathy

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35 percent were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg-1·min-1 at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

A safety and feasibility study of cell therapy in dilated cardiomyopathy.

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35% were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg⁻¹·min⁻¹ at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1â (3.8X) and TNF-á (6.0X). IFN-ã was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Bone marrow cell transplant does not prevent or reverse murine liver cirrhosis.

We tested the effect of bone marrow cell (BMC) transplantation in either preventing or reversing cirrhosis on an experimental model of chronic liver disease. Female Wistar rats were fed a liquid alcohol diet and received intraperitoneal injections of carbon tetrachloride (CCl4) over 15 weeks. Ten animals (cell-treated group) received five injections of BMCs during the cirrhosis induction protocol (on the 4th, 6th, 8th, 10th, and 12th weeks) and four animals received the cells after liver injury was established through tail vein. Nine animals (nontreated group) were submitted to the previously described protocols; however, they received vehicle injections. Analyses were performed to verify whether the infusion of cells was effective in preventing the development of cirrhosis in our model of induction, and if the cells could reverse cirrhosis once it was established. Hepatic architecture and fibrotic septa were analyzed in liver slices stained with hematoxilin & eosin and Sirius red, respectively. Fibrosis quantification was measured by Sirius red histomorphometry. Indirect immunofluorescence was performed to detect the amount of tissue transglutaminase 2. Blood analyses were performed to assess liver injury and function by the assessment of alanine aminotransferase and albumin. Ultrasound was performed to analyze the portal vein caliber and presence of ascitis. Cirrhosis features (regenerative nodules and fibrous septa) were observed in histopathology after 15 weeks of continuous hepatic injury in nontreated and cell-treated groups. Collagen content, immunofluorescence analysis, and biochemical and ultrasound parameters were similar in nontreated and cell-treated groups; however, both groups showed significant differences compared to a normal control group. Cell infusions with bone marrow-derived cells seem to be ineffective in improving morphofunctional parameters of the liver when applied to chronic cases either during or after establishment of the hepatic lesion.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5 percent, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 ± 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 ± 0.02 cm, P < 0.01) vs control (0.16 ± 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 ± 2.64 percent, P < 0.001; EtOH + CCl4/8wR: 10.4 ± 1.36 percent, P < 0.05) vs control (2.2 ± 1.21 percent). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 ± 1.3 percent, P < 0.01; collagen III: 1.3 ± 0.2 percent, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 ± 0.06 percent, P < 0.05; collagen III: 1.5 ± 0.8 percent, P < 0.01) vs control (collagen I: 0.38 ± 0.11 percent; collagen III: 0.25 ± 0.06 percent). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 ± 8 percent, P < 0.01; EtOH + CCl4/8wR: 58.8 ± 21 percent, P < 0.01) vs control (7.9 ± 0.8 percent). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

A novel form of cellular communication among thymic epithelial cells: intercellular calcium wave propagation.

We here describe intercellular calcium waves as a novel form of cellular communication among thymic epithelial cells. We first characterized the mechanical induction of intercellular calcium waves in different thymic epithelial cell preparations: cortical 1-4C18 and medullary 3-10 thymic epithelial cell lines and primary cultures of thymic "nurse" cells. All thymic epithelial preparations responded with intercellular calcium wave propagation after mechanical stimulation. In general, the propagation efficacy of intercellular calcium waves in these cells was high, reaching 80-100% of the cells within a given confocal microscopic field, with a mean velocity of 6-10 microm/s and mean amplitude of 1.4- to 1.7-fold the basal calcium level. As evaluated by heptanol and suramin treatment, our results suggest the participation of both gap junctions and P2 receptors in the propagation of intercellular calcium waves in thymic nurse cells and the more prominent participation of gap junctions in thymic epithelial cell lines. Finally, in cocultures, the transmission of intercellular calcium wave was not observed between the mechanically stimulated thymic epithelial cell and adherent thymocytes, suggesting that intercellular calcium wave propagation is limited to thymic epithelial cells and does not affect the neighboring thymocytes. In conclusion, these data describe for the first time intercellular calcium waves in thymic epithelial cells and the participation of both gap junctions and P2 receptors in their propagation.

Modulation of gap junction mediated intercellular communication in TM3 Leydig cells.

