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D C Spray

Publications and source records attributed to D C Spray.

At least 127 records · Page 7Linked to original sources
PubMed1991

Characterization of gap junctions between cultured leptomeningeal cells.

Leptomeningeal cells in intact meninges or dissociated and cultured for 2 h to several weeks were dye-coupled (Lucifer yellow), and voltage-clamped pairs of freshly dissociated leptomeningeal cells were well coupled electrically. Unitary conductances of junctional channels were predominantly 40-90 pS. Junctional conductance was reversibly reduced by 2 mM halothane, 1 mM heptanol and 100% CO2 and was increased by 1 mM 8 Br-cAMP. Two gap junction proteins, connexin 26 and connexin 43, were identified between leptomeningeal cells using immunocytochemical methods; Northern blot analyses of RNA isolated from cultured leptomeningeal cells showed specific hybridization to cDNAs encoding connexins 26 and 43, but not to a cDNA encoding connexin 32. These studies demonstrate co-expression of two connexins in a single cell type in the nervous system; biophysical properties do not differ significantly from those of astrocytes and cardiac myocytes, which express only connexin 43.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo.

Gap junction channels provide a pathway for exchange of ions and small molecules between coupled cells, and this exchange is believed to be critical for normal tissue growth and development. As a test for a role of gap junction-mediated intercellular communication in control of cell growth, we have compared growth rates of communication-deficient human tumor cells (SKHep1) with clones stably transfected with cDNA encoding the rat liver gap junction protein connexin 32. In culture, growth rates for parental and transfected clones were similar. However, when sizes of tumors were evaluated following injection of these clones into athymic nude mice, growth rates for two well-coupled clones were significantly lower than for communication-deficient or poorly coupled clones. This study demonstrates that growth rate of these tumor cells in situ is negatively correlated with strength of intercellular communication.

Animals↗
PubMed1991

Functional analysis of human cardiac gap junction channel mutants.

The connexins form a family of membrane spanning proteins that assemble into gap junction channels. The biophysical properties of these channels are dependent upon the constituent connexin isoform. To begin identifying the molecular basis for gap junction channel behavior in the human heart, a tissue that expresses connexin43, we used site-directed mutagenesis to generate mutant cDNAs of human connexin43 with shortened cytoplasmic tail domains. Premature stop codons were inserted, resulting in proteins corresponding in length to the mammalian isoforms connexin32 and connexin26, which are expressed primarily in liver. All constructs restore intercellular coupling when they are transfected into SKHep1 cells, a human hepatoma line that is communication deficient. Whereas wild-type connexin43 transfectants display two distinct unitary conductance values of about 60 and 100 pS, transfectants expressing the mutant proteins, from which 80 and 138 amino acids have been deleted, exhibit markedly different single-channel properties, with unitary conductance values of about 160 and 50 pS, respectively. Junctional conductance of channels composed of wild-type connexin43 is less voltage-sensitive compared with transfectants expressing wild-type connexin32. However, neither of the connexin43 truncation mutants alters this relative voltage insensitivity. These results suggest that the cytoplasmic tail domain is an important determinant of the unitary conductance event of gap junction channels but not their voltage dependence. Furthermore, since the mutant connexins are missing several consensus phosphorylation sites, modification of these particular sites may not be required for membrane insertion or assembly of human connexin43 into functional channels.

Biological Transport↗
PubMed1991

Intercellular communication through gap junctions: a potential role in pharmacomechanical coupling and syncytial tissue contraction in vascular smooth muscle isolated from the human corpus cavernosum.

Kinetic and steady-state protocols were used to examine the effects of disruption of intercellular communication with heptanol, on contractile responses elicited by activation of the alpha 1-adrenergic receptor in human corporal vascular smooth muscle. For the steady-state studies, strips of corporal tissue from 19 patients were submaximally precontracted with phenylephrine (PE) and then relaxed by the cumulative addition of heptanol. Heptanol completely and reversibly relaxed all tissues studied in a concentration-dependent manner. The heptanol concentration response data were then computer fit to the general logistic equation to obtain pEC50 (negative logarithm of the concentration that elicits one-half of the maximal effect) and slope factor values, with Emax (maximal relaxation) set to 100%. The mean pEC50 and slope factor values, respectively, were 2.86 +/- 0.04 and 1.86 +/- 0.17. Furthermore, kinetic studies on corporal tissues from a subset of the patient population (11 patients) revealed that preincubation of tissues with 2 mM heptanol caused a significant decrease in both the rate and magnitude of PE-induced contractions in all tissues studied, without affecting the rate constant for onset of contraction (k(obs)). The present results on intact tissue extend our previous observations on cultured corporal cells, and support the supposition that intercellular communication through gap junctions may play an important role in the initiation, maintenance and modulation of alpha 1-adrenergic contractions in human vascular smooth muscle.

