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PubMed2005

Differential localization of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 and CD47 and its molecular mechanisms in cultured hippocampal neurons.

Polarized localization of membrane proteins to axons or dendrites is important for a variety of neuronal functions, including neurite outgrowth and synaptogenesis during neural development. Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) and its ligand cluster of differentiation 47 (CD47), both of which are members of the Ig superfamily of proteins, are thought to constitute an intercellular communication system in the CNS, although the physiological functions of this CD47-SHPS-1 system remain unknown. To provide insight into these functions, we have now examined the localization of SHPS-1 and CD47 in cultured hippocampal neurons. Endogenous SHPS-1 was detected at the surface of both axons and dendrites, whereas endogenous CD47 was localized predominantly to the surface of dendrites. Forced expression of these two proteins confirmed their distinct localizations. The extracellular regions of SHPS-1 and CD47 were responsible, at least in part, for their axonal and dendritic localizations, respectively; however, the axonal localization of SHPS-1 was not mediated by any one of the three Ig domains in its extracellular region. Overexpression of SHPS-1 and CD47 in distinct neurons resulted in marked accumulation of these proteins at sites of contact between SHPS-1-expressing axons and CD47-expressing dendrites. Such contact sites exhibited an enlarged structure but did not contain the synaptic marker protein vesicle-associated membrane protein-2. These results suggest that differential localization of SHPS-1 and CD47 at axons and dendrites generates a directional intercellular communication system that potentially contributes to regulation of synaptogenesis and the formation of neural networks.

Animals↗
PubMed1990

An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily.

The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster. In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation. Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted. Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function. Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity. Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser. A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles. Ubiquitous expression of sgg transcripts was found during embryonic stages. A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.

Amino Acid Sequence↗
PubMed2002

Altered pattern of vascular connexin expression in atherosclerotic plaques.

Paracrine cell-to-cell interactions are crucial events during atherogenesis. However, little is known about the role of direct intercellular communication via gap junctions during this process. We have investigated the expression pattern of 3 vascular gap junction proteins (connexins) in mouse and human atherosclerotic plaques. Low density lipoprotein receptor-deficient mice were fed a high-fat diet for 0, 6, 10, or 14 weeks to induce different stages of atherosclerosis. Connexin37 (Cx37) and Cx40 were detected in the endothelium, and Cx43 was detected in the media of nondiseased aortas. In early atheromas, endothelial and medial connexin expression remained unchanged, and "islets" of Cx43 in smooth muscle cells and Cx37 in macrophages were observed in the neointima. In advanced atheromas, Cx37 was detected in medial smooth muscle cells and in macrophages in the lipid core but not in the endothelium covering the plaques. Cx40 could also no longer be detected in the endothelium covering the plaques. Cx43, on the other hand, was detected in the endothelium covering the shoulder of the plaques and also sparsely in neointimal smooth muscle cells. Similar results were obtained for human carotid arteries. In conclusion, vascular connexins are differentially expressed by atheroma-associated cells within lesions. These observations suggest a role for gap junctional intercellular communication during atherogenesis.

Animals↗
PubMed2002

Transient decrease in gap junction expression during compensatory renal growth in mice.

OBJECTIVES: To examine gap junction expression during compensatory renal growth. Compensatory renal growth is a normal response to the removal of renal cells. Gap junctional intercellular communication plays important roles in homeostatic regulation, differentiation, and synchronization of cellular electronic and metabolic functions. METHODS: The right ureters of 5-week-old ddy mice were dorsally incised and unilaterally obstructed, and the animals were killed at varying intervals. The control group underwent a sham operation. Freeze-fractured kidney tissue samples were studied using electron microscopy. The amount of proliferative cell nuclear antigen-positive cells and connexin 43 (Cx43) protein was studied by immunohistochemistry and Western blotting, respectively. RESULTS: Proliferative cell nuclear antigen-positive cells in the renal tubules increased on days 1 and 2 after surgery that obstructed the ureter, and decreased to normal levels by day 14. The number of gap junctions significantly decreased on days 1 and 2, and then gradually increased to normal levels from days 3 to 14 after surgery. The amount of Cx43 protein in the renal tubules decreased until day 2 and recovered to the same level as that of the control by day 14 after surgery. Significantly, a hyperphosphorylated band of Cx43 in the control kidney was not detected in the operated kidney. CONCLUSIONS: These data suggest that the gap junctional intercellular communication of renal tubular cells during compensatory renal growth after unilateral ureteral obstruction could be temporarily reduced concomitant with a decrease of the expression of a phosphorylated Cx43 protein in renal cortical tubular cells after unilateral ureteral obstruction of the contralateral kidney.

