Biomedical subjects

N M Kumar

Publications and source records attributed to N M Kumar.

At least 37 records · Page 2Linked to original sources

PubMed1996

Induction of interleukin-1 and interleukin-8 mRNAs and proteins by TGF beta 1 in rat lung alveolar epithelial cells.

The transforming growth factor-beta (TGF-beta) has been shown to increase in lung injury and in fibrotic states of the lung. In the current study, we sought to investigate whether TGF beta 1 induced the expression of IL-1 alpha and IL-8 in rat alveolar epithelial cells. We evaluated TGF beta 1, IL-1 alpha, and IL-8 expression by immunofluorescence in silica-injured and saline-treated control rat lungs. Antibodies to IL-1 alpha, IL-8, and TGF beta 1 showed intense staining in silica-injured lungs as compared to saline-instilled lungs. Primary isolated type II cells from silica-injured lungs showed increased expression of IL-1 alpha as compared to saline-instilled lungs. To evaluate the effects of TGF beta 1, we treated an immortalized rat type II cell-derived cell line (LM5) with 100 pg/ml of TGF beta 1 in serum-free medium for 0-24 hours and analyzed the expression of IL-1 alpha and IL-8 mRNAs and proteins using semiquantitative RT-PCR, Northern blot analysis, Western blot analysis, and immunohistochemistry. Densitometric analysis of Northern blots showed modest constitutive expression of IL-1 alpha gene in untreated control LM5 cells. TGF beta 1 treatment resulted in an increase in IL-1 alpha mRNA, that reached maximum levels (4-fold) by 2 hours and remained elevated for 4-16 hours, with a subsequent decline by 24 hours. Similarly, Northern blot and RT-PCR analysis demonstrated that TGF beta 1 treatment resulted in maximum induction of IL-8 mRNA (6- 8.5-fold) within 1-4 hours. The levels remained elevated for up to 24 hours afterwards. Western blot analysis results further confirmed the expression of both IL-1 alpha and IL-8 proteins by LM5 cells. TGF beta 1 treatment resulted in increased expression of both IL-1 alpha and IL-8 proteins. Immunofluorescence studies demonstrated increased staining of IL-1 alpha by TGF beta 1 as compared to untreated cells. These results suggest that TGF beta 1 may regulate IL-1 alpha and IL-8 expression in alveolar epithelial cells and contribute to polymorphonuclear leukocyte recruitment and lung injury in clinical states with increased TGF beta 1.

Animals↗

PubMed1996

TNF alpha inhibits Schwann cell proliferation, connexin46 expression, and gap junctional communication.

Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-alpha or the mitogen neu differentiation factor (NDF)-beta. TNF alpha inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNF alpha also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both Po and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDF beta initially had little effect on cell division although it reduced junctional coupling within 8 h. However, by 48 h, NDF beta stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU+) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNF alpha may be a signal which terminates proliferation as well as junctional communication.

Animals↗

PubMed1996

Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization.

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.

Ethanol↗

PubMed1996

Distribution of alveolar type II cells in neonatal and adult rat lung revealed by RT-PCR in situ.

Type II pneumocytes in newborn lungs are more uniformly distributed, whereas in adult lungs they are located at alveolar corners. We used morphometry and reverse transcription-polymerase chain reaction in situ hybridization of surfactant protein C mRNA to determine the patterns of type II cell distribution in random lung sections from Sprague-Dawley rats at various neonatal stages and adults. There was a progressive increase in the percentage of type II cells at alveolar corners from 30% at 1 day to 51, 62, 78, and 81% at 3, 5, and 7 days old and adult rats, respectively. There were statistically significant differences (P < 0.001) in the localization of type II cells from the nearest alveolar corner in the 1-day-old compared with 7-day-old and adult rat lungs. These results show that rat type II cells localize to the alveolar corners within the first 7 days postnatally and provide a system for study of factors that regulate alveolar epithelial cell distribution.

Aging↗

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

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↗

PubMed1995

Differential modulation of integrin receptors and extracellular matrix laminin by transforming growth factor-beta 1 in rat alveolar epithelial cells.

The transforming growth factors-beta (TGFs-beta) family of genes plays important roles in cell growth and differentiation in many cell types. TGF beta modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGF beta is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGF beta may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGF beta on modulation of laminin and its integrin receptors alpha 6 beta 1 and alpha 3 beta 1 in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGF beta 1 (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin beta 2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGF beta 1 induced dose-dependent increases in alpha 6 and beta 1 mRNA levels. TGF beta 1 also increased the expression of laminin beta 2 chain mRNA at 12-16 h poststimulation. In contrast, TGF beta 1 decreased alpha 3 mRNA expression. Immunoprecipitation studies of TGF beta 1-treated cells showed increased surface expression of both alpha 6 and beta 1 protein while surface expression of the alpha 3 integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGF beta 1 may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.

Animals↗

PubMed1995

Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers.

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.

Animals↗

PubMed1995

Synthesis and assembly of human beta 1 gap junctions in BHK cells by DNA transfection with the human beta 1 cDNA.

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.

Animals↗

PubMed1994

Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation.

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.

Animals↗

PubMed1994

Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins.

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.

Amino Acid Sequence↗

PubMed1994

Non-communicating human and murine carcinoma cells produce alpha 1 gap junction mRNA.

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.

Animals↗

PubMed1993

Cloning and expression of a major rat lens membrane protein, MP20.