Adult↗
PubMed1991

Connexin32 gap junction channels in stably transfected cells: unitary conductance.

Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class.

Biophysical Phenomena↗
PubMed1991

Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties.

Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (gj) to transjunctional voltages (Vjs) of either polarity, with the ratio of minimal to maximal gj (gmin/gmax) being approximately 0.1 at the highest Vjs. Steady-state gj values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which gj was reduced by 50%, was approximately 25-30 mV; A, the Boltzmann parameter describing voltage dependence, was approximately 0.06 (corresponding to an energy difference between states of approximately 1 kCal/mol and to approximately 2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (tau greater than 5 s at 20-40 mV, tau = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (alpha) was approximately 30% lower than that of the closing rate constant (beta) over the Vj range 0-70 mV; at higher voltages, voltage sensitivity of alpha and beta saturated. The kinetic response of gj to a paradigm in which gj was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state.

Biophysical Phenomena↗
PubMed1991

Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.

Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved.

Animals↗
PubMed1991

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).

Animals↗
PubMed1991

Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver.

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to "oval" cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

Animals↗
PubMed1991

Humoral factors reduce gap junction sensitivity to cytoplasmic pH. I. Organ ablation studies.

The sensitivity of gap junctions connecting crayfish lateral axons to uncoupling by axoplasmic acidification was studied after altering the hormonal balance of animals by 1) ablation of eyestalks or sinus glands or 2) inducing long-lasting defensive posturing behavior (stress). Internal pH (pHi) was measured with microelectrodes, and junctional resistance (Rj) was calculated from input and transfer resistances. In isolated nerve cords from intact animals, the maximal Rj (Rjmax) reached after acidification varied diurnally (Rjmax approximately 10 and 0.6 M omega at 0900 and 1800 h, respectively). Basal Rj (20-30 k omega) did not change during the 24-h period. Organ ablation (eyestalks or sinus glands) or stress rendered gap junctions less sensitive to uncoupling by low pHi within 1 h or 2 days; recovery toward control values had different time courses. The reduced pH sensitivity of crayfish junctions seen after eyestalk ablation is attributable to stress in its early phase (lasting 1-2 days) and to ablation of the endocrine organs in its late phase (2-7 days). No striking structural differences accompanied these changes, indicating that the altered properties are not due to major changes in gap junction expression.

Animals↗
PubMed1991

Expression of connexin43 in the developing rat heart.

Connexin43 is the predominant gap junction protein expressed in the heart. To determine the relation between cardiac maturation and gap junction gene expression, the developmental profiles of connexin43 mRNA and protein were examined in the rat heart. Connexin43 mRNA levels accumulate progressively (eightfold) during embryonic and early neonatal stages, accompanied by a parallel, but temporally delayed, accumulation of connexin43 protein (15-fold). As the heart matures further, both mRNA and protein levels subsequently decline, to about 50% and 30% of their maximum levels, respectively. These observations suggest that increases in intercellular coupling that characterize cardiac development do not depend solely on modulation of connexin43 gene expression, but rather are likely to involve organization of gap junction channels into the intercalated disc.

Animals↗
PubMed1991

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

1. 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 decreased with imposed transmembrane voltages exceeding +/- 20 mV. A residual voltage-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. 2. These results are generally similar to those described by Young, Cohn 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 distinct channel populations in the incorporated membrane fractions. Our results suggest that a population of larger conductance channels (greater than or equal to 150 pS) contributes the voltage-dependent component of the membrane conductance, while smaller channels (unitary conductance about 50-150 pS) contribute the voltage-independent component. The biophysical properties 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. 3. These findings indicate that connexin32 comprises the larger, voltage-dependent channels seen in the bilayer experiments in which liver junctional membranes are incorporated.

Animals↗
PubMed1991

Gap junctions between cultured astrocytes: immunocytochemical, molecular, and electrophysiological analysis.