Long-term modulation of intercellular communication via gap junctions was investigated in TM3 Leydig cells, under low and high confluence states, and upon treatment of the cells for different times with activators of protein kinase A (PKA) and protein kinase C (PKC). Cells in low confluence were readily coupled, as determined by transfer of the dye Lucifer Yellow; on reaching confluence, the cells uncoupled. Western blots and RT-PCR revealed that connexin 43 (Cx43) was abundantly expressed in TM3 Leydig cells and its expression was decreased after the cells achieved confluence. Stimulation of PKA or PKC induced a decrease in cell-cell communication. Staurosporin, an inhibitor of protein kinases, increased coupling and was able to prevent and reverse the uncoupling actions of dibutyryl cAMP and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Under modulation by confluence, Cx43 was localized to the appositional membranes when cells were coupled and was mainly in the cytoplasm when they were uncoupled. In addition, cAMP and TPA reduced the surface membrane labeling for Cx43, whereas staurosporin increased it. These data show a strong correlation between functional coupling and the membrane distribution of Cx43, implying that this connexin has an important role in intercellular communication between TM3 cells. Furthermore, increased testosterone secretion in response to luteinizing hormone was accompanied by a decrease in intercellular communication, suggesting that gap junction mediated coupling may be a modulator of hormone secretion in TM3 cells.

Short term regulation of cell-cell communication in TM3 Leydig cells. A perforated patch study.

Determination of the junctional conductance (g(j)) in TM3 Leydig cells by the dual whole cell patch clamp technique (DWCPC) shows that coupling undergoes a rapid and irreversible run down. Addition of ATP or cAMP derivatives to the pipette solution has been shown to prevent this phenomenon in several tissues, but this same treatment is unable to inhibit run down in Leydig cells. Because the run down in junctional conductance may pose serious problems to the interpretation of results, we also measured g(j) by using the double perforated patch clamp technique (DPPT). Access to the cell interior was achieved by adding 200 microgram/ml of nystatin to the pipette solution. With this method, run down in g(j) was greatly reduced, amounting to no more than 5% of the initial value. Exposure of the cells, under DWCPC or DPPT, to dibutyryl cAMP or to tumor promoting agent (TPA) led to a decrease in cell to cell communication. Staurosporine, a PKC inhibitor, increased g(j) and was able to prevent and reverse the uncoupling action of cAMP or TPA. Our results indicate that cell-cell communication in Leydig cells is down regulated by both protein kinases A and C, interacting in a complex manner.

Sera from chronic chagasic patients with complex cardiac arrhythmias depress electrogenesis and conduction in isolated rabbit hearts.

BACKGROUND: Immune dysfunction has long been proposed as a mechanism for the etiopathogenesis of the chronic phase of Chagas' disease. Antibodies of chagasic patients have been shown to interfere with electric and mechanical activity of embryonic myocardial cells in culture. Here, we demonstrate that antibodies derived from a group of chronic chagasic patients are able to induce disturbances in the electrogenesis and conduction in isolated adult rabbit hearts. METHODS AND RESULTS: Sera from chronic chagasic patients with complex cardiac arrhythmias (ChA+) decreased heart rate (from 131+/-26 to 98+/-37 bpm [mean+/-SD]; n=6; P<.05) in isolated rabbit hearts when perfused at a dilution of 1:100 (vol:vol) by the Langendorff method. Sera from another experimental group of four chronic chagasic patients without complex arrhythmias (ChA-) and two control groups composed of five Wolff-Parkinson-White (WPW) syndrome patients and five orthopedic surgery patients did not affect heart rate when tested under similar conditions. In addition, sera from five of six ChA+ patients and from one WPW patient induced AV conduction blockade. Effects of the sera from ChA+ patients are due to their IgG fractions. Both serum and IgG effects are blocked by atropine (10 micromol/L). CONCLUSIONS: Antibodies of ChA+ patients decrease heart rate and induce AV conduction block in isolated adult rabbit hearts through activation of muscarinic receptors.

Properties of chicken lens MIP channels reconstituted into planar lipid bilayers.

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V0 = 18.5 mV, n = 4.5 and gmin/gmax = 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels.

Are there functional gap junctions or junctional hemichannels in macrophages?

The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin-43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch-clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.

Biophysical properties of the human cardiac gap junction channel

1. Gap junction channels interconnect cells of the pacemaking, conduction and contraction elements of the heart and also endothelial and smooth muscle cells of vasculature, thereby providing pathways for electrotonic current spread and for second messenger diffusion. The major gap junction protein in the cardiovascular system is connexin43. 2. When human connexin43 is stably expressed in pairs of a communication-deficient cell line (SKHep1) channels are produced with unitary conductance (gamma j), lipophile sensitivity and voltage-dependent gating similar to those of mammalian systems in which connexin43 is endogenously expressed. 3. At moderate transjunctional voltages (Vj), two gamma j values dominated the recordings, about 60 and 90 pS with CsCl patch solution. The smaller channel size is favored by phosphorylating treatments and the larger channel, by dephosphorylating treatments. 4. Human connexin43 mutants truncated at the carboxy termini display a change in gamma j while a point mutation in the third transmembrane spanning domain appears to change channel selectivity. 5. Voltage dependence of the human connexin43 channel is marked at Vjs, above +/- 50 mV, but large residual conductance remains (due probably to a voltage-insensitive substate) even at the largest Vj values; kinetic but not steady-state behavior is affected by phosphorylation state

Gap junctions between human corpus cavernosum smooth muscle cells: gating properties and unitary conductance.