Adaptation, Physiological↗
PubMed1997

Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells.

Mechanical stimulation was used to initiate Ca2+ waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca2+ to adjacent cells and to assess alternative Ca2+ signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca2+ wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca2+ wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18beta-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca2+ wave. In contrast, treatment with suramin, a P2-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca2+ wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca2+ wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.

Animals↗
PubMed1991

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

Animals↗
PubMed1995

Reappearance and long-term maintenance of connexin32 in proliferated adult rat hepatocytes: use of serum-free L-15 medium supplemented with EGF and DMSO.

Intercellular communication, especially gap junctional communication, is thought to be one of the highly differentiated functions of hepatocytes. In primary cultures of rat hepatocytes, it has been considered that the maintenance and the reinduction of differentiated functions is very difficult. In the present study, we succeeded in inducing the gap junctional protein connexin32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor (EGF) and dimethylsulfoxide (DMSO). When the hepatocytes were cultured in L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2:95% air incubator, the cells proliferated. Fluorescence immunocytochemistry showed spots immunoreactive to Cx32 on the cell membranes between adjacent cells until day 3, but only a few Cx32-positive spots were found after day 4. Western and northern blot analyses also showed that the amounts of both the protein and mRNA of Cx32 in the cells decreased with time in culture. However, when the cells were treated with 2% DMSO from day 4, the immunoreactive spots reappeared on the cell membranes from day 6 and both their number and intensity gradually increased. The reappearance of Cx32 was accompanied by increases in both the protein and mRNA of Cx32. Furthermore, the expression of Cx32 was well maintained, together with extensive gap junctional intercellular communication, for more than 4 weeks. In addition, ultrastructurally, many gap junctional structures were observed between the hepatocytes, and the antibodies to Cx32 were shown to bind to those structures. This culture system may be useful for studies of the reconstruction of the gap junctional structure, the intracellular pathways of the proteins, and the regulation of synthesis and processing in differentiated hepatocytes.

Animals↗
PubMed2005

Involvement of the integrin-linked kinase pathway in hexachlorobenzene-induced gender-specific rat hepatocarcinogenesis.

Overexpression of the integrin-linked kinase (ILK) pathway disrupts cell-cell interactions, an epigenetic event leading to epithelial cell transformation. Female rats exposed to hexachlorobenzene (HCB) for 5 consecutive days and sampled 45 days later show a decrease in liver gap junctional intercellular communication. We hypothesized that HCB also alters E-cadherin expression and that this alteration is mediated by the ILK pathway. Hepatic ILK levels were markedly increased in HCB-treated female rats. Cytoplasmic/membrane levels of protein kinase B (Akt), a target of ILK, and its phosphorylated active form were decreased in treated female rats. Flow cytometric analysis showed a concomitant increase in nuclear Akt levels. Both ILK and Akt can phosphorylate glycogen synthetase kinase-3beta (GSK3beta), rendering it inactive. Phosphorylated-GSK3beta levels were higher in treated females and resulted in a twofold decrease in the activity of GSK3beta. The inactivation of GSK3beta in HCB-treated female rats resulted in the nuclear translocation of beta-catenin, as demonstrated by both immunocytochemistry and flow cytometric analyses. Western blot analysis showed an 84% decrease in E-cadherin levels in HCB-treated rats as compared to controls, and this decrease was not mediated by Snail activation. Mimicking the activation of ILK with specific GSK3beta inhibitors resulted in downregulation of E-cadherin levels but had no effect on Cx32 expression in the MH(1)C(1) cells. Overall, these results indicate that hepatic E-cadherin is downregulated as a result of an overexpression of the ILK pathway. The concomitant HCB-induced downregulation of intercellular communication does not occur as a result of either E-cadherin downregulation or GSK3beta inactivation.

