Hormonal modulation of gap junctions in rat ovarian follicles

Summary Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24 h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.     

Hormonal modulation of ovarian interstitial cells with particular reference to gap junctions

Thin-section and freeze-fracture studies on the rat ovarian interstitial cells revealed reductions in the size and the number of gap junctions after pituitary ablation. Small gap junctions, however, persist as long as 90 days after hypophysectomy even though regressive cytoplasmic changes are completed 75 d earlier. Administration of exogenous human chorionic gonadotrophin (HCG) results in the restoration of the normal interstitial cell morphology which is accompanied by amplification of junctional membrane. Within 24 h of hormone application, gap junction growth is characterized by the appearance of formation plaques. These studies suggest that the effect of hormone on interstitial cell gap junctions is to modulate the junctional surface area.     

Hormonal control of uterine contraction: Characterization of cyclic AMP-dependent membrane properties in the myometrium

A mitochondria-free membrane fraction prepared from rat myometrium accumulated ⁴⁵Ca²⁺ in the presence of oxalic acid and ATP. The rate of transport of Ca²⁺ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca²⁺-transport. ATPase activity was stimulated by Mg²⁺; slight additional stimulation was obtained in the presence of Na⁺ and K⁺ but not in the presence of Ca²⁺. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca²⁺-transport suggest it has little to do with Ca²⁺ accumulation by the membranes.Cyclic AMP-induced increase in Ca²⁺-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10⁻⁴ M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca²⁺-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.     

Hormonal control of uterine contraction. Characterization od cyclic AMP-dependent membrane properties in the myometrium.

A mitochondria-free membrane fraction prepared from rat myometrium accumulated 45Ca2+ in the presence of oxalic acid and ATP. The rate of transport of Ca2+ into the membranous vesicles was increased by greater than 50% in the presence of 3',5'-cyclic AMP, but not by 2',3'-cyclic AMP or 5'AMP. Membrane ATPase activity was stimulated by Mg2+; slight additional stimulation was obtained in the presence of Na+ and K+ but not in the presence of Ca+2. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca2+-transport suggest it has little to do with Ca2+ accumulation by the membranes. Cyclic AMP-induced increase in Ca2+-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10(-4)M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca2+-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.     

Supramolecular dynamics of gap junctions

During the life cycle of a membrane protein its molecular structure may change and for aggregated proteins this process may be observed on the supramolecular level. Here we demonstrate that this is the case for gap junction channels which maintain cell-cell communication. Freshly synthesized connexins are integrated as hexamers (connexons) into the plasma membrane where they form plaques after pairing with connexons of an attached cell. We inhibited protein trafficking by applying the fungal metabolite brefeldin A (BFA), quantified cell-cell coupling by calcein transfer and fluorescence-activated flow cytometry, and examined the degradation and formation of gap junction plaques by indirect immunofluorescence and immunogold labeling. Under control conditions 50% of the detected plaques were ubiquitylated and less than 10% showed a two-dimensional crystalline packing. One hour after BFA reversal about 60% of the plaques were crystalline and ubiquitylation dropped to 14%. Label for ubiquitin was predominantly found on non-crystalline plaques. We, therefore, conclude that newly formed gap junction plaques are of crystalline morphology which changes to a pleomorphic structure when individual channels are modified during their aging process. This dynamic in plaque morphology correlates with channel inactivation and plaque ubiquitylation.     

Molecular organization of gap junctions

Highly purified gap junction fractions from heart and liver contain a single major protein component. The proteins isolated from different organs have apparent molecular weights of 26,000-30,000. Peptide mapping and partial sequencing show close homology of the hepatic junctional protein of different species. In contrast, no homologies can be detected when polypeptides from different tissues of the rat were compared by peptide mapping. Preliminary results from partial sequencing, however, show that the amino terminal regions of the liver and heart proteins are related to one another. Sequencing has not yet revealed any such homologies between the lens and the other junction proteins.     

Role of gap junctions in CO(2) chemoreception and respiratory control

Gap junctions are composed of connexins, which are organized into intercellular channels that form transmembrane pathways between neurons (cell-cell coupling), and in some cases, neurons and glia, for exchange of ions and small molecules (metabolic coupling) and ionic current (electrical coupling). Cell-cell coupling via gap junctions has been identified in brain stem neurons that function in CO(2)/H(+) chemoreception and respiratory rhythmogenesis; however, the exact roles of gap junctions in respiratory control are undetermined. Here we review the methods commonly used to study gap junctions in the mammalian brain stem under in vitro and in vivo conditions and briefly summarize the anatomical, pharmacological, and electrophysiological evidence to date supporting roles for cell-cell coupling in respiratory rhythmogenesis and central chemoreception. Specific research questions related to the role of gap junctions in respiratory control are suggested for future research.     

Hepatic gap junctions in the hepatocarcinogen-resistant DRH rat

Although the gap junction or connexin (Cx) is considered to be a tumor-suppressor, it is also required for tumor promotion. Therefore, we examined hepatic gap junctions in hepatocarcinogen-resistant (DRH) rats. Specifically, we investigated gap junction structure and Cx32 expression during normal conditions and in response to a hepatocarcinogen, 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). On a basal diet without 3'-MeDAB, hepatic gap junctions and Cx32 protein expression were greater in DRH rats than in control Donryu rats, as evidenced by morphometry, immunohistochemistry and immunoblotting. On a diet containing 3'-MeDAB, gap junctions and expressed Cx32 were increased significantly in Donryu rats, but not in DRH rats. In this condition, Donryu rats lost weight but DRH rats increased relative liver weight. After 3'-MeDAB treatment, cathepsin D expression in hepatocytes was significantly increased only in Donryu rats, indicating that DRH rats were less susceptible to 3'-MeDAB. The abundance of mitogen-activated protein kinase, some constituent of which might be associated with the degree of Cx protein phosphorylation, was reduced to a greater extent in Donryu than in DRH rats after 3'-MeDAB treatment. The resistance of DRH rats to carcinogenesis may be due partially to their stabilized gap junctions, which could coordinate metabolic coupling to evade 3'-MeDAB toxicity.     

Emerging role of gap junctions in epilepsy

This review highlights the contribution of gap junctions to the pathophysiology of epilepsy. The tissue expression and spatiotemporal regulation of connexins is discussed, and the phenotypes of specific connexin knockouts are considered. Electrophysiologic studies have implicated gap junctions in the generation of very fast oscillations preceding seizures. Gap junction inhibitors have shown powerful anticonvulsant effects, to date primarily in in vitro studies. Specific inhibition of gap junctions in vivo along with more detailed human tissue studies are needed to understand more fully the role of gap junctions in epileptogenesis.     

Hormonal regulation of gap junction differentiation

Thin-section, tracer, and freeze-cleave experiments on hypophysectomized Rana pipiens larvae reveal that gap junctions form between differentiating ependymoglial cells in response to thyroid hormone. These junctions assemble in large particle-free areas of the plasma membrane known as formation plaques. Between 20 and 40 h after hormone application, formation plaque area increases approximately 26-fold while gap junction area rises about 20-fold. The differentiation of these junctions requires the synthesis of new protein and probably RNA as well. On the basis of inhibitor experiments, it can be reported that formation plaques develop at about 16-20 h after hormone treatment and stages in the construction of gap junctions appear 4-8 h later. These studies suggest that gap junction subunits are synthesized and inserted into formation plaque membrane during the differentiation of the anuran ependymoglial cells.