Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication

The naturally occurring stilbene/alexin trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is a promising agent for the prevention of cancer. We investigated the effect of resveratrol on gap-junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells because inhibition of GJIC is an important mechanism of tumor promotion. Seventeen to 50 microM resveratrol increased GJIC significantly by a factor of 1.3 compared with solvent vehicle controls, when the WB-F344 cells were exposed to resveratrol for 6 h. Most tumor promoters, including the phorbol ester TPA and the insecticide DDT, block GJIC. Resveratrol at 17-50 microM also significantly prevented down-regulation of GJIC by TPA and DDT, by a factor of 2.7 and 1.8, respectively. This recovery of GJIC from TPA inhibition was partly correlated with hindered hyperphosphorylation of Cx43. In conclusion, resveratrol was found to enhance GJIC and counteract the effects of tumor promoters on GJIC, and this is likely a mechanism that contributes to the antipromotional and anticarcinogenic properties of resveratrol.     

Changes in calcium concentrations in subcellular compartments of rat submandibular gland acinar cells induced by cholinergic stimulation

 The distribution of anti-Müllerian hormone (AMH) and laminin (Ln) α5 chain in differentiating rat testis and ovary were studied by immunohistochemistry. In the incipient embryonic male gonad a weak reaction for Ln α5 chain, but not for AMH, was detected. With further prenatal development, Ln α5 chain rapidly disappeared from the basement membrane (BM) of the incipient testicular cords in parallel with the appearance of AMH in the Sertoli cells. After birth, Ln α5 chain reappeared in the BMs of the cords with the decline and disappearance of AMH from the respective Sertoli cells. In the corresponding stages of the ovary, Ln α5 chain was present in the BM of the prenatal gonadal cords and in postnatal primordial follicles. The cells of those epithelia were negative for AMH. With the growth of the follicles, Ln α5 chain disappeared from the BM when AMH appeared in the epithelial follicular cells. The present results show that male and female gonadal epithelia negative for Ln α5 chain were positive for AMH, and that epithelia positive for Ln α5 chain were negative for AMH. Thus, epithelial Ln α5 chain and AMH as a product of the same cell seemed to exclude each other. The results require an explanation why Ln α5 chain has to be excluded from the BM of the epithelia during the secretion of AMH chain through the basal cell membrane to the surrounding tissues where it executes its important biological functions. These observations suggest a hypothesis that the production of both components is regulated by the same gene and factor system. Accepted: 14 January 1999     

Insulin-stimulated protein synthesis in submandibular acinar cells: interactions with adrenergic and cholinergic agonists

The effects of insulin and secretory agonists on amino acid incorporation into submandibular gland proteins were studied using isolated acinar cell aggregates. Insulin stimulated the incorporation of 3H-leucine into TCA-precipitable proteins in a rapid, dose-dependent manner (half-maximal response at 1 nM). Isoproterenol, a beta-adrenergic agonist, also stimulated amino acid incorporation, and this effect was mimicked by both dibutyryl cAMP and IBMX, a phosphodiesterase inhibitor. Although insulin further stimulated incorporation in the presence of isoproterenol and IBMX, no additional increase in the rate of synthesis was observed after stimulation by dibutyryl cAMP. High concentrations of carbamylcholine, a cholinergic agonist, inhibited both basal and insulin-stimulated incorporation. At low concentrations, however, carbamylcholine stimulated synthesis, and the effects of insulin and carbamylcholine were additive. A23187, a calcium ionophore, also inhibited 3H-leucine incorporation and insulin stimulation, but in contrast to carbamylcholine, low concentrations of A23187 neither inhibited nor enhanced the rate of synthesis. Thus, protein synthesis in the rat submandibular gland is regulated by both insulin and neurotransmitters. Whereas beta-adrenergic stimulation appears to be mediated through cAMP, the intracellular signals mediating the actions of insulin and cholinergic agonists remain to be elucidated.     