Administration, Oral↗
PubMed2000

Impaired conduction of vasodilation along arterioles in connexin40-deficient mice.

Connexins have been hypothesized to play an important role in intercellular communication within the vascular wall and may provide a mechanistic explanation for conduction of vasomotor responses. To test this hypothesis, we studied the transmission of vasomotor responses in the intact skeletal muscle microcirculation of connexin40-deficient mice (Cx40(-/-)). Arterioles were locally stimulated with hyperpolarizing dilators (acetylcholine [ACh] as well as bradykinin [Bk]) or depolarizing K(+) solution, and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.32 mm upstream. Arterial pressure was elevated 25% in Cx40(-/-) mice (94+/-5 versus 75+/-4 mm Hg). Vessels selected for study had equivalent basal diameter and vasomotor tone in both genotypes of mice. Vasomotion was present in small arterioles of both genotypes, but its intensity was exaggerated in Cx40(-/-) mice. ACh and Bk induced dilation (33% and 53%, respectively, of maximal response) at the site of application that was of similar magnitude in both genotypes. These dilations were observed to spread upstream within <1 second without significant attenuation in Cx40(+/+) mice. However, spreading was severely attenuated in Cx40(-/-) animals (11+/-4% versus 35+/-7% with ACh and 38+/-5% versus 60+/-7% with Bk in Cx40(-/-) and Cx40(+/+), respectively; P<0.05). In contrast, conducted vasoconstrictions, induced by K(+) solution decreased equally with distance in both genotypes. These results support a significant role for Cx40 in vascular intercellular communication. Our observations indicate that Cx40 is required for normal transmission of endothelium-dependent vasodilator responses and may underlie altered vasomotion patterns.

Acetylcholine↗
PubMed2000

Immunolocalization of the gap junction protein Connexin43 in the interstitial cells of Cajal in the normal and Hirschsprung's disease bowel.

BACKGROUND: Interstitial cells of Cajal (ICC) are pacemaker cells between gastrointestinal smooth muscles; they generate spontaneous slow waves of the smooth muscle layers and mediate neurotransmission. The cellular network of ICC is connected by Gap junctions to each other and to the smooth muscle cells. Although there have been several studies reporting distribution of ICC in the normal bowel and pathological conditions such as Hirschsprung's disease, there is little information on the crucial role of Gap junctions in the intercellular communication in the gut musculature. The aim of this study was to investigate the immunolocalization of the Gap junction protein Connexin43 in the normal and Hirschsprung's disease (HD) bowel using whole-mount preparation technique and confocal laser scanning microscopy. METHODS: Full-thickness bowel specimens were collected at pull-through operation from 8 patients diagnosed as having HD. Normal control large bowel specimens were collected from 12 patients during bladder augmentation operation. Whole-mount preparation was performed on all specimens and double immunostaining was carried out using anti c-kit and antiConnexin43 antibodies. The immunolocalization was detected with the help of confocal laser scanning microscopy. RESULTS: Connexin43 immunoreactivity appeared in and between the c-kit-positive cells and along the smooth muscle fibers of the normal bowel and ganglionic part of HD bowel. In the aganglionic part of HD bowel there was no expression of Connexin43. In the transitional zone of HD the Connexin43 staining was weak and colocalized only in the processes of the c-kit-positive Cajal cells. CONCLUSIONS: Results of this study show for the first time that Gap junctional protein Connexin43 is present in the ICCs, which form a 3-dimensional network in the normal bowel wall. The lack of expression of Connexin43 in the aganglionic bowel and reduced expression in the transitional zone of HD suggest that the impaired intercellular communication between ICCs and smooth muscle cells may partly be responsible for the motility dysfunction in HD.

Child↗
PubMed2005

Developmental regulation of the direct interaction between the intracellular loop of connexin 45.6 and the C terminus of major intrinsic protein (aquaporin-0).

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K(D(app)) of 7.5 and 10.3 microm, respectively. The K(D(app)) for the full-length loop region is 7.7 microm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.

Animals↗
PubMed2005

Differential expression and localisation of connexin-37 and connexin-43 in follicles of different stages in the 4-week-old mouse ovary.