We previously showed that corpus cavernosum smooth muscle cells are connected via gap junctions in situ and in culture and that a major protein component of these gap junctions is connexin43. To characterize the physiological properties of the gap junctions between corpus cavernosum smooth muscle cells, we now demonstrate that the cells are dye and electrically coupled and describe some of the gating properties of these gap junctional channels at macroscopic and single-channel levels. Junctional conductance (gj) between corporal smooth muscle cells was moderately voltage sensitive; was reduced rapidly, reversibly, and completely by halothane; and was increased by treatment with a tumor-promoting phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] and decreased by isoproterenol. Histograms of unitary junctional currents revealed multiple conductance peaks with events of approximately 90 pS being the most abundant. TPA and phenylephrine produced large increases in relative frequencies of the smaller events, whereas isoproterenol and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) slightly increased the relative frequencies of the larger events. None of the tested drugs substantially affected the steady-state voltage dependence of gj. These second messenger systems also affected expression of connexin43 by corpus cavernosum smooth muscle cells, as judged by immunoblots. At 6 h of treatment, both TPA- and 8-BrcAMP-treated cultures showed markedly elevated levels of connexin43, whereas at 24 h, the level of connexin43 in TPA-treated cultures had returned to control levels. Together, these data indicate that second messenger molecules involved in penile erection produce changes in gap junction expression and function; it is plausible that these changes could be physiologically relevant in altering and propagating changes in vasomotor tone.

Properties of channels from rat liver gap junction membrane fractions incorporated into planar lipid bilayers

Rat membrane fractions highly enriched for gap junctions can be incorporated into planar lipid bilayers exhibiting channel currents with both voltage-dependent and independent components. Voltage dependence, however, is only one of the characteristics of liver gap junction channels. Other features include poor ionic selectivity and sensitivity to calcium, pH, octanol and to some intracellularly applied antibodies. To further test the junctional nature of channels from membrane fractions highly enriched in gap junctions incorporated into lipid bilayers we studied the sensitivity of these channels to uncoupling agents and determined channel selectivity properties. We found the incorporated channels to be insensitive to calcium and octanol, and in most cases to pH in the range of 5-7, suggesting that either these agents do not interact directly with the junctional channels or that the corresponding gating regions are inactivated during the isolation and reconstitution procedures. Attempts to block channel activity using polyclonal and monoclonal connexin 32 antibodies were generally unsuccessful, although one antibody (a monoclonal directed against the carboxy terminus portion of connexin32) blocked channel activity. Selectivity measurements indicated that the incorporated channels were slightly cation selective (PNa=Pk > PCl) and were permeable to large ions. These results further support the idea that functional connexin32 gap junction channels are present in channel activity recorded from rat liver junctional membranes incorporated into planar bilayers

Complex channel activity recorded from rat liver GAP junctional membranes incorporated into lipid bilayers

Channels from isolated liver junctional membranes were incorporated into lipid bilayers and studied under voltage clamp conditions. Detergent treatment of junctional membrane fragments greatly increased the incidence of channel incorporation but did not noticeably alter the properties of the incorporated channels. Incorporation resulted in channel activity displaying an approximately symmetric voltage dependence in which conductance was decrease with imposed transmembrane voltage exceeding ñ 20 mV. A residual coltage-independent conductance was also detected in membranes in which liver junctional membranes were incorporated. The magnitude of this voltage-insensitive component varied from less than 20% to more than 75% of the total conductance. These results are generally similar to those described by Young, Chn and Gilula(Cell, 48:733-743, 1987) in incorporation experiments following detergent treatment of isolated gap junction membranes. However, we interpret these data as indicating the existence of distint channel populations in the incorporated membrane fractions. Our results suggest that a population of larger conductance channels (* 150 pS) contributes the voltage-dependent component of the membrane conductance, while smailler channels (unitary conductance abouth 50-150 pS) contribute the voltage-independent component. The biophysical proprieties of the larger channel are comparable to those seen in communication-deficient cells transfected with connexin32, confirming a report describing conductance of bilayers in which electroeluted 27-kDa liver gap junction protein was inserted. These findings indicate that connexin32 comprises the larger, voltage-dependent channels seen in the bilayer experiments in which liver junctional membranes are incorporated