Potentiation by propofol of the response of rat submandibular acinar cells to purinergic agonists

The effect of propofol (2,6-diisopropylphenol) on the intracellular concentration of calcium ([Ca(2+)](i)) and on the response of rat submandibular acini to purinergic agonists was studied. By itself, propofol (60 to 200 microM) slowly increased the [Ca(2+)](i) without affecting the production of inositol phosphates. The increase of the [Ca(2+)](i) involved for about 50% the mobilization of thapsigargin-sensitive intracellular calcium pools. The rest of the calcium originated from a pool distinct from mitochondria. Propofol also increased the uptake of extracellular calcium but not manganese by a mechanism inhibited by nickel. The variation of the [Ca(2+)](i) by propofol provoked a decrease of cell volume measured by light scattering. Propofol increased the effect of a maximal concentration of extracellular ATP on the [Ca(2+)](i). This interaction could be observed when propofol and ATP were added simultaneously to the medium but not when propofol had been removed from the medium before adding ATP. Among ATP analogs, propofol only increased the response to benzoyl-ATP (Bz-ATP). The blockade of P2X(7) receptors with oxidized ATP or Coomassie blue did not prevent the interaction between propofol and ATP. The effect of propofol could also be observed even when the concentration of ATP(4-) was decreased by extracellular magnesium to such a level that only P2X(4) receptors could possibly be activated by the nucleotide. Propofol had no effect on the uptake of manganese, the formation of pores and the activation of phospholipase D in response to a P2X(7) agonist. These results exclude an interaction with this receptor.It is concluded that, in rat submandibular acini, propofol can increase the [Ca(2+)](i) and decrease the cell volume. Propofol can also modulate the activation of P2X(4) receptors by extracellular nucleotides. These effects are observed at concentrations of propofol reached during the induction of anesthesia and might explain why hypersalivation has been reported as one of the side-effects of propofol.     

Effects of cholinergic and α-adrenergic agonists on the monovalent ion content of rat submandibular gland acinar cells studied by X-ray microanalysis

 The effects of cholinergic and α-adrenergic stimulation (in vivo and in vitro) on the monovalent ion content of rat submandibular gland acinar cells were evaluated at the subcellular level by X-ray microanalysis. Fragments of glands or enzymatically dispersed acini were slam-frozen and cut into ultrathin cryosections. Spectra were collected from secretory granules, nucleus, the basal cytoplasm containing endoplasmic reticulum and the apical cytoplasm identified between secretory granules. No significant changes in Na and Cl content were observed after the isolation of acini, but the K concentration decreased compared with cells from in situ glands. The Cl and K content in all four compartments studied decreased significantly after cholinergic stimulation both in vivo and in vitro but in a more restricted fashion after α-adrenergic stimulation. Our findings indicate that: (1) the physiological mechanisms regulating the monovalent ion composition of submandibular cells are relatively well preserved in isolated acinar cells; (2) the results from in vivo experiments are in good agreement with those from in vitro experiments; and (3) the effects of cholinergic and α-adrenergic stimulation on the K⁺ and Cl^–efflux at the subcellular level are similar but the response is generally less with α-adrenergic stimulation. Accepted: 24 April 1997     

The modulation of gap-junctional intercellular communication by lipid rafts

Lipid rafts are specific microdomains of plasma membrane which are enriched in cholesterol and sphingolipids. These domains seem to favour the interactions of particular proteins and the regulation of signalling pathways in the cells. Recent data have shown that among the proteins, which are preferentially localized in lipid rafts, are connexins that are the structural proteins of gap junctions. Since gap junctional intercellular communication is involved in various cellular processes and pathologies such as cancer, we were interested to review the various observations concerning this specific localization of connexins in lipid rafts and its consequences on gap junctional intercellular communication capacity. In particular, we will focus our discussion on the role of the lipid raft-connexin connection in cancer progression. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Effects of cholinergic and adrenergic agonists on the secretion of fluid and protein by submandibular glands of the hamster and the rat

1. Significant differences were observed between the hamster and the rat in terms of the secretion of fluid and protein from submandibular glands in response to pilocarpine, phenylephrine and isoproterenol.2. In both the rat and the hamster the secretory responses induced by pilocarpine, phenylephrine and isoproterenol were inhibited by pretreatment with 4-DAMP, prazosin and metoprolol, respectively.3. These results suggest that the submandibular glands of the hamster and the rat have M₃-cholino-receptors, as well as α₁- and β₁-adrenoceptors, and that these receptors play different roles in the secretion of fluid and protein from hamster and rat submandibular glands.     