Mammalian female fertility is critically dependent on timely coordinated intercellular communication between follicle cells. Gap junctions are small, membrane-bound, aqueous channels that couple adjacent cells ionically and gap junction proteins, connexin-37 (Cx37) and connexin-43 (Cx43) both participate in sustaining proper growth and maturation of the oocyte. The purpose of the present study was to localise Cx37 and Cx43 in sections of ovarian mouse follicles of different developmental stage, and compare their relative expression using immunofluorescence and confocal microscopy. Cx37 exclusively localised to oocytes of all follicle stages, and the estimated number of Cx37 gap junctions per mum oocyte circumference in follicles of stage 2 to stage 6 increased in parallel to oocyte and follicle growth, reaching a maximum in the pre-antral stage. Acetylated alpha-tubulin and Cx37 double labelling revealed that the majority of Cx37 gap junctions, irrespective of follicle stage, were located on the outer surface of the oocyte cytoskeleton. Cx43 was detected in granulosa cells of all follicle stages, beginning with distinct spots in resting primordial follicles. The estimated number of Cx43 gap junctions per granulosa cell increased significantly with follicle development, in particular in the transition from pre-antral to the antral stage. A subpopulation of Cx43 in the outer part of the zona pellucida was identified and it is suggested these Cx43 gap junctions connect cumulus trans-zonal projections. Together, these data suggests specific roles for Cx37 and Cx43 in folliculogenesis and offers new insights in patterns of intercellular communication of the mouse follicle.

Animals↗
PubMed1999

Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes.

Nonparenchymal cells, particularly Kupffer cells, might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. This intercellular communication via the exchange of soluble factors was investigated in primary rat Kupffer cells and hepatocytes. Freshly isolated rat Kupffer cells were seeded onto cell culture inserts and cocultured with 5 day old serum-free rat hepatocyte monolayer cultures at a ratio of 1:1 for 2 days. Hepatocyte cultures, Kupffer cell cultures or cocultures were treated with 0.1 ng/ml-10 microg/ml lipopolysaccharide (LPS). Within this concentration range, no significant toxicity was observed in either cell type. In LPS-exposed cocultures, tumor necrosis factor alpha (TNFalpha) levels rose up to 5 ng/ml within 5 h; nitric oxide (NO) levels increased up to 70 microM within 48 h of treatment, both in a dose-dependent fashion. The release of negative (albumin) and positive (alpha1-acid-glycoprotein) acute phase proteins from the hepatocytes was strongly down- and up-regulated, respectively. The simultaneous treatment of the cocultures with phenobarbital and LPS (10 ng/ml) or 3-methylcholanthrene and LPS (10 ng/ml) resulted in a strong down-regulation (85%) of the phenobarbital-induced cytochrome P450 (CYP) isoform CYP2B1 in the hepatocytes whereas the 3-methylcholanthrene-induced isoform CYP1A1 was only weakly affected (15%). This specific down-regulation of CYP2B1 was mediated exclusively by TNFalpha, released from the Kupffer cells. It was not linked with NO release from or inducible NO synthase activity in the hepatocytes. The TNFalpha release was not affected by the two xenobiotics. Acetaminophen tested in these cocultures showed no direct interaction with the Kupffer cells. The use of liver cell cocultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.

Acetaminophen↗
PubMed2000

Human hemangiosarcomas have a common polymorphism but no mutations in the connexin37 gene.

Gap junctional intercellular communication is often impaired in cancers, and the genes which encode the connexin gap junction proteins are considered to be tumor-suppressor genes. In this study, we analyzed the presence of mutations in the connexin 37 (Cx37) gene in 22 human hepatic angiosarcomas, 6 and 4 of which were associated with exposure to vinyl chloride and Thorotrast, respectively. The other 12 samples were from patients with no history of exposure to these 2 agents. In 9 samples, a proline (ACC) to serine (ACT) amino acid change in codon 319 was detected. However, DNA from non-tumorigenic tissue of the same patients also showed this amino acid change, suggesting that this is a polymorphism rather than a mutation. Subsequent analysis of 84 DNA samples from normal donors revealed the frequencies of Pro/Pro, Pro/Ser and Ser/Ser alleles to be 65.5%, 23.8% and 10.7%, respectively, while among the group of angiosarcoma patients the corresponding figures were 59.1%, 31.8% and 9. 1%, respectively. Thus, there was no correlation between the polymorphism at codon 319 and hepatic angiosarcoma occurrence. However, among the 6 cases of vinyl chloride-associated angiosarcoma, the percentages of the polymorphic alleles were 33.3%, 66.7% and 0%, respectively. While the number of samples was too small to allow us to conclude that the Ser319 allele in Cx37 predisposes to this rare type of human cancer, it may be noted that codon 319 is located at the cytoplasmic tail of Cx37, where most regulatory sequences reside, and that it could be a site of phosphorylation for some protein kinases, which may in turn affect the function of Cx37, including intercellular communication.