The properties of astroglial gap junction channels and the protein that constitutes the channels were characterized by immunocytochemical, molecular biological, and physiological techniques. Comparative immunocytochemical labeling utilizing different antibodies specific for liver connexin 32 and connexin 26 and antibodies to peptides corresponding to carboxy-terminal sequences of the heart gap junction protein (connexin 43) indicates that the predominant gap junction protein in astrocytes is connexin 43. The expression of this connexin in cultured astrocytes was also established by Western and Northern blot analyses. Cultured astrocytes expressed connexin 43 mRNA and did not contain detectable levels of the mRNAs encoding connexin 32 or connexin 26. Further, the cells contained the same primary connexin 43 translation product and the same phosphorylated forms as heart. Finally, electrophysiological recordings under voltage-clamp conditions revealed that astrocyte cell pairs were moderately well coupled, with an average junctional conductance of about 13 nS. Single-channel recordings indicated a unitary junctional conductance of about 50-60 pS, which is of the same order as that found in cultured rat cardiac myocytes, where the channel properties of connexin 43 were first described. Thus, physiological properties of gap junction channels appear to be determined by the connexin expressed, independent of the tissue type.

Animals↗
PubMed1990

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.

Amino Acid Sequence↗
PubMed1990

Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32.

The gene family encoding gap junction proteins (connexins) consists of several known members, and multiple connexins are frequently coexpressed by coupled cells. To characterize the channel properties of the major rat liver gap junction protein (connexin 32) in isolation from other gap junction proteins, we have introduced the cDNA encoding it into a human hepatoma cell line (SKHep1) in which we have identified a gap junction deficiency. In this cell line, dye coupling was absent and junctional conductance was near zero. Connexins and connexin 32 mRNA were not detectable by immunocytochemistry and Northern blot analysis. After transfection and selection, cells were strongly coupled with regard to dye and electrical current, and connexin 32 mRNA and punctate connexin 32-immunoreactive membrane contacts were abundant. Functional gap junction channels were still expressed after 19 passages of the cells, indicating stable transfection. When junctional conductance was rendered reversibly low by exposing the cells to agents that uncouple other cell types, currents through single gap junction channels could be observed. The unitary conductance of these expressed channels was about 120-150 pS, a value that is distinctly larger than in heart cells, which express a different gap junction protein.

Animals↗
PubMed1990

Molecular characterization and functional expression of the human cardiac gap junction channel.

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.

Amino Acid Sequence↗
PubMed1990

Structure-activity relations of the cardiac gap junction channel.

Cardiac gap junction channels play the important roles of synchronizing pacemaker cells and allowing impulse propagation along the conduction system and throughout the ventricular myocardium. These channels, which support current flow in both longitudinal and tranverse directions, are permeable to anions and cations with radii less than approximately 0.5 nm and in rat heart have unitary conductances on the order of 50 pS. This unitary conductance is consistent with channel geometry described by a right cylindrical pore with diameter large enough for the brilliantly fluorescent dye molecule lucifer yellow to pass between cells. These channels, like others in biological systems, are opened and closed by various treatments, a process termed gating. Cytoplasmic acidification reduces junctional conductance (gj), an effect that is apparently potentiated by elevated myoplasmic Ca ions. Reduced gj also occurs in response to a variety of lipophilic molecules, including halothane, heptanol, and unsaturated fatty acids; the mechanism of action may involve disruption of the protein-lipid microenvironment of the gap junction channel. Arachidonic acid uncouples, and this effect is partially, but incompletely, blocked by an inhibitor of the lipoxygenase metabolic pathways. Cyclooxygenase inhibitors have no protective effects. Certain cyclic nucleotides can rapidly increase gj [adenosine 3',5'-cyclic monophosphate (cAMP)] or slightly decrease it [guanosine 3',5'-cyclic monophosphate (cGMP)], and agents that use these cyclic nucleotides as second messengers (isoproterenol and perhaps carbachol, respectively) produce consistent effects. Agents expected to cause protein kinase C activation (tumor-promoting phorbol esters and diacylglycerol) increase gj rapidly. The gap junction protein from rat heart has been cloned and sequenced. From the primary sequence for the protein, plausible sites of action within the putative cytoplasmic domains are proposed for each of these treatments. In response to gating stimuli that close the channel (halothane, CO2, heptanol), unitary channel conductance is unchanged, suggesting that these agents act by reducing open time probability. Together, these properties constitute the beginnings of our endeavor to define pharmacological agents that are potentially useful in therapeutic manipulation of synchronous discharge, conduction velocity, and isochronous wavefront propagation in cardiac tissue.

Animals↗