Apoptosis and inhibition of gap-junctional intercellular communication induced by LA-12, a novel hydrophobic platinum(IV) complex

A new hydrophobic platinum(IV) complex, LA-12, a very efficient anticancer drug lacking cross-resistance with cisplatin (CDDP), is now being tested in clinical trials. Here we investigated the apoptogenic activity of LA-12 and its effect on gap-junctional intercellular communication (GJIC) in the rat liver epithelial cell line WB-F344. LA-12 induced apoptosis much more efficiently than did CDDP due to a combination of rapid penetration into the cell and attack on DNA, leading to fast activation of p53 and caspase-3. Exposure of WB-F344 cells to LA-12 led to rapid induction of the time- and dose-dependent decrease in GJIC. On the molecular level, loss of GJIC induced by LA-12 was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated by the use of inhibitors of ERK activation. Inhibition of GJIC was linked to rapid hyperphosphorylation of connexin-43 and disappearance of connexon clusters from membranes, which was not observed in the case of CDDP.     

Effects of phenolics in Empire apples on hydrogen peroxide-induced inhibition of gap-junctional intercellular communication

The present study investigated antioxidant and antitumor-promoting activities of major phenolic phytochemicals of apples. The contents of each antioxidant in Empire apples was quantified and their contributions to total antioxidant activity of apples were determined using assay for inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced superoxide radical generation in cell culture model and expressed in vitamin C equivalent antioxidant capacity (VCEAC). The estimated contribution of major phenolics and vitamin C to total anitoxidant capacity of 100 g fresh Empire apples is as follows: quercetin (60.05 VCEAC) > chlorogenic acid (12.32) > phloretin (7.41) > procyanidin B2 (7.22) > vitamin C (6.61) > epicatechin (5.10) in superoxide radical scavenging assay. Recent reports suggest that the mechanism of carcinogenic process of hydrogen peroxide (H2O2) may be associated with the inhibition of gap-junctional intercellular communication (GJIC), which is involved in tumor promotion process. Apple extracts showed the protective effects against the inhibition of GJIC by H2O2 in a dose-dependent manner. Quercetin exerted the strongest protective effects among major antioxidants in apples on H2O2-induced inhibition of GJIC, following epicatechin, procyanidin B2, and vitamin C, while chlorogenic acid and phloretin had no effects. Our results indicate that cancer chemopreventive activity of apples is associated with the combined antioxidant capacity and antitumor-promoting activities of diverse antioxidants.     

Effects of pilocarpine on the secretory acinar cells in human submandibular glands

Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10(-5) M pilocarpine activated the outward rectifying-current, which was predominantly K(+) selective in the whole cell patch clamp study. The pilocarpine increased [Ca(2+)](i) in a concentration-dependent manner in the range of 10(-6) M to 10(-4) M. We found that both increases of [Ca(2+)](i) and outward rectifying- K(+) current were inhibited by 10(-5) M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca(2+)](i) transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K(+) channels, increasing [Ca(2+)](i) via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.     

Inhibition of gap-junctional intercellular communication between epithelial cells transformed by the activated H-ras-1 oncogene

In order to study the effects of an activated H-ras-1 oncogene on gap-junctional intercellular communication, we introduced the EJ/T24 H-ras-1 oncogene into cells of the epithelial Clone 9-3 cell line. Gap-junctional intercellular communication was significantly reduced in H-ras-1-transformed Clone 9-3 derivatives; this result shows that transformation by the activated H-ras-1 oncogene can inhibit gap-junctional intercellular communication. We postulate that the activated H-ras-1 oncogene product could mediate this effect through a change in the phosphorylation of the major gap-junction protein.     

Regulation by clozapine of calcium handling by rat submandibular acinar cells

The effect of clozapine on the intracellular concentration of calcium ([Ca2+](i)) in rat submandibular acinar cells was tested. By itself clozapine had no effect on the mobilization of intracellular pools of calcium or on the uptake of extracellular calcium. It inhibited the increase of the [Ca2+](i) in response to carbachol (half-maximal inhibitory concentrations, IC(50)=100nM) and to norepinephrine and epinephrine (IC(50)=10nM) without affecting the response to substance P, extracellular ATP or thapsigargin. Clozapine inhibited the uptake of extracellular calcium in response to epinephrine but not to substance P, ATP or thapsigargin. It also decreased the production of inositol phosphates elicited by epinephrine but not by substance P or fluoride. It is concluded that, by itself, clozapine has no effect on the [Ca2+](i) in rat salivary acinar cells. It selectively inhibits muscarinic and adrenergic receptors in the acinar plasma membrane.     