Carcinogens↗
PubMed2000

Risk assessment for possible carcinogens: a critical look.

Our overall understanding of mechanisms of toxicology in relation to human disease, with prevention of disease as a major objective, depends in part on the development of an adequate number of ways to assess risks, both short term and long term. Despite the cost, the long duration of the test, and some pitfalls, the long-term animal tests remain, to date, the only reliable assay for possible carcinogens. Recent work has concentrated increasingly on the development of short-term tests to replace the long-term tests. Such a development would be most welcome from several points of view. To date, a variety of approaches have been or are being used. These include (1) activation to an alkylating agent with DNA as the most important target, generating possible mutations in DNA and DNA damage with or without repair, (2) induction of cell proliferation, at least a few cycles, with DNA synthesis as the major target, again favoring mutations, and (3) decrease in cell-to-cell communication (gap-junctional intercellular communication) as a supposed test for promotion. None of these proposed assays are reliable indices for possible carcinogenic effects of chemicals or other agents; the scientific basis for this negative conclusion is discussed.

Carcinogenicity Tests↗
PubMed1985

Structure-function relationships in pancreatic islets: support for intraislet modulation of insulin secretion.

Pancreatic islet B cell function was studied in vitro using three structurally different preparations of islet tissues: isolated, intact islets, dispersed islet cells attached singly to microcarrier beads, and reaggregated islet cells. Mechanisms of intercellular communication are eliminated with single cell preparations, whereas in aggregates cell to cell communications are reestablished and a defined microenvironment restored. Perifusion studies measured nonstimulated and glucose- and arginine-stimulated insulin release from the three islet tissues. Insulin secretion rates were expressed as a function of cellular DNA content, permitting direct comparison between tissues. During perifusion with low (2.8 or 5.5 mM) glucose concentrations, secretion rates of single islet cells were up to 6-fold greater (P less than 0.001) than those of intact islets. Perifusion of islet cells with 2.8 mM glucose and 100 or 500 pg glucagon/ml had no effect whereas GH-release-inhibiting factor (330 and 1000 pg/ml) decreased nonstimulated insulin secretion rates by 15% (P less than 0.05). After reaggregation, basal insulin secretion rates were restored toward those of intact islets. Glucose (5.5-30 mM) and L-arginine (5-20 mM) elicited first phase insulin responses from single islet cells that were not significantly different from those observed with intact islets; in contrast, second phase responses of single islets to glucose were approximately 50% those seen with intact islets, and their second phase responses to arginine were absent. Single islet cell first and second phase insulin responses to 5.5 mM glucose were enhanced 2.2-fold (P less than 0.01) and 2.8-fold (P less than 0.05), respectively, in the presence of exogenous glucagon, resulting in secretory profiles characteristic of intact islets. Reaggregation of single islet cells was associated with markedly increased first and second phase insulin responses to both glucose and arginine stimulation. These data show that disruption of the islet microanatomy results in alteration of insulin secretory responses and that these effects can be reversed, in part by exogenous glucagon and GH-release-inhibiting factor, and by reaggregation. Although different mechanisms appear important for nonstimulated, first and second phase insulin release, the findings support a role for both direct intercellular communication and hormonal secretion by islet A and D cells in the modulation of B cell function.

Animals↗
PubMed1998

Molecular studies of human connexin 43 (Cx43) expression in isolated corporal tissue strips and cultured corporal smooth muscle cells.