The lens contains a major membrane protein with an apparent molecular weight of 18 kDa and which has been referred to as MP18 [Louis et al. (1989). J. Biol. Chem. 264, 19967-73]. We have cloned a rat MP20 cDNA that appears to be identical to this previously described protein based on sequence homology. The predicted protein sequence has characteristics typical of an integral membrane protein and a molecular mass of 19637. The transcripts for MP20 appear to be lens specific as indicated by RNA analysis; Southern blot analysis indicates that MP20 is coded for by a single gene with possibly a single intron in its coding sequence. The expression of transcripts for several lens membrane proteins (MP20, MP26 and alpha 3-gap junctions) was compared in 1.5 month bovine fetal and 4-6 month post-natal calf lenses. The transcript levels for these proteins were more abundant in fetal lenses than in calf lenses indicating a possible developmental regulation of the transcripts for these three lens-specific membrane proteins. The MP20 cDNA was expressed in a heterologous mammalian cell culture system in which the majority of the protein was integrated into membranous structures localized near the nucleus; there was little incorporation of MP20 into the cell plasma membrane as detected by immunofluorescence. This system should prove useful for both the isolation and purification of MP20, and will enable study of its properties under defined conditions.

Amino Acid Sequence↗

PubMed1993

Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro.

This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.

Alkaline Phosphatase↗

PubMed1993

Differential regulation of multiple gap junction transcripts and proteins during rat liver regeneration.

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.

Animals↗

PubMed1993

The developmental expression of three mammalian lens fiber cell membrane proteins.

PURPOSE: The expression of the lens fiber cell integral membrane proteins MP20, the alpha 3 gap junction protein (connexin 46), and MP26 (MIP) was determined both during development of the bovine fetal lens and in different animal species. METHODS: The relative levels of the mRNA species coding for these proteins was determined by Northern blotting; this was correlated with the amounts of these proteins as determined by Western immunoblotting. RESULTS: RNA extracted from fetal calf lenses at 3-4, 5-6, and 7-8 mo gestation, and from postnatal calf lenses, showed a progressive decrease of the mRNA species coding for MP20 (20-fold), alpha 3 gap junction protein (22-fold), and MP26 (12-fold) with development. This compared with a 6-fold decrease in total RNA and a 15-fold decrease in poly-A(+) RNA over the same period. In contrast to these significant changes in the levels of mRNA, there were no significant changes in the concentrations of these proteins over the same period. Whereas the mRNA coding for each of these proteins were concentrated in the lens cortical regions. MP20 and MP26 protein concentrations in cortical and nuclear regions were very similar. Both protein and mRNA coding for MP20, alpha 3 gap junction, and MP26 were detected in calf, sheep, pig, and rat lenses. CONCLUSIONS: These results indicate that the expression of the mRNA coding for the lens fiber cell integral membrane proteins MP20, alpha 3 gap junction, and MP26 is regulated and may be coordinated. That the decrease in mRNA concentrations is not correlated with changes in the concentrations of these lens membrane proteins indicates their unique stability.

Animals↗

PubMed1992

Molecular biology and genetics of gap junction channels.

Gap junctional communication between cells provides a mechanism for the movement of molecular information between cells via the unit of gap junction structure and function, the gap junctional channel. In the past five years, there has been rapid progress in identifying and characterizing a multigene family that is responsible for producing the gap junction polypeptides that are responsible for generating gap junctional channel oligomers between cells. The products of these genes have been referred to as connexins, and the multigene family can be categorized into two classes at present, the alpha class and the beta class. Members of these two classes can be distinguished on the basis of their primary sequence and overall predicted topological organization. The gap junction genes map to different chromosomes in both mice and humans, and these genes are utilized on a cell specific basis. Furthermore, these genes can be developmentally regulated, and multiple genes can be co-expressed simultaneously by the same cell type. Efforts to understand the precise structure-function relationship of the products of these different genes is now being approached by utilizing various expression systems. Criteria that can be used as a basis for determining membership in the multigene family is presented and discussed, as well as the rationale for using a nomenclature system for the gap junction multigene family that is based on genetic and structural relationships rather than the molecular size of the deduced protein products.

Amino Acid Sequence↗

PubMed1992

Differential expression of gap junction mRNAs and proteins in the developing murine kidney and in experimentally induced nephric mesenchymes.

The expression of three gap junction (GJ) proteins, alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26), and their transcripts were examined during the ontogeny of the mouse and rat kidney. These proteins were expressed in two non-overlapping patterns. The alpha 1 GJ protein was first observed in mesenchymal cells in the 12-day mouse kidney. By day 14 and thereafter, the alpha 1 protein was detected in the transient S-shaped bodies, but not in the podocytes of the maturing glomeruli. After birth the antigen was retained in a small subset of secretory tubules. The beta 1 and beta 2 GJ proteins were similar in their developmental patterns. They were first detected in a small subset of secretory tubules in the subcortical zone of day 17 embryos. These tubules were identified by immunohistochemical markers to be proximal. At birth, practically all proximal tubules expressed the two antigens. This analysis of GJ proteins was consistent with the results of S1 nuclease protection assays showing that, while the alpha 1 mRNA appeared early during kidney development and declined around birth, the two beta mRNAs appeared later and became intensified during the last days of intrauterine development. In experimentally induced metanephric mesenchymes, a transient expression of the alpha 1 GJ protein was seen during the segregation of the tubular anlagen. beta 1 and beta 2 GJ proteins were not detected in such induced mesenchymes cultivated up to 7 days. These observations provide evidence for the cell-specific utilization of different GJ genes during different stages of kidney organogenesis. The alpha 1 gene is activated during the early segregation of the secretory tubule and might contribute to its compartmentalization, while the beta 1 and beta 2 gene products are not detected until advanced stages of development. The latter gene products might be correlated with the physiological activity of the proximal tubules in vivo, as they are not expressed in experimentally induced tubules detectable with markers for proximal tubules.

Animals↗