Cytodifferentiation of the acinar cells of the rat submandibular gland

The present study examines the cytodifferentiation of the acinar exocrine cells of the rat submandibular gland (SMG) at the ultrastructural level. Submandibular glands from rats at 14 days of gestation through 12 weeks postpartum were examined. The acinar cells of the SMG begin to develop at 15–16 days of gestation and are not fully differentiated until 3–4 weeks after birth. The earliest cells show multiple Golgi zones and a few strands of widely dilated rough endoplasmic reticulum (rer). Subsequently, the cells show an orderly sequence of organelle changes and rearrangement which leads to fully differentiated exocrine cells. A series of five morphologically distinct secretory granules is observed during differentiation and these granules serve as markers of the functional maturity of the cells. Attention is given in this study to the development of the apical-basal polarity of functional organelles typically seen in exocrine cells and its relationship to secretory granule production. The findings of the current study are compared with similar reports in the literature on the developing pancreas and parotid gland of the rat. It is concluded that different developmental pathways are followed to attain a similar functional capacity in the three organs.     

Uptake and metabolism of d‐glucose in isolated acinar and ductal cells from rat submandibular glands

The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d ‐glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d ‐[U‐¹⁴C]glucose and its non‐metabolised analogue 3‐O‐[¹⁴C‐methyl]‐d ‐glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d ‐glucose and 3‐O‐methyl‐d ‐glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d ‐glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d ‐glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca²⁺ or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin‐sensitive, cytochalasin B‐sensitive and phloridzin‐sensitive transport of d ‐glucose across the plasma membrane. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.     

Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet

Parotid glands of experimental animals fed a liquid diet are reported to show atrophy (Hall and Schneyer 1964; Wilborn and Schneyer 1970; Hand and Ho 1981; Scott et al. 1990; Scott and Gunn 1991). To clarify whether apoptosis and proliferation of acinar cells participate in atrophy of rat parotid glands induced by liquid diet, rats were fed a liquid diet and compared to pellet-fed controls. Parotid glands were removed at 3, 7, 14 or 21?days, weighed, and examined using transmission electron microscopy (TEM), and studied immunohistochemically for cleaved-caspase-3 (Casp-3), a marker of apoptotic cells, and 5-bromo-2′-deoxyuridine (BrdU), a marker for proliferating cells. Body weights of experimental rats fed liquid diets were not significantly different from controls fed pellet diets; however weights of experimental parotid glands were smaller than those of controls. In the experimental parotid glands, structures like apoptotic bodies were histologically observed in acini at each time point; more Casp-3-positive acinar cells were identified in experimental parotid glands than in the controls on days 3, 7, and 14. Experimental glands showed fewer BrdU-positive acinar cells at each time point. TEM confirmed typical apoptotic acinar cells in the atrophic glands. These findings suggest that increased acinar cell apoptosis and reduced acinar cell proliferation occur in atrophic parotid glands of rats fed a liquid diet.     

Effects of cholinergic and adrenergic agonists on the secretion of fluid and protein by submandibular glands of the guinea-pig and the mouse

Significant differences were observed between the guinea-pig and the mouse in terms of the secretion of fluid, protein and secretory granules from submandibular glands in response to pilocarpine, phenylephrine and isoproterenol. In both the guinea-pig and the mouse, the secretory responses induced by pilocarpine, phenylephrine and isoproterenol were inhibited by pretreatment with 4-DAMP, phentolamine and propranolol, respectively. The results suggest that the submandibular glands of the guinea-pig and the mouse have M₃-cholinoreceptors, as well as α- and β-adrenoceptors, and that these receptors play different roles in the secretion of fluid, protein and secretory granules from guinea-pig and mouse submandibular glands.     

Comparative effects of cytochalasin D on the protein discharge induced by alpha- and beta-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via alpha-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2 X 10(-6) M cytochalasin D when the discharge resulted from cholinergic, alpha- or beta-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.