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Publications and source records attributed to N B Gilula.
Electron cryo-microscopy and image analysis of frozen-hydrated, two-dimensional crystals of gap junction membrane channels formed by recombinant alpha 1 connexin (Cx43) reveal a ring of transmembrane alpha-helices that lines the aqueous pore and a second ring of alpha-helices in close contact with the membrane lipids.
Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.
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To examine the possible role of gap junctions in mouse skin tumor progression, we generated a panel of mouse skin tissue samples exhibiting normal, hyperplastic, or neoplastic changes and characterized the expression of the gap-junction genes connexin 43 (Cx43) and connexin 26 (Cx26) by in situ hybridization and immunohistochemical analyses. In normal skin, these two gap junction genes were differentially expressed; Cx43 was found predominantly in the less differentiated lower spinous layers, whereas Cx26 was found in terminally differentiating upper spinous and granular layers. In hyperplastic epidermis exhibiting an expansion of the differentiated upper layer, i.e., epidermis with a thickened granular layer or in which the granular layer was replaced with keratinocytes exhibiting tricholemmal differentiation, expression of Cx43 and Cx26 remained segregated in the lower and upper spinous layers, respectively. However, in papillomas, Cx26 was localized in the lower but not upper spinous layer, an expression pattern identical to that of Cx43. In addition, the overall expression levels of both Cx43 and Cx26 appeared to be greatly elevated in the papillomas. It is interesting that such marked alteration in the pattern of Cx26 expression occurred within the context of hyperplastic changes histologically identical to those seen in the nonpapillomous hyperplasias. Interestingly, in neoplastic skin lesions containing a squamous cell carcinoma, Cx43 and Cx26 expression was extinguished. Moreover, expression of Cx43 was also significantly reduced in adjacent apparently nonneoplastic tissues. Overall, these observations show that perturbations in gap-junction gene expression are associated with skin hyperplasia and neoplasia. Such findings suggest a possible role for gap junctions in the malignant conversion of mouse epidermal cells.
The objective of the present study was to examine the effects of the antiprogesterone RU486 on the expression levels of multiple gap junction (GJ) gene products and, in detail, on alpha 1 (connexin43 [Cx43]), in various regions of the rat implantation chamber during experimentally induced preterm labor at mid/late stages of gestation. Vaginal bleeding, but not expulsion of concepti, was observed in a majority of animals 24 h after a single injection of RU486 at Day 15 of gestation, and it persisted until animals were killed 48 h later. The bleeding was completely suppressed by R 5020, a synthetic progesterone with a high affinity for the progesterone receptor (PR). Various components of the implantation chamber (uterus, mesometrial stroma, placenta) and ovaries were isolated 24 and 48 h postinjection and analyzed for alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26) connexin expression by immunohistochemistry and by Northern blots (alpha 1 only). Alpha 1 Connexin was present at high levels in the myometrium following the inhibition of progesterone action by RU486; accordingly, this effect was completely suppressed by R 5020. The blocking of the PR also had a dramatic effect on expression levels, size, and distribution of junctional plaques composed of beta 1 and beta 2 connexins in polarized luminal and glandular epithelium. The average size of junctional plaques was significantly reduced in the luminal epithelium after RU486 administration. This effect was inhibited in the presence of R 5020. At the RNA level, the alpha 1 transcript was markedly elevated in the uterus and in the ovaries 24 and 48 h after administration of RU486. An elevation was also observed in the mesometrial stroma, while no increase was detected in the placenta. The RU486-induced alpha 1 mRNA steady-state levels in various tissues were completely suppressed by R 5020. These results demonstrate the modulation of multiple connexins in various cell types of the implantation chamber upon blocking of PR action. The expression profile of the myometrial and epithelial GJs was similar to that previously observed in the estrogen-treated rat uterus.
Enriched subcellular fractions of double membrane gap junctions (plaques) from rat livers were treated under reducing conditions with high salt and non-ionic detergent concentrations at high pH to obtain a preparation of structural 80-90 A complexes of oligomers (connexons). The isolated oligomers were chromatographically purified, and subsequently characterized immunologically, morphologically by electron microscopy, hydrodynamically by gel filtration and ultracentrifugation, spectroscopically by circular dichroism, and chemically via cross-linking studies. The physical characteristics of these isolated gap junction complexes were compared to those of native membrane-bound gap junctions in rat liver. These analyses indicate that the isolated complex (connexon) principally contains a hexameric arrangement of gap junction protein to form a single membrane hemi-channel.