Intercellular communication plays an important role in erectile function. The goal of this study, therefore, was two-fold. Firstly, to determine if cultured corporal smooth muscle cells provide a valid model system for evaluating the role of junctional communication to erectile physiology, and secondly, to explore the possibility that there may be age-related alterations in Cx43 mRNA expression. Human corpus cavernosum tissue was obtained from 31 patients with a mean age of 58 (range 27-89), while cell cultures were developed from 21 distinct patients with a mean age of 57 (range 26-59). Northern blots revealed that mRNA for Cx43 was expressed at detectable levels in all samples examined. It migrated as a transcript with an apparent size of 3.1 Kb. Western blots revealed the presence of multiple bands of Cx43 protein in both tissues and cells. However, Cx43 protein in tissue predominantly migrated as a 45 kDa band, while the Cx43 from cultured cells predominantly migrated as 41 kDa band. Cx43 mRNA expression was similarly heterogeneous in both frozen tissues and cultured cells. An approximately 3-5-fold increase in Cx43 mRNA levels was observed in cultured cells relative to frozen tissue, but the expression of Cx43 mRNA was not further altered upon passaging (p1-5). When Cx43 mRNA levels were normalized, and expressed as a ratio of the Cx43/beta-tubulin mRNA, there was a significant negative correlation between patient age and Cx43 levels on frozen tissues, but not on cultured cells. We conclude that: (1) There is similar heterogeneity/variability in Cx43 mRNA levels in frozen tissues and cultured cells derived from human corpus cavernosum. (2) That the expression of Cx43 mRNA in cultured cells is sufficiently stable, and similar to, expression levels in tissue as to provide a valid and physiologically relevant model system for further studying the role(s) of Cx43 in the regulation of penile erection. (3) There is a statistically significant, albeit modest, negative correlation between the Cx43/beta-tubulin ratio and patient age in frozen corporal tissue strips, but not on cultured corporal smooth muscle cells. Such observations provide further evidence for the plasticity of intercellular communication in the erectile process. Moreover, the similarities in the apparent regulation of Cx43 mRNA levels and that of the putative 'housekeeping' gene beta-tubulin, may suggest that Cx43 is constitutively synthesized in this tissue.

Adult↗
PubMed2003

Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action.

Follicle-stimulating hormone (FSH) controls the development of follicle-enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication, intracellular signaling, and up-regulation of steroidogenesis; the entire spectrum of genes regulated by FSH is not yet fully characterized. We have established monoclonal rat FSH-responsive granulosa cell lines that express FSH receptors at 20-fold higher rates than with primary cells, and thus increased the probability of yielding a distinct spectrum of genes modulated by FSH. Using Affymetrix DNA microarrays, we discovered 11 genes not reported earlier to be up-regulated by FSH and 9 genes not reported earlier to be down-regulated by FSH. Modulation of signal transduction associated with G-protein signaling, phosphorylation of proteins, and intracellular-extracellular ion balance was suggested by up-regulation of decay accelerating factor GPI-form precursor (DAF), membrane interacting protein RGS16, protein tyrosine phosphatase (PTPase), oxidative stress-inducible protein tyrosine phosphatase (OSIPTPase), and down-regulation of rat prostatic acid phosphatase (rPAP), Na+, K+-ATPase, and protein phosphatase 1beta. Elevation in granzyme-like proteins 1 and 3, and natural killer (NK) cell protease 1 (NKP-1) along with reduction in carboxypeptidase E indicates possible FSH-mediated preparation of the cells for apoptosis. Up-regulation of vascular endothelial growth factors indicates the ability of FSH to produce angiogenic factors upon their maturation; whereas, reduction in insulin-like growth factor binding protein (IGFBP3) indicates its increased potential to promote p53-induced apoptosis. Striking similarities in FSH modulation of gene expression were found in primary cultures of human granulosa cells obtained from IVF patients although these cells expressed only 1% of FSH receptor compared with immortalized rat cells, as indicated by microarray technique, which probably is in the normal range of expression of this receptor in nontransformed cells. These findings should increase our understanding of the mechanism of FSH action in stimulating development of the ovarian follicular cells, of intracellular and intercellular communication, and of increasing the potential of ovarian follicular cells to undergo apoptosis during the process of selection of the dominant follicle.

Animals↗