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N B Gilula

Publications and source records attributed to N B Gilula.

At least 19 recordsLinked to original sources
PubMed1998

Gap junctional coupling in lenses lacking alpha3 connexin.

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing alpha3 (Cx46) and alpha8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous alpha3 knockout (-/-), heterozygous (+/-), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that alpha8 remained at similar levels in the three classes of lenses, whereas alpha3 was approximately 50% of the normal level in the +/- lenses, and it was absent from the -/- lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In -/- lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (approximately -65 mV). The DF remained coupled, but the conductance was reduced to 30-35% of normal. However, the gap junctions in the DF of alpha3 -/- lenses remained sensitive to pH. We conclude that alpha3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

Animals↗
PubMed1998

Gap junction-mediated cell-cell communication modulates mouse neural crest migration.

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and alpha1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the alpha1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium- an elevation in the CMV43 mice vs. a reduction in the alpha1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest- derived tissues. Overall, these findings suggest that gap junction communication mediated by alpha1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of alpha1 connexin function.

Animals↗
PubMed1998

Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide.

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16-24 carbons of which 16-18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Delta9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.

Amidohydrolases↗
PubMed1998

Connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access.

We reported previously (Falk, M. M., Kumar, N. M., and Gilula, N. B. (1994) J. Cell Biol. 127, 343-355) that the membrane integration of polytopic connexin polypeptides can be accompanied by an inappropriate cleavage that generates amino-terminal truncated connexins. While this cleavage was not detected in vivo, translation in standard cell-free translation/translocation systems resulted in the complete cleavage of all newly integrated connexins. Partial cleavage occurred in heterologous expression systems that correlated with the expression level. Here we report that the transmembrane topology of connexins generated in microsomal membranes was identical to the topology of functional connexins in plasma membranes. Characterization of the cleavage site and reaction showed that the connexins were processed by signal peptidase immediately downstream of their first transmembrane domain in a reaction similar to the removal of signal peptides from pre-proteins. Increasing the length and hydrophobic character of the signal anchor sequence of connexins completely prevented the aberrant cleavage. This result indicates that their signal anchor sequence was falsely recognized and positioned as a cleavable signal peptide within the endoplasmic reticulum translocon, and that this mispositioning enabled signal peptidase to access the cleavage sites. The results provide direct evidence for the involvement of unknown cellular factors in the membrane integration process of connexins.

Amino Acid Sequence↗
PubMed1998

Modulation of gap junction expression during transient hyperplasia of rat epidermis.

Retinoids and phorbol esters have profound effects on proliferation and differentiation of epidermal keratinocytes when applied topically on rodent skin. Since both agents also modulate gap junction (GJ)-mediated cell-cell communication, we have examined the effects of all-trans retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of alpha1 (Cx43) and beta2 (Cx26) connexins, the two major gap junction gene products in mature rat epidermis. In fully differentiated, mature epidermis, alpha1 is expressed in the lower, less differentiated portion, while beta2 is localized in upper, more differentiated layers. Dorsal skin of 21-day old rats was treated topically with a single dose of RA, TPA or vehicle alone and used for histological and molecular analyses at different time points. Keratinocytes in interfollicular epidermis were examined for proliferation and differentiation using specific antibodies for keratins (K10, K14) and proliferating cell nuclear antigen (PCNA). An increase in epidermal thickness was noticed within 4 hours after the application of RA or TPA. This increase, however, appeared to be primarily due to hypertrophy, since no substantial changes were observed in the proliferative index of epidermal keratinocytes. PCNA immunoreactivity significantly increased after 8 hours treatment of RA or TPA, suggesting a hyperproliferative growth response. Epidermal hyperplasia was confirmed by monitoring the expression patterns of K10 and K14 in RA- or TPA-treated skin. RA-induced hyperplasia lasted longer as compared to TPA induction. Changes in keratin phenotypes were paralleled by an increase in alpha1 and beta2 connexin expression as well as their colocalization in same epidermal layers. Differences in hyperplastic growth response kinetics were also confirmed at the connexin level, with beta2 antigen sustained for longer and at higher levels in suprabasal layers of RA-treated skin. Overall, this type of connexin expression resembled that observed in the non-differentiated rat epidermis during embryonic development. An increase in alpha1 and beta2 connexin abundance was also observed at the protein and RNA levels. At 96 hours after RA or TPA treatment, expression of both connexins was similar to that of the control epidermis. Taken together, these findings suggest that a higher level of GJ-mediated cell-cell communication, is required for the maintenance of homeostasis during periods of rapid epidermal growth and differentiation.

Animals↗
PubMed1997

The sleep-inducing lipid oleamide deconvolutes gap junction communication and calcium wave transmission in glial cells.

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction-mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18beta-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.

Animals↗
PubMed1997

Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme activity that degrades neuromodulatory fatty acid amides, including oleamide and anandamide. A single 2.5-kb FAAH mRNA is distributed throughout the rat CNS and accumulates progressively between embryonic day 14 and postnatal day 10, remains high until postnatal day 30, then decreases into adulthood. FAAH enzymatic activity, as measured in dissected brain regions, was well correlated with the distribution of its messenger RNA. In situ hybridization revealed profound distribution of FAAH mRNA in neuronal cells throughout the CNS. The most prominent signals were detected in the neocortex, hippocampal formation, amygdala, and cerebellum. The FAAH distribution in the CNS suggests that degradation of neuromodulatory fatty acid amides at their sites of action influences their effects on sleep, euphoria, and analgesia.

Amidohydrolases↗
PubMed1997

Disruption of alpha3 connexin gene leads to proteolysis and cataractogenesis in mice.

Gap junction channels formed by alpha3 (Cx46) and alpha8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of alpha3 connexin in vivo, the alpha3 connexin gene was disrupted in mice. Although the absence of alpha3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted alpha3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell-cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.

Aging↗
PubMed1997

Cell-free synthesis and assembly of connexins into functional gap junction membrane channels.

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.

Cell-Free System↗
PubMed1997

Projection structure of a gap junction membrane channel at 7 A resolution.

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.

Animals↗
PubMed1996

Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.

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.

Amides↗
PubMed1996

On signal sequence polymorphisms and diseases of distribution.

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.

Base Sequence↗
PubMed1996

Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia.

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.

Animals↗
PubMed1996

Gap junction regulation during preterm labor in the rat: multiple effects of the antiprogesterone RU486.

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.

Amino Acid Sequence↗
PubMed1995

Physical characterization of gap junction membrane connexons (hemi-channels) isolated from rat liver.

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.

Animals↗
PubMed1995

Chemical characterization of a family of brain lipids that induce sleep.

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.

Animals↗
PubMed1995

Analysis of multiple gap junction gene products in the rodent and human mammary gland.

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.

Adult↗