A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compounds compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognized class of biological signaling molecules.
The expression and localization of three different connexins (alpha 1, Cx43; beta 1, Cx32; and beta 2, Cx26) were analyzed in human, mouse, and rat mammary glands by PCR analysis of reverse-transcribed RNA (RT-PCR) and indirect immunohistochemistry. For the rodent mammary gland, the study included different physiological stages of development during nonpregnancy, pregnancy, lactation, and postweaning. RT-PCR amplification revealed a constitutive expression of RNA for alpha 1 connexin in all three species. In contrast, both beta 1 and beta 2 transcripts were expressed only during lactation in the rodent mammary gland. Specifically, immunohistochemistry showed that the expression of all three connexins was restricted to specific cell types, and it varied according to the physiological activity of the organ. In particular, alpha 1 antigen was detected only between myoepithelial cells, the contractile cells surrounding alveoli and ductal systems in the mammary gland, while beta 1 and beta 2 antigens were localized solely at the basolateral border of alveolar secretory cells. The level of beta 1 and beta 2 connexins was increased in the rodent mammary gland during lactation. No staining for either beta connexin was detected in human mammary gland or in that of nonpregnant, pregnant, or postweaning rodents. The inducible coexpression of beta 1 and beta 2 antigens in luminal cells of the lactating rodent suggests a possible role for these connexins in the coordination of the secretory epithelium.
The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions.
Gap junctional communication is important in many physiological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human beta 1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional beta 1 connexin was detected only in cells transfected with beta 1 connexin cDNA in the correct orientation (beta 1-BHK). Analysis of the cells by light microscopic immunocytochemistry indicated that beta 1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomerization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.
The effects of estrogen (E2) and progesterone (P) were examined on the expression levels of multiple gap junction (GJ) gene products (alpha 1 = Cx43, beta 1 = Cx32, beta 2 = Cx26) in the uterus and ovaries of immature rats by immunohistochemistry, electron microscopy and northern blot analysis. E2 induced the expression of alpha 1 connexin in the uterus (specifically in the myometrium and in endometrial stroma proximal to luminal epithelium) and ovaries. The E2-induced alpha 1 expression was completely suppressed by P in the uterus, but only partly in ovaries. Steroid hormones also modulated the quantity, size, and distribution of beta 1 and beta 2 containing junctional plaques along lateral cell borders in polarized luminal and glandular uterine epithelia. Small GJs were detected at basolateral regions in proliferative luminal epithelium following administration of E2. In contrast, large GJs were localized at subapical-lateral cell borders of the secretory epithelium following P-treatment. The co-administration of E2 + P had a synergistic effect on beta 1 and beta 2 expression in the luminal epithelium, but an inhibitory effect on beta 2 expression in glandular epithelium. Myometrial GJs were detected in freeze-fracture replicas as aggregates containing regularly arranged particles with particle free zones. In contrast, GJs in secretory epithelium contained particles which were arranged in a non-crystalline fashion. These GJs contained domains of mixed and segregated beta 1 and beta 2 antigens within a single plaque as revealed by laser scanning confocal microscopy analysis of immuno-double-labeled secretory epithelium. The demonstration of segregated antigens within a single GJ plaque indicates the possibility of multiple channel populations formed by homo-oligomeric connexons. These results suggest that different connexins can be differentially regulated by steroid hormones in different cell types, and that the same steroid hormone can have different effects on the same connexin in different cell types.
Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.
The developmental regulation of gap junctions was analyzed in the developing rat epidermis by immunohistochemical and ultrastructural methods. The molecular composition of gap junction plaques was examined by laser scanning confocal microscopy following immuno-double labeling with monoclonal and polyclonal antibodies specific for alpha 1 (Cx43) and beta 2 (Cx26) connexins, respectively. During early fetal development (embryonic period), gap junctions were identified as large junctional plaques consisting of alpha 1 and beta 2 connexins. Ultrastructurally, gap junctions were detected in the two-layered epidermis between the subapical borders of peridermal cells, at the periderm/basal layer interface, and between the basal cells. The first "switch" in the utilization of alpha 1 and beta 2 connexins was observed at the onset of epidermal stratification, when beta 2 expression was down-regulated in the periderm and in the upper part of the intermedium. Gap junctions were also detected ultrastructurally in all layers of the stratified, nondifferentiated epidermis at E16. Junctional sizes included small plaques (0.05 micron 2) in the periderm, medium-size plaques (1 micron 2) in the upper part of the intermediate layer, and very large plaques (25 microns 2) in the basal layer. The second "switch" in the utilization of gap junction components coincided with epidermal differentiation (> E18), when beta 2 was preferentially expressed in the differentiated granular and upper spinous layers. alpha 1 connexin was present in the less differentiated spinous layer and in the proliferating basal layer. Gap junctions were no longer detectable in the periderm following differentiation (keratinization) of the epidermis (E18-E20). An analysis of immuno-double-stained sections by laser scanning confocal microscopy revealed domains of potentially mixed and segregated antigens within large junction plaques. These results indicated that large gap junction plaques (> 1 micron in size) can contain segregated domains of connexons, which contain a single protein (homooligomer).
Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.
The gap junctional communication capacity of six human carcinoma-derived tumorigenic cell lines (pancreatic, pharyngeal and cervical), two murine carcinoma-derived tumorigenic cell lines (bladder-derived and Ehrlich ascites) and one monkey non-tumorigenic cell line (kidney epithelium) have been compared by the dye-transfer technique. All the tumorigenic cell lines were communication defective, while the non-tumorigenic cell line was not. Moreover, six of the eight tumorigenic cell lines expressed a gap junction transcript coding for the connexin 43 (alpha 1). Finally, gap junction plaques were not detected between tumorigenic cells by immunofluorescence staining. Consequently, these data suggest that communication defects in tumorigenic cells may result from either abnormally low levels of translation of junction mRNA or alterations in the assembly of junction protein into cell surface plaques, and not from failure to produce junction transcripts. Furthermore, since induction of alpha 1 expression has been associated with dedifferentiation processes, the presence of alpha 1 mRNA might be a characteristic property of certain tumorigenic cells that originate from cells that do not normally express alpha 1 mRNA.
OBJECTIVE: The purpose of this study was to determine if gap junctions are a necessary component of the human laboring uterus and if their presence in myometrium is a prerequisite for both term and preterm labor. STUDY DESIGN: We obtained 27 human myometrial samples at cesarean section or nongravid hysterectomy. Gap junction formation was analyzed in a blind fashion by freeze fracture and indirect immunofluorescence. Six samples were obtained from term patients with no labor, six from term patients in labor, six from preterm patients with no labor, six from patients in preterm labor, and three from nongravid hysterectomy specimens. RESULTS: Gap junction structures were identified in the human myometrium of patients in term and in preterm labor but not in the other patient samples. In addition, evidence was obtained for the expression of (alpha 1) gap junction ribonucleic acid and (alpha 1) gap junction protein in term samples of human myometrium. CONCLUSION: Gap junctions are a necessary component of the human myometrium during term and preterm labor. The formation of gap junctions may be a final common event for the development of labor, and inhibition of gap junction activity could be a novel approach for the treatment of preterm labor.
The mRNA and protein expression of alpha 1 (connexin 43), beta 1 (connexin 32), and beta 2 (connexin 26) gap junction genes were examined in the regenerating rat liver after 70% partial hepatectomy (PH). Expression of beta 1 and beta 2 steady-state mRNA levels changed minimally until 12 h after PH when both transcripts decreased to approximately 15% of baseline values. A similar decrease in assembled connexin levels was detected by immunoblot and indirect immunofluorescence at 18 h after PH. Both transcripts simultaneously increased between 24 and 42 h and again rapidly decreased by 48 h post-PH. beta 1 and beta 2 assembled gap junction protein expression increased at 48 h post-PH and rapidly decreased by 56 h. By 72 to 84 h post-PH, beta 1 and beta 2 mRNA and assembled protein expression returned to near baseline levels and were maintained. Interestingly, inhibition of protein synthesis with cycloheximide completely inhibited disappearance of the beta 2 transcript, in contrast to beta 1 mRNA which was unaffected. Nuclear run-on assays showed no change in transcriptional rates for either gene during the regenerative period. However, both beta 1 and beta 2 transcripts exhibited significantly decreased mRNA half-lives at 12 h post-PH (3.8 and 3.7 h, respectively) relative to those at 0 h (10.9 and 6.1 h, respectively). Surprisingly, although the transcriptional rate for alpha 1 was similar to that observed for beta 2, no alpha 1 transcripts were detectable by northern or RNase protection analysis. The results suggest that in the regenerating rat liver, beta 1 and beta 2 gap junction genes are not regulated at the transcriptional level. Rather, the cyclical modulation of their steady-state transcripts is regulated primarily by posttranscriptional events of which mRNA stability is at least one critical factor in